USO095281.23B2

(12) Patent (10) Patent No.: US 9,528,123 B2 Raemaekers et al. (45) Date of Patent: *Dec. 27, 2016

(54) DSRNAAS CONTROL AGENT FOREIGN PATENT DOCUMENTS Applicant: DEVGEN N.V., Zwijnaarde (BE) WO 99.32619 A1 7, 1999 (71) WO 99.53050 A1 10, 1999 WO OO/O1846 A2 1, 2000 (72) Inventors: Romaan Raemaekers, De Pinte (BE); WO WOOO,5537.6 A1 9, 2000 Laurent Kubler, Beynost (FR); Els WO WO O1/O9301 A2 2, 2001 Vanbleu, Berlare (BE); Thierry Andre WO 01,34815 A1 5, 2001 Olivier Eddy Bogaert, Kortrijk (BE) WO O1/37654 A2 5, 2001 WO WOO 1/71042 A2 9, 2001 WO 01f88121 A1 11, 2001 (73) Assignee: Devgen NV (BE) WO O2/46432 A2 6, 2002 WO 03/004644 A1 1, 2003 (*) Notice: Subject to any disclaimer, the term of this WO 2004/001013 A2 12/2003 patent is extended or adjusted under 35 WO 2005/O19408 A2 3, 2005 U.S.C. 154(b) by 0 days. WO 2005/047300 A2 5, 2005 WO 2005/049841 A1 6, 2005 This patent is Subject to a terminal dis WO 2005.11.0068 A2 11/2005 claimer. WO WO 2005,116204 A1 12/2005 WO WO 2007/083193 A2 7/2007 (21) Appl. No.: 14/470,868 OTHER PUBLICATIONS (22) Filed: Aug. 27, 2014 Stapleton et al (2003). Accession No. AY069131; deposited Jan. (65) Prior Publication Data 2003.* Genbank Submission; NCBI; Accession No. ABL02283. Newton et US 2014/O373.197 A1 Dec. 18, 2014 al.; Nov. 19, 2010. Genbank Submission; NCBI; Accession No. AM106685. Dillon et al.; Dec. 23, 2005. Related U.S. Application Data Genbank Submission; NCBI; Accession No. AR508074. (62) Division of application No. 12/087,536, filed as Homburger et al.; Sep. 22, 2004. application No. PCT/EP2007/000286 on Jan. 12, Genbank Submission; NCBI: Accession No. BT001619. Stapleton 2007, now abandoned. et al.; Nov. 15, 2002. Genbank Submission; NCBI; Accession No. CB602554. Srinivasan et al.; Apr. 4, 2003. (60) Provisional application No. 60/875,356, filed on Dec. Genbank Submission; NCBI; Accession No. CK811880. Siviero et 18, 2006, provisional application No. 60/837,910, al.; Dec. 1, 2004. filed on Aug. 16, 2006, provisional application No. Genbank Submission; NCBI. Accession No. DN200332. Hunter et 60/771,160, filed on Feb. 7, 2006, provisional al.; Feb. 25, 2005. application No. 60/758,191, filed on Jan. 12, 2006. Genbank Submission; NCBI; Accession No. FW658194. Naito et al.; Apr. 18, 2001. (51) Int. C. Baumann et al., Sequence analysis of DNA fragments from the CI2N 5/82 (2006.01) genome of the primary endosymbiont of the whitefly Bemisia AOIN 57/16 (2006.01) tabaci. Curr Microbiol. Jan. 2004:48(1):77-81. AOIN 6L/00 (2006.01) Roberts et al., Loss of SEC-23 in Caenorhabditis elegans causes C07K I4/435 (2006.01) defects in oogenesis, morphogenesis, and extracellular matrix secre CI2N IS/IT3 (2010.01) tion. Mol Biol Cell. Nov. 2003;14(11):4414-26. Epub Aug. 7, 2003. (52) U.S. C. Robertson et al., Diversity of odourant binding proteins revealed by CPC ...... CI2N 15/8286 (2013.01); A0IN 57/16 an expressed sequence tag project on male Manduca sexta moth (2013.01); A0IN 61/00 (2013.01); C07K antennae. Insect Mol Biol. Nov. 1999;8(4):501-18. 14/4354 (2013.01); C07K 14/43527 (2013.01); Severson et al., Linkage map organization of expressed sequence CI2N 15/113 (2013.01); C12N 15/8282 tags and sequence tagged sites in the mosquito, Aedes aegypti. (2013.01); C12N 15/8285 (2013.01); C12N Insect Mol Biol. Aug. 2002; 11(4):371-8. 23 10/14 (2013.01) (Continued) (58) Field of Classification Search None Primary Examiner — Medina A Ibrahim See application file for complete search history. (74) Attorney, Agent, or Firm — Yoshimi D. Barron (56) References Cited (57) ABSTRACT The present invention relates to methods for controlling U.S. PATENT DOCUMENTS infestation using double stranded RNA molecules. The 6,703,491 B1 3/2004 Homburger et al. invention provides methods for making transgenic plants 2003/O1500 17 A1 8, 2003 Mesa et al. that express the double stranded RNA molecules, as well as 2005/0287570 A1 12, 2005 Mounts pesticidal agents and commodity products produced by the 2006, 0021087 A1 1/2006 Baum et al...... 800,279 2006/0272049 A1 11/2006 Waterhouse et al...... 800,279 inventive plants. 2008/O113351 A1 5, 2008 Naito et al. 2009,0285784 A1 1 1/2009 Raemaekers et al. 30 Claims, 16 Drawing Sheets US 9,528,123 B2 Page 2

(56) References Cited Fire, RNA-triggered gene silencing. TIG. Sep. 1999; 15(9):358-63. Koyama, Artificial rearing and nutritional physiology of the planthoppers and leafhoppers (: Delphacidae and OTHER PUBLICATIONS Deltocephalidae) on a holidic diet. Japan Agricultural Research Soares et al., Capillary feeding of specific dsRNA induces silencing of the isac gene in ymphal Ixodes scapularis ticks. Insect Mol Biol. Quarterly, 1988:22(1):20-27. Aug. 2005;14(4):443-52. Rice et al., EMBOSS: the European Molecular Biology Open Timmons et al., Ingestion of bacterially expressed dsRNAS can Software Suite. TIG. Jun. 2000:16(6):276-7. produce specific and potent genetic interference in Caenorhabditis Sharp, RNA interference—2001. Genes Dev. Mar. 1, elegans. Gene. Jan. 24, 2001:263(1-2): 103-12. 2001; 15(5):485-90. Zhu et al., Ingested RNA interference for managing the populations Whyard et al., Ingested double-stranded RNAS can act as of the Colorado , decemlineata. Pest specific . Insect Biochem Mol Biol. Nov. Manag Sci. Feb. 2011:67(2): 175-82. Epub Nov. 8, 2010. 2009;39(11):824-32. Epub Oct. 6, 2009. Genbank Submission; NCBI. Accession No. AMO48926; Longhorn; Thomas et al. The Plant Journal (2001) 25(4), pp. 417-425. Jul. 16, 2005. Genbank Submission; NCBI; Accession No. CO334556, 2004. Genbank Submission; NCBI. Accession No. Q4GXU7; Longhornet Hamada et al., Effects on RNA interference in gene expression al.; Nov. 28, 2006. (RNAi) in cultured mammalian cells of mismatches and the intro Clough et al., Floral dip: a simplified method for Agrobacterium duction of chemical modifications at the 3'-ends of siRNAs. Anti mediated transformation of Arabidopsis thaliana. Plant J. Dec. sense Nucleic Acid Drug Dev, Oct. 2002; 12(5):301-9. 1998:16(6):735-43. Qiu et al. A computational study of off-target effects of RNA Febvay et al., Influence of the amino acid balance on the improve interference. Nucleic Acids Res. Mar. 30, 2005:33(6):1834-47. ment of an artificial diet for a biotype of Acyrthosiphon pisum (Homoptera: Aphididae). Can. J. Zool. 1988;66(11):2449-2453. * cited by examiner U.S. Patent Dec. 27, 2016 Sheet 1 of 16 US 9,528,123 B2

120 . i...I100 ...... -- target 6 . . : -8...target 7 on 80 s -- target 10 60 ox- target 1 40 ... target 14 - -8-glp dsRNA o 20 -a-diet only ; : s asS. O. 9.

days on diet

FGURE 1-LO

OO . -- target s'ss's&-target 2 80 - ox target 3 60 - : ... target 15 -- target 16 40 . -e-glp dsRNA

FGURE 2-D U.S. Patent Dec. 27, 2016 Sheet 2 of 16 US 9,528,123 B2

3 O

days on diet

FGURE 3-D

3 i20 : 100 six-0-mm-0-mm-0 r . &A 8 -- untreated control g 80 X. ^, .8. LD014 w -- LD014 F1 60 - Nex- : ox-LD014 Cl as 40 --LD014 F2 --LD014 C2 s 20 -\-- “”” 9. as O - s 9. O 2 3 4 5 6 7 8 days on diet

FGURE 4-D U.S. Patent Dec. 27, 2016 Sheet 3 of 16 US 9,528,123 B2

------

-0-0 ugfu ~8- ugful -&-0, ugful

- V - v. 3:... 0,0 ugful ~%-0,001 ugful -8-0,000 ugful ...a... 0.0000 ugfu

------re-or------O 2 3 4 5 6 7 8 9 O 2 3 4 5 days on diet

FIGURE 5-LD (a)

-- ugfu -- 0,1 ugfu 3. 0,0 ugful -- 0.001 ugful -e-0,0001 ugful -a-0,00001 ugul

------8------Brrerrerrer-km-- O 1 2 3 4 5 6 7 8 9 O 1 2 3 4 5 days on diet

FIGURE 5-LD (b) U.S. Patent Dec. 27, 2016 Sheet 4 of 16 US 9,528,123 B2

-0-0 ugful -- ugful --0.1 ugful 4.4.4.4. 0.01 ugful --0,001 ugful -e-0,0001 ugful -a-0,00001 ug/ul

O 1 2 3 4 5 6 7 8 9 O 2 3 4 5 days on diet

FIGURE 5-LD (c)

-- 0,1 ugful

60 - rx - W - Y 0.0 ugfui --0.001 ug?ul 40 - -e-0,0001 ugful 2O war. 0.00001 ugui

O ...... x:...e. s O 2 3 4 5 6 7 8 9 O 2 3 4 5 days on diet

FIGURE 5-LD (d) U.S. Patent Dec. 27, 2016 Sheet S of 16 US 9,528,123 B2

-0-0 Ugful x- ugful -- 0,1 ugfu 3.0,01 ugful --0,001 ugui -e-0,0001 uglut || -a-0,00001 ugui recip----ex------... O 1 2 3 4 5 6 7 8 9 O 1 2 3 4 5 days on diet

isssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss

20 ---r -0-0 ugful -- ugful --0, Ligful coco 0.01 ugui --0,001 ugful i.e. 0.0001 ugul -A-0,00001 uglul

------FIGURE 5-LD (f) U.S. Patent Dec. 27, 2016 Sheet 6 of 16 US 9,528,123 B2

-- 0,1 ugful x· 0.01 ugful -- 0.001 ugful -e-0,0001 ugul i-A-0.00001 ugut

300 :

---. Ougi: -- 0. ugli -- 0.31 tigiu -x-0.001 gul : ck-0,000 ugu : ce:0.0000 gul:

3 4. 5 8. 7 8 9. 10 2 33 A. 5

FIGURE 5-LD (h) U.S. Patent Dec. 27, 2016 Sheet 7 of 16 US 9,528,123 B2

:

Atep :::: as Aep Exxx

:::::::::

E::::::::::::: x:

*XXXXXXXXXXXXXXXXXXXXX.

Snipe O go equiriu

FIGURE 6-D U.S. Patent Dec. 27, 2016 Sheet 8 of 16 US 9,528,123 B2

f 20 40 68 80 O Mortality

FIGURE 7-D U.S. Patent Dec. 27, 2016 Sheet 9 of 16 US 9,528,123 B2

days on ea

FIGURE 1-PC (a)

at gets age 3

-3-gic dsNA --8.5% on X.

days on eaf

FIGURE 1-PC (b) U.S. Patent Dec. 27, 2016 Sheet 10 of 16 US 9,528,123 B2

00 -

80

--0. gfu -A-001 ugu -x-0.001 jgful 40 - :-x-0,000 ugu

20

days on leaf

FIGURE 2-PC (a)

OO

80

:-0-0 ugful 6 - :--0.1 ugu -- 0:0 ugu :-X-000 tugu i-x-0.0001 tags

FIGURE 2-PC (b) U.S. Patent Dec. 27, 2016 Sheet 11 of 16 US 9,528,123 B2

2.

80 - -in-target it -a-target 5 s :-x-target 16 --gip dsRNA -0- intreated

days on leaf

FGURE EV

8 -- targe; it : -- target 18 : 8 - -A-target 16 : ress-gp dsRNA : --- intfeated : 4.

2

days of leaf FIGURE 2-EV (a) U.S. Patent Dec. 27, 2016 Sheet 12 of 16 US 9,528,123 B2

FIGURE2-EV (b) U.S. Patent Dec. 27, 2016 Sheet 13 of 16 US 9,528,123 B2

8

70

60 . is diet only S 3 target 14,

days on diet FIGURE - C

diet only target 27 eatient FEGURE MP U.S. Patent Dec. 27, 2016 Sheet 14 of 16 US 9,528,123 B2

&S:X 3 S -x- diet only -a-glp 9. i-A-N 002 -(- NL003 -(-NL005 -- NL010

s------

~8- diet only -8-gfp

------e.------,

days on diet

FIGURE 1-NL (b) U.S. Patent Dec. 27, 2016 Sheet 15 of 16 US 9,528,123 B2

-- diet only -8-gfp dsRNA

-- NL014

FIGURE 1-NL (c)

go 100 x S-SX. """"""""""""""""""""""""""""""""""": -8- diet only -8-gfp dsRNA S 60. s st -- NL013 : -0. NL015 40 -v- SKS-X-sur- KY NO2 e s s E

days on diet

FIGURE 1-Ni (d) U.S. Patent Dec. 27, 2016 Sheet 16 of 16 US 9,528,123 B2

20 ------:

OO

-8-1 ugful -a- 0.2 ugfu -0-0.08 ugful

FIGURE 2-N- US 9,528,123 B2 1. 2 DSRNAAS INSECT CONTROL AGENT belonging to the family, especially potato (Sola num tuberosum), but also ( lycopersicum), CROSS REFERENCE TO RELATED (Solanum melongena), (Solanum cap APPLICATIONS sicum), and nightshade (for example, Solanum aculeastrum, S. bulbocastianum, S. cardiophyllum, S. douglasii, S. dulca This application is a divisional application of and claims mara, S. lanceolatum, S. robustum, and S. triquetrum), priority to U.S. patent application Ser. No. 12/087,536 filed particularly the control of coleopteran pests. on Jan. 13, 2009 which is a national stage filing under 35 Biological control using extract from neem seed has been U.S.C. S371 of International Application No. PCT/EP2007/ shown to work against coleopteran pests of vegetables. 000286, filed on Jan. 12, 2007, which claims benefit of 10 Commercially available neem-based insecticides have 60/758,191, filed on Jan. 12, 2006, and claims benefit of azadirachtin as the primary active ingredient. These insec 60/771,160, filed on Feb. 7, 2006, and claims benefit of ticides are applicable to a broad spectrum of . They 60/837,910, filed on Aug. 16, 2006, and claims benefit of act as , azadirachtin prevents insects 60/875,356, filed on Dec. 18, 2006, the contents of each of from molting by inhibiting production of an insect hormone, which are herein incorporated by reference in their entire 15 ecdysone. ties. Biological control using protein Cry3A from varieties tenebrionis and San diego, and SEQUENCE LISTING derived insecticidal proteins are alternatives to chemical A Sequence Listing in ASCII text format, submitted under control. The Bt toxin protein is effective in controlling 37 CFRS1.821, entitled “80388.txt”, 736 kilobytes in size, larvae either as formulations sprayed generated on Aug. 26, 2014 and filed via EFS-Web is onto the foliage or expressed in the leaves of potatoes. provided in lieu of a paper copy. This sequence listing is An alternative biological agent is dsRNA. Over the last hereby incorporated by reference into the specification for few years, down-regulation of genes (also referred to as its disclosures. 25 'gene silencing’) in multicellular organisms by means of RNA interference or “RNAi has become a well-established FIELD OF THE INVENTION technique. RNA interference or “RNAi is a process of sequence The present invention relates to the field of double specific down-regulation of gene expression (also referred to stranded RNA (dsRNA)-mediated gene silencing in insect 30 as "gene silencing or “RNA-mediated gene silencing) species. More particularly, the present invention relates to initiated by double-stranded RNA (dsRNA) that is comple genetic constructs designed for the expression of dsRNA mentary in sequence to a region of the target gene to be corresponding to novel target genes. These constructs are down-regulated (Fire, A. Trends Genet. Vol. 15, 358-363, particularly useful in RNAi-mediated plant . The 1999: Sharp, P. A. Genes Dev. Vol. 15, 485-490, 2001). invention further relates to methods for controlling insects, 35 Over the last few years, down-regulation of target genes methods for preventing insect infestation and methods for in multicellular organisms by means of RNA interference down-regulating gene expression in insects using RNAi. (RNAi) has become a well established technique. Reference The invention also relates to transgenic plants resistant to may be made to International Applications WO 99/32619 insect infestation. (Carnegie Institution) and WO 00/01846 (by Applicant). 40 DsRNA gene silencing finds application in many different BACKGROUND TO THE INVENTION areas, such as for example dsRNA mediated gene silencing in clinical applications (WO2004/001013) and in plants. In The environment is replete with pests and numerous plants, dsRNA constructs useful for gene silencing have also methods have attempted to control pests infestations of been designed to be cleaved and to be processed into short plants. Commercial crops are often the targets of insect 45 interfering RNAs (siRNAs). attack. Substantial progress has been made in the last few RNAi has also been proposed as a means of protecting decades towards developing more efficient methods and plants against plant parasitic , i.e. by expressing compositions for controlling insect infestation in plants. in the plant (e.g. in the entire plant, or in a part, tissue or cell Chemical have been very effective in eradicat of a plant) one or more nucleotide sequences that form a ing pest infestation. However, there are several disadvan 50 dsRNA fragment that corresponds to a target gene in the tages to using chemical pesticidal agents. Not only are they plant parasitic that is essential for its growth, potentially detrimental to the environment, but they are not reproduction and/or survival. Reference may be made to the selective and are harmful to various crops and non-target International Application WO 00/01846 (by Applicant) and fauna. Chemical pesticides persist in the environment and U.S. Pat. No. 6,506,559 (based on WO99/32619). generally are slow to be metabolized, if at all. They accu 55 Although the technique of RNAi has been generally mulate in the food chain, and particularly in the higher known in the art in plants, C. elegans and mammalian cells predator species where they can act as mutagens and/or for some years, to date little is known about the use of RNAi carcinogens to cause irreversible and deleterious genetic to down-regulate gene expression in insects. Since the filing modifications. There has thus been continued controversy in and publication of the WO 00/01846 and WO 99/32619 the use of chemical insecticides to combat crop pests. They 60 applications, only few other applications have been pub can rapidly develop resistance against these insecticides lished that relate to the use of RNAi to protect plants against because of repetitive usage of the same or of insects. These include the International Applications WO insecticides having the same mode of action, and because 01/37654 (DNA Plant Technologies), WO 2005/019408 accumulation also results in the development of resistance to (Bar Ilan University), WO 2005/049841 (CSIRO, Bayer the agents in species higher up the evolutionary ladder. 65 Cropscience), WO 05/047300 (University of Utah Research Control of insect pests on agronomically important crops foundation), and the US application 2003/0015.0017 (Mesa is important, particularly insect pests which damage plants et al.). US 9,528,123 B2 3 4 The present invention provides target genes and con 2354, 2359, 2364, 2366, 2368, 2370, 2372,2384 to 2460, structs useful in the RNAi-mediated insect pest control, 2461, 2466, 2471, 2476, 2481 or 2486, or the complement especially the control of insect plant pathogens. The present thereof, invention also provides methods for controlling insect pest (ii) sequences which are at least 70%, preferably at least infestation by repressing, delaying, or otherwise reducing 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, target gene expression within a particular insect pest. 97%, 98% or 99% identical to a sequence represented by any of SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49 DESCRIPTION OF THE INVENTION to 158, 159, 160-163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 240 to 247, 249, 251, 253, 255, The present invention describes a novel non-compound, 10 257, 259,275 to 472, 473,478,483,488, 493,498, 503, 508 non-protein based approach for the control of insect crop to 513,515,517,519, 521,533 to 575, 576,581,586, 591, pests. The active ingredient is a nucleic acid, a double 596,601, 603, 605, 607, 609, 621 to 767, 768, 773,778,783, stranded RNA (dsRNA), which can be used as an insecti 788, 793, 795, 797,799,801, 813 to 862, 863, 868,873, 878, cidal formulation. In another embodiment, the dsRNA can 883, 888,890, 892, 894, 896,908 to 1040, 1041, 1046, 1051, 15 1056, 1061, 1066 to 1071, 1073, 1075, 1077, 1079, 1081, be expressed constitutively in the host plant, plant part, plant 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, cell or seed to protect the plant against chewing insects 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161 to 1571, especially coleopterans Such as . The sequence of the 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, dsRNA corresponds to part or whole of an essential insect 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, gene and causes downregulation of the insect target via RNA 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, interference (RNAi). As a result of the downregulation of 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730 to 2039, mRNA, the dsRNA prevents expression of the target insect 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080, protein and hence causes death, growth arrest or sterility of 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120 to the insect. 2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, The methods of the invention can find practical applica 25 2370, 2372,2384 to 2460, 2461, 2466, 2471, 2476,2481 or tion in any area of technology where it is desirable to inhibit 2486, or the complement thereof, and viability, growth, development or reproduction of the insect, (iii) sequences comprising at least 17 contiguous nucleo or to decrease pathogenicity or infectivity of the insect. The tides of any of the sequences represented by SEQID NOS 1. methods of the invention further find practical application 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, 160-163, where it is desirable to specifically down-regulate expres 30 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, sion of one or more target genes in an insect. Particularly 230, 240 to 247, 249, 251, 253, 255, 257, 259, 275 to 472, useful practical applications include, but are not limited to, 473,478,483,488, 493,498, 503, 508 to 513,515,517,519, protecting plants against insect pest infestation. 521,533 to 575, 576,581,586,591,596,601, 603, 605, 607, In accordance with one embodiment the invention relates 609, 621 to 767, 768,773,778,783,788, 793, 795, 797,799, 35 801, 813 to 862, 863, 868,873, 878, 883, 888,890, 892, 894, to a method for controlling insect growth on a cell or an 896, 908 to 1040, 1041, 1046, 1051, 1056, 1061, 1066 to organism, or for preventing insect infestation of a cell or an 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, organism Susceptible to insect infection, comprising con 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, tacting insects with a double-stranded RNA, wherein the 1107, 1109, 1111, 1113, 1161 to 1571, 1572, 1577, 1582, double-stranded RNA comprises annealed complementary 40 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, Strands, one of which has a nucleotide sequence which is 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, complementary to at least part of the nucleotide sequence of 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, an insect target gene, whereby the double-stranded RNA is 1698, 1700, 1702, 1704, 1730 to 2039, 2040, 2045, 2050, taken up by the insect and thereby controls growth or 2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, prevents infestation. 45 2100, 2102, 2104, 2106, 2108, 2120 to 2338, 2339, 2344, The present invention therefore provides isolated novel 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372,2384 to nucleotide sequences of insect target genes, said isolated 2460, 2461,2466, 2471,2476,2481 or 2486, or the comple nucleotide sequences comprising at least one nucleic acid ment thereof, sequence selected from the group comprising: or wherein said nucleic acid sequence is an orthologue of (i) sequences represented by any of SEQID NOS 1, 3, 5, 50 a gene comprising at least 17 contiguous nucleotides of any 7, 9, 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, 160-163, 168, of SEQID NOs 49 to 158, 275 to 472, 533 to 575, 621 to 173, 178, 183, 188, 193, 198,203, 208, 215, 220, 225, 230, 767, 813 to 862, 908 to 1040, 1161 to 1571, 1730 to 2039, 240 to 247, 249, 251, 253, 255, 257, 259, 275 to 472, 473, 2120 to 2338, 2384 to 2460, or a complement thereof, 478,483,488, 493,498, 503, 508 to 513,515,517,519, 521, said nucleic acid sequences being useful for preparing the 533 to 575, 576,581,586,591,596,601, 603, 605, 607, 609, 55 double stranded RNAs of the invention for controlling insect 621 to 767, 768,773,778,783,788,793, 795, 797,799,801, growth. 813 to 862, 863, 868,873, 878, 883, 888,890, 892, 894, 896, “Controlling pests' as used in the present invention means 908 to 1040, 1041, 1046, 1051, 1056, 1061, 1066 to 1071, killing pests, or preventing pests to develop, or to grow or 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, preventing pests to infect or infest. Controlling pests as used 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 60 herein also encompasses controlling pest progeny (develop 1109, 1111, 1113, 1161 to 1571, 1572, 1577, 1582, 1587, ment of eggs). Controlling pests as used herein also encom 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, passes inhibiting viability, growth, development or repro 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, duction of the pest, or to decrease pathogenicity or 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, infectivity of the pest. The compounds and/or compositions 1700, 1702, 1704, 1730 to 2039, 2040, 2045, 2050, 2055, 65 described herein, may be used to keep an organism healthy 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, and may be used curatively, preventively or systematically 2102, 2104, 2106, 2108, 2120 to 2338, 2339, 2344, 2349, to control pests or to avoid pest growth or development or US 9,528,123 B2 5 6 infection or infestation. Particular pests envisaged in the In a more preferred aspect, the invention provides a present invention are plant pathogenic insect pests. "Con method for down-regulating expression of at least one target trolling insects' as used herein thus also encompasses con gene in a target organism (which is capable of ingesting a trolling insect progeny (such as development of eggs). host cell, or extracts thereof) comprising feeding a hostplant, Controlling insects as used herein also encompasses inhib plant part, plant cell or seed to the target organism which iting viability, growth, development or reproduction of the hostplant, plant part, plant cell or seed expresses a double insect, or decreasing pathogenicity or infectivity of the Stranded RNA molecule comprising a nucleotide sequence insect. In the present invention, controlling insects may complementary to or representing the RNA equivalent of at inhibit a biological activity in a insect, resulting in one or least part of the nucleotide sequence of the at least one target more of the following attributes: reduction in feeding by the 10 gene, whereby the ingestion of the host cell, host plant, plant insect, reduction in viability of the insect, death of the insect, part, plant cell or seed by the target organism causes and/or inhibition of differentiation and development of the insect, leads to down-regulation of expression of the at least one absence of or reduced capacity for sexual reproduction by target gene. the insect, muscle formation, juvenile hormone formation, The invention provides for use of a plant, plant part, plant juvenile hormone regulation, ion regulation and transport, 15 cell or seed as defined herein for down regulation of expres maintenance of cell membrane potential, amino acid bio sion of an insect target gene. In more detailed terms, the synthesis, amino acid degradation, sperm formation, phero invention provides for use of a host cell as defined herein mone synthesis, pheromone sensing, antennae formation, and/or an RNA molecule comprising a nucleotide sequence wing formation, leg formation, development and differen that is the RNA complement of or that represents the RNA tiation, egg formation, larval maturation, digestive enzyme equivalent of at least part of the nucleotide sequence of a formation, haemolymph synthesis, haemolymph mainte target gene from a target organism, as produced by tran nance, neurotransmission, cell division, energy metabolism, Scription of a nucleic acid molecule in a plant, plant part, respiration, apoptosis, and any component of a eukaryotic plant cell or seed, for instance in the manufacture of a cells cytoskeletal structure. Such as, for example, actins and commodity product, for down regulation of expression of a tubulins. The compounds and/or compositions described 25 target gene. Suitable target genes and target organisms in herein, may be used to keep an organism healthy and may be respect of the invention are discussed below in further detail. used curatively, preventively or systematically to control a According to one embodiment, the methods of the inven insect or to avoid insect growth or development or infection tion rely on a GMO approach wherein the double-stranded or infestation. Thus, the invention may allow previously RNA is expressed by a cell or an organism infested with or Susceptible organisms to develop resistance against infesta 30 susceptible to infestation by insects. Preferably, said cell is tion by the insect organism. a plant cell or said organism is a plant. The expression "complementary to at least part of as The present invention thus also relates to a method for used herein means that the nucleotide sequence is fully producing a plant resistant to a plant pathogenic insect, complementary to the nucleotide sequence of the target over comprising: more than two nucleotides, for instance over at least 15, 16, 35 transforming a plant cell with a recombinant construct 17, 18, 19, 20, 21, 22, 23, 24 or more contiguous nucleo comprising at least one regulatory sequence operably tides. linked to a sequence complementary to at least part of According to a further embodiment, the invention relates (a) a nucleotide sequence of a target insect gene to a method for down-regulating expression of a target gene Selected from the group consisting of: in an insect, comprising contacting said insect with a double 40 (i) sequences which are at least 75% identical to a stranded RNA, wherein the double-stranded RNA comprises sequence represented by any of SEQID NOs 1, 3, 5, annealed complementary Strands, one of which has a nucleo 7, 9, 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, tide sequence which is complementary to at least part of the 160-163, 168, 173, 178, 183, 188, 193, 198, 203, nucleotide sequence of the insect target gene to be down 208, 215, 220, 225, 230, 247, 249, 251, 253, 255, regulated, whereby the double-stranded RNA is taken up 45 257, 259, 275 to 472, 473, 478, 483, 488, 493, 498, into the insect and thereby down-regulates expression of the 503, 513,515,517,519, 521,533 to 575, 576, 581, insect target gene. 586, 591, 596,601, 603, 605, 607, 609, 621 to 767, Whenever the term 'a' is used within the context of “a 768,773,778,783,788, 793, 795, 797,799,801, 813 target gene', this means “at least one' target gene. The same to 862, 863, 868, 873, 878, 883, 888, 890, 892, 894, applies for 'a' target organism meaning “at least one target 50 896, 908 to 1040, 1041, 1046, 1051, 1056, 1061, organism, and “a” RNA molecule or host cell meaning “at 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, least one' RNA molecule or host cell. This is also detailed 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, further below. 1103, 1105, 1107, 1109, 1111, 1113, 1161 to 1571, According to one embodiment, the methods of the inven 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, tion rely on uptake by the insect of double-stranded RNA 55 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, present outside of the insect (e. g. by feeding) and does not 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, require expression of double-stranded RNA within cells of 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, the insect. In addition, the present invention also encom 1702, 1704, 1730 to 2039, 2040, 2045, 2050, 2055, passes methods as described above wherein the insect is 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, contacted with a composition comprising the double 60 2100, 2102, 2104, 2106, 2108, 2120 to 2338, 2339, stranded RNA. 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, The invention further provides a method for down-regu 2372,2384 to 2460, 2461, 2466, 2471,2476 or 2481, lating expression of at least one target gene in a target or the complement thereof, organism (which is capable of ingesting a plant, plant part, (ii) sequences comprising at least 17 contiguous plant cell or seeds) comprising feeding a plant, plant part, 65 nucleotides of any of SEQ ID Nos 1, 3, 5, 7, 9, 11, plant cell or seed to the target organism which plant, plant 13, 15, 17, 19, 21, 23, 49 to 158, 159, 160-163, 168, part, plant cell or seed expresses double-stranded RNA. 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, US 9,528,123 B2 7 8 225, 230, 247, 249, 251, 253, 255, 257, 259, 275 to In preferred, but non-limiting, embodiments and methods 472, 473, 478, 483, 488, 493, 498, 503, 513, 515, of the invention the insect is chosen from the group con 517,519, 521,533 to 575, 576, 581,586,591, 596, sisting of an insect which is a plant pest, such as but not 601, 603, 605, 607, 609, 621 to 767, 768, 773, 778, limited to Nilaparvata spp. (e.g. N. lugens (brown planthop 783, 788, 793, 795, 797, 799, 801, 813 to 862, 863, per)); Laodelphax spp. (e.g. L. striatellus (Small brown 868, 873, 878, 883, 888, 890, 892, 894, 896, 908 to planthopper)); Nephotettix spp. (e.g. N. virescens or N. 1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, cincticeps (green leafhopper), or N. nigropictus (rice leaf 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, hopper)); Sogatella spp. (e.g. S. fircifera (white-backed 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, planthopper)); Blissus spp. (e.g. B. leucopterus leucopterus 10 (chinch bug)); Scotinophora spp. (e.g. S. vermidulate (rice 1107, 1109, 1111, 1113, 1161 to 1571, 1572, 1577, blackbug)); Acrosternum spp. (e.g. A. hilare (green Stink 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, bug)); Parnara spp. (e.g. P. guttata (rice skipper)); Chilo 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, spp. (e.g. C. suppressalis (rice striped stem borer), C. 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, auricilius (gold-fringed stem borer), or C. polychlysus (dark 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 15 headed stem borer)); Chilotraea spp. (e.g. C. polychrysa 1730 to 2039, 2040, 2045, 2050, 2055, 2060, 2065, (rice stalk borer); Sesamia spp. (e.g. S. inferens (pink rice 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, borer); Tryporyza spp. (e.g. T. innotata (white rice borer), or 2104, 2106, 2108, 2120 to 2338, 2339, 2344, 2349, T. incertulas (yellow rice borer)). Cnaphalocrocis spp. (e.g. 2354, 2359, 2364, 2366, 2368, 2370, 2372,2384 to C. medinalis (rice leafroller)); Agromyza spp. (e.g. A. oryzae 2460, 2461, 2466, 2471, 2476 or 2481, or the (leafminer), or A. parvicornis (corn blot leafminer)); Dia complement thereof, and traea spp. (e.g. D. Saccharalis (Sugarcane borer), or D. (iii) sequences comprising a sense strand comprising a grandiosella (Southwestern corn borer)); Narnaga spp. (e.g. nucleotide sequence of (i) and an antisense Strand N. aenescens (green rice caterpillar)); Xanthodes spp. (e.g. comprising the complement of said nucleotide X. transverse (green caterpillar)); Spodoptera spp. (e.g. S. sequence of (i), wherein the transcript encoded by 25 frugiperda (fall armyworm), S. exigua (beet armyworm), S. said nucleotide sequence is capable of forming a littoralis (climbing cutworm) or S. praefica (western yel double-stranded RNA, lowstriped armyworm)); Mythinna spp. (e.g. Mythmna or (b) a nucleotide sequence which is an insect orthologue (Pseudaletia) seperata (armyworm)); Helicoverpa spp. (e.g. of a gene comprising at least 17 contiguous nucleotides H. Zea (corn earworm)); Colaspis spp. (e.g. C. brunnea of any of SEQ ID Nos 49 to 158, 275 to 472, 533 to 30 (grape colaspis)): Lissorhoptrus spp. (e.g. L. Oryzophilus 575, 621 to 767,813 to 862, 908 to 1040, 1161 to 1571, (rice water )); Echinocnemus spp. (e.g. E. Squamos 1730 to 2039, 2120 to 2338, 2384 to 2460, or the (rice plant weevil)); Diclodispa spp. (e.g. D. armigera (rice complement thereof; hispa)); Oulema spp. (e.g. O. Oryzae (); Sitophilus regenerating a plant from the transformed plant cell; and spp. (e.g. S. oryzae (rice weevil)); Pachydiplosis spp. (e.g. P growing the transformed plant under conditions Suitable 35 Oryzae (rice gall midge)); Hydrellia spp. (e.g. H. griseola for the expression of the recombinant construct, said (Small rice leafminer), or H. Sasakii (rice stem maggot)); grown transformed plant resistant to plant pathogenic Chlorops spp. (e.g. C. Oryzae (stem maggot)); Diabrotica insects compared to an untransformed plant. spp. (e.g. D. virgifera virgifera (western corn rootworm), D. The insect can be any insect, meaning any organism barberi (northern corn rootworm), D. undecimpunctata belonging to the Kingdom , more specific to the 40 howardi (southern corn rootworm), D. virgifera zeae (Mexi Phylum Arthropoda, and to the Class Insecta or the Class can corn rootworm); D. balteata (banded cucumber beetle)); Arachnida. The methods of the invention are applicable to Ostrinia spp. (e.g. O. nubilalis (European corn borer)); all insects and that are susceptible to gene silencing by RNA Agrotis spp. (e.g. A. ipsilon (black cutworm)); Elasmopalpus interference and that are capable of internalising double spp. (e.g. E. lignosellus (lesser cornstalk borer)); Melanotus stranded RNA from their immediate environment. The 45 spp. (wireworms); Cyclocephala spp. (e.g. C. borealis invention is also applicable to the insect at any stage in its (northern masked chafer), or C. immaculate (southern development. Because insects have a non-living exoskel masked chafer)); Popillia spp. (e.g. P. japonica (Japanese eton, they cannot grow at a uniform rate and rather grow in beetle)); Chaetocnema spp. (e.g. C. pullicaria (corn flea stages by periodically shedding their exoskeleton. This beetle)); Sphenophorus spp. (e.g. S. maidis (maize billbug)); process is referred to as moulting or ecdysis. The stages 50 Rhopalosiphum spp. (e.g. R. maidis (corn leaf aphid)); between moults are referred to as “” and these stages Anuraphis spp. (e.g. A. maidiradicis (corn root aphid)); may be targeted according to the invention. Also, insect eggs Melanoplus spp. (e.g. M. femurrubrum (redlegged grasshop or live young may also be targeted according to the present per) M. differentialis (differential grasshopper) or M. san invention. All stages in the developmental cycle, which guinipes (migratory grasshopper)); Hvlemya spp. (e.g. H. includes metamorphosis in the pterygotes, may be targeted 55 platura (seedcorn maggot)); Anaphothrips spp. (e.g. A. according to the present invention. Thus, individual stages obscrurus (grass thrips)); Solenopsis spp. (e.g. S. milesta Such as larvae, pupae, nymph etc stages of development may (thief ant)); or spp. (e.g. T. urticae (twospotted spider ), all be targeted. T. cinnabarinus ( spider mite); Helicoverpa spp. In one embodiment of the invention, the insect may (e.g. H. Zea (cotton bollworm), or H. armigera (American belong to the following orders: , Araneae, Anoplura, 60 bollworm)); Pectinophora spp. (e.g. P. gossypiella (pink Coleoptera, Collembola, Dermaptera, Dictyoptera, Diplura, bollworm)); Earias spp. (e.g. E. vittella (spotted bollworm)); Diptera, Embioptera, Ephemeroptera, Grylloblatodea, Heliothis spp. (e.g. H. virescens (tobacco budworm)); Hemiptera, Homoptera, , Isoptera, Lepidop Anthonomus spp. (e.g. A. grandis (boll weevil)); Pseudato tera, Mallophaga, Mecoptera, , Odonata, Orthop moscelis spp. (e.g. P. seriatus (cotton fleahopper)); Trialeu tera, Phasmida, Plecoptera, Protura, Psocoptera, Siphonap 65 rodes spp. (e.g. T. abutiloneus (banded-winged whitefly) T. tera, Siphunculata, Thysanura, Strepsiptera, Thysanoptera, vaporariorum (greenhouse whitefly)); Bemisia spp. (e.g. B. Trichoptera, and Zoraptera. argentifolii (silverleaf whitefly): Aphis spp. (e.g. A. gossypii US 9,528,123 B2 9 10 (cotton aphid)); Lygus spp. (e.g. L. lineolaris (tarnished Anthonomus spp. (e.g. A. grandis (boll weevil)); Phaedon plant bug) or L. hesperus (western tarnished plant bug)); spp. (e.g. P. cochleariae (mustard leaf beetle)): Epilachna Euschistus spp. (e.g. E. conspersus (consperse Stink bug)); spp. (e.g. E. varivetis (mexican bean beetle)); Tribolium spp. Chlorochroa spp. (e.g. C. sayi (Say Stinkbug)); Nezara spp. (e.g. T. castaneum (red floor beetle)); Diabrotica spp. (e.g. (e.g. N. viridula (Southern green Stinkbug)); Thrips spp. (e.g. 5 D. virgifera virgifera (western corn rootworm), D. barberi T. tabaci (onion thrips)); Frankliniella spp. (e.g. F. fisca (northern corn rootworm), D. undecimpunctata howardi (tobacco thrips), or F. Occidentalis (western flower thrips)); (Southern corn rootworm), D. virgifera zeae (Mexican corn Leptinotarsa spp. (e.g. L. decemlineata (Colorado potato rootworm); Ostrinia spp. (e.g. O. nubilalis (European corn beetle), L. juncta (false potato beetle), or L. texana (Texan borer)); Anaphothrips spp. (e.g. A. Obscrurus (grass thrips)); false potato beetle)); Lema spp. (e.g. L. trilineata (three 10 Pectinophora spp. (e.g. P. gossypiella (pink bollworm)); lined potato beetle)); Epitrix spp. (e.g. E. cucumeris (potato Heliothis spp. (e.g. H. virescens (tobacco budworm)); Tri flea beetle), E. hirtipennis (flea beetle), or E. tuberis (tuber aleurodes spp. (e.g. T. abutiloneus (banded-winged white flea beetle)); Epicauta spp. (e.g. E. vittata (striped blister ) T vaporariorum (greenhouse whitefly)); Bemisia spp. beetle)); Phaedon spp. (e.g. P. cochleariae (mustard leaf (e.g. B. argentifolii (silverleaf whitefly): Aphis spp. (e.g. A. beetle)); Epillachna spp. (e.g. E. varivetis (mexican bean 15 gossypii (cotton aphid)); Lygus spp. (e.g. L. lineolaris (tar beetle)); Acheta spp. (e.g. A. domesticus (house cricket)); nished plant bug) or L. hesperus (western tarnished plant Empoasca spp. (e.g. E. fabae (potato leafhopper)); Myzus bug)); Euschistus spp. (e.g. E. conspersus (consperse Stink spp. (e.g. M. persicae (green peach aphid)); Paratrioza spp. bug)); Chlorochroa spp. (e.g. C. sayi (Say Stinkbug)); (e.g. P. cockerelli (psyllid)); Conoderus spp. (e.g. C. falli Nezara spp. (e.g. N. viridula (Southern green Stinkbug)); (Southern potato wireworm), or C. vespertinus (tobacco Thrips spp. (e.g. T. tabaci (onion thrips)): Frankliniella spp. wireworm)); Phthorinaea spp. (e.g. P. operculella (potato (e.g. F. fisca (tobacco thrips), or F. Occidentalis (western tuberworm)); Macrosiphum spp. (e.g. M. euphorbiae (potato flower thrips)); Acheta spp. (e.g. A. domesticus (house aphid)); Thyanta spp. (e.g. T. pallidovirens (redshouldered cricket)); Myzus spp. (e.g. M. persicae (green peach aphid)); Stinkbug)); Phthorinaea spp. (e.g. P. operculella (potato Macrosiphum spp. (e.g. M. euphorbiae (potato aphid)); tuberworm)); Helicoverpa spp. (e.g. H. Zea (tomato fruit 25 Blissus spp. (e.g. B. leucopterus leucopterus (chinch bug)); worm); Keiferia spp. (e.g. K. lycopersicella (tomato pin Acrosternum spp. (e.g. A. hilare (green Stink bug)); worm)); Limonius spp. (wireworms); Manduca spp. (e.g. M. Chilotraea spp. (e.g. C. polychrysa (rice stalk borer)); sexta (tobacco hornworm), or M. quinquemaculata (tomato Lissorhoptrus spp. (e.g. L. Oryzophilus (rice water weevil)); hornworm)); Liriomyza spp. (e.g. L. sativae, L. trifoli or L. Rhopalosiphum spp. (e.g. R. maidis (corn leaf aphid)); and huidobrensis (leafminer)); Drosophila spp. (e.g. D. melano 30 Anuraphis spp. (e.g. A. maidiradicis (corn root aphid)). gaster, D. vakuba, D. pseudoobscura or D. simulans); Cara According to a more specific embodiment, the methods of bus spp. (e.g. C. granulatus); Chironomus spp. (e.g. C. the invention are applicable for Leptinotarsa species. Lep tentanus); Ctenocephalides spp. (e.g. C. felis (cat flea)); tinotarsa belong to the family of Chrysomelidae or leaf Diaprepes spp. (e.g. D. abbreviatus (root weevil)); Ips spp. beetles. Chrysomelid beetles such as Flea Beetles and Corn (e.g. I. pini (pine engraver)); Tribolium spp. (e.g. T. cas 35 Rootworms and Curculionids such as Alfalfa are taneum (red floor beetle)); Glossina spp. (e.g. G. morsitans particularly important pests. Flea Beetles include a large (tsetse fly)); Anopheles spp. (e.g. A. gambiae (malaria mos number of small leaf feeding beetles that feed on the leaves quito)); Helicoverpa spp. (e.g. H. armigera (African Boll of a number of grasses, cereals and herbs. Flea Beetles worm)); Acyrthosiphon spp. (e.g. A. pisum (pea aphid)); include a large number of genera (e.g., Attica, Apphthona, Apis spp. (e.g. A. melifera ( )); Homalodisca spp. 40 Argopistes, Disonycha, Epitrix, Longitarsus, Prodagri (e.g. H. coagulate (glassy-winged sharpshooter)); Aedes comela, Systema, and Phyllotreta). The Flea Beetle, Phyl spp. (e.g. Ae. aegypti (yellow fever mosquito)); Bombyx spp. lotreta cruciferae, also known as the Rape Flea Beetle, is a (e.g. B. mori (silkworm)); Locusta spp. (e.g. L. migratoria particularly important pest. Corn rootworms include species (migratory locust)); Boophilus spp. (e.g. B. microplus (cattle found in the Diabrotica (e.g., D. undecimpunctata tick)); Acanthoscurria spp. (e.g. A. gonesiana (red-haired 45 undecimpunctata, D. undecimpunctata howardii, D. longi chololate bird eater)); Diploptera spp. (e.g. D. punctata cornis, D. virgifera and D. balteata). Corn rootworms cause (pacific beetle cockroach)); Heliconius spp. (e.g. H. erato extensive damage to corn and curcubits. The Western Spot (red passion flower butterfly) or H. melpomene (postman ted Cucumber Beetle, D. undecimpunctata undecimpunc butterfly)); Curculio spp. (e.g. C. glandium (acorn weevil)); tata, is a pest of curcubits in the western U.S. Alfalfa weevils Plutella spp. (e.g. P. xylostella (diamondback moth)); 50 (also known as clover weevils) belong to the genus, Hypera Amblyomma spp. (e.g. A. variegatum (cattle tick)); Anteraea (H. postica, H. brunneipennis, H. nigrirostris, H. punctata spp. (e.g. A. yamamai (silkmoth)); and Armigeres spp. (e.g. and H. meles), and are considered an important pest of A. Subalbatus); legumes. The Egyptian alfalfa weevil, H. brunneipennis, is Preferred plant pathogenic insects according to the inven an important pest of alfalfa in the western U.S. tion are plant pest are selected from the group consisting of 55 There are more than 30 Leptinotarsa species. The present Leptinotarsa spp. (e.g. L. decemlineata (Colorado potato invention thus encompasses methods for controlling Lepti beetle), L. juncta (false potato beetle), or L. texana (Texan notarsa species, more specific methods for killing insects, or false potato beetle)); Nilaparvata spp. (e.g. N. lugens (brown preventing Leptinotarsa insects to develop or to grow, or planthopper)); Laodelphax spp. (e.g. L. striatellus (Small preventing insects to infect or infest. Specific Leptinotarsa brown planthopper)); Nephotettix spp. (e.g. N. virescens or 60 species to control according to the invention include Colo N. cincticeps (green leafhopper), or N. nigropictus (rice rado Potato Beetle (Leptinotarsa decemlineata (Say) and leafhopper)); Sogatella spp. (e.g. S. furcifera (white-backed False Potato Beetle (Leptinotarsa juncta (Say). planthopper)); Chilo spp. (e.g. C. suppressalis (rice striped CPB is a (serious) pest on our domestic potato (Solanum stem borer), C. auricilius (gold-fringed stem borer), or C. tuberosum), other cultivated and wild tuber bearing and polychrysus (dark-headed stem borer)); Sesamia spp. (e.g. S. 65 non-tuber bearing potato species (e.g. S. demissium, S. inferens (pink rice borer)); Tryporyza spp. (e.g. T. innotata phureja a.o.) and other Solanaceous (nightshades) plant (white rice borer), or T incertulas (yellow rice borer)); species including: US 9,528,123 B2 11 12 (a) the crop species tomato (several Lycopersicon spe The present invention extends to methods as described cies), eggplant (Solanum melongena), peppers (several Cap herein, wherein the insect is Acheta domesticus (house sicum species), tobacco (several Nicotiana species including cricket) and the plant is any plant as described herein or any ornamentals) and ground cherry (Physalis species); organic matter. (b) the weed/herb species, horse nettle (S. carolinense), In terms of “susceptible organisms, which benefit from common nightshade (S. dulcamara), belladonna (Atropa the present invention, any organism which is Susceptible to species), thorn apple (datura species), henbane (Hyoscya pest infestation is included. Preferably plants may benefit mus species) and buffalo burr (S. rostratum). from the present invention by protection from infestation by FPB is primarily found on horse nettle, but also occurs on plant pest organisms. common nightshade, ground cherry, and husk tomato (Ph 10 In a preferred embodiment the Susceptible organism is a lysalis species). plant and the pest is a plant pathogenic insect. In this The term “insect’ encompasses insects of all types and at embodiment the insect is contacted with the RNA molecule all stages of development, including egg, larval or nymphal, by expressing the dsRNA molecule in a plant, plant part, pupal and adult stages. 15 plant cell or plant seed that is infested with or susceptible to The present invention extends to methods as described infestation with the plant pathogenic pest. herein, wherein the insect is Leptinotarsa decemlineata In this context the term “plant' encompasses any plant (Colorado potato beetle) and the plant is potato, eggplant, material that it is desired to treat to prevent or reduce insect tomato, pepper, tobacco, ground cherry or rice, corn or growth and/or insect infestation. This includes, inter alia, COtton. whole plants, seedlings, propagation or reproductive mate The present invention extends to methods as described rial Such as seeds, cuttings, grafts, explants, etc. and also herein, wherein the insect is Phaedon cochleariae (mustard plant cell and tissue cultures. The plant material should leaf beetle) and the plant is mustard, chinese cabbage, turnip express, or have the capability to express, the RNA molecule greens, collard greens or bok choy. comprising at least one nucleotide sequence that is the RNA The present invention extends to methods as described 25 complement of or that represents the RNA equivalent of at herein, wherein the insect is Epilachna varivetis (Mexican least part of the nucleotide sequence of the sense Strand of bean beetle) and the plants are beans, field beans, garden at least one target gene of the pest organism, Such that the beans, Snap beans, lima beans, mung beans, string beans, RNA molecule is taken up by a pest upon plant-pest inter black-eyed beans, Velvet beans, soybeans, cowpeas, pigeon action, said RNA molecule being capable of inhibiting the peas, clover or alfalfa. 30 target gene or down-regulating expression of the target gene by RNA interference. The present invention extends to methods as described The target gene may be any of the target genes herein herein, wherein the insect is Anthonomus grandis (cotton described, for instance a target gene that is essential for the boll weevil) and the plant is cotton. viability, growth, development or reproduction of the pest. The present invention extends to methods as described 35 The present invention relates to any gene of interest in the herein, wherein the insect is Tribolium castaneum (red flour insect (which may be referred to herein as the “target gene') beetle) and the plant is in the form of stored grain products that can be down-regulated. Such as flour, cereals, meal, crackers, beans, spices, pasta, The terms “down-regulation of gene expression' and cake mix, dried pet food, dried flowers, chocolate, nuts, "inhibition of gene expression' are used interchangeably seeds, and even dried museum specimens. 40 and refer to a measurable or observable reduction in gene The present invention extends to methods as described expression or a complete abolition of detectable gene herein, wherein the insect is Myzus persicae (green peach expression, at the level of protein product and/or mRNA aphid) and the plant is a tree such as Prunus, particularly product from the target gene. Preferably the down-regula peach, apricot and plum; a vegetable crop of the families tion does not substantially directly inhibit the expression of Solanaceae, Chenopodiaceae, Compositae, Cruciferae, and 45 other genes of the insect. The down-regulation effect of the Cucurbitaceae, including but not limited to, artichoke, dsRNA on gene expression may be calculated as being at asparagus, bean, beets, broccoli, Brussels sprouts, cabbage, least 30%, 40%, 50%, 60%, preferably 70%, 80% or even carrot, cauliflower, cantaloupe, celery, corn, cucumber, fen more preferably 90% or 95% when compared with normal nel, kale, kohlrabi, turnip, eggplant, lettuce, mustard, okra, gene expression. Depending on the nature of the target gene, parsley, parsnip, pea, pepper, potato, radish, spinach, Squash, 50 down-regulation or inhibition of gene expression in cells of an insect can be confirmed by phenotypic analysis of the cell tomato, turnip, watercress, and watermelon; a field crops or the whole insect or by measurement of mRNA or protein Such as, but not limited to, tobacco, Sugar beet, and Sun expression using molecular techniques such as RNA solu flower; a flower crop or other ornamental plant. tion hybridization, PCR, nuclease protection, Northern The present invention extends to methods as described 55 hybridization, reverse transcription, gene expression moni herein, wherein the insect is Nilaparvata lugens and the toring with a microarray, antibody binding, enzyme-linked plant is a rice plant. immunosorbent assay (ELISA), Western blotting, radioim The present invention extends to methods as described munoassay (RIA), other immunoassays, or fluorescence herein, wherein the insect is Chilo suppressalis (rice striped activated cell analysis (FACS). stem borer) and the plant is a rice plant, bareley, Sorghum, 60 The “target gene' may be essentially any gene that is maize, wheat or a grass. desirable to be inhibited because it interferes with growth or The present invention extends to methods as described pathogenicity or infectivity of the insect. For instance, if the herein, wherein the insect is Plutella xylostella (Diamond method of the invention is to be used to prevent insect back moth) and the plant is a Brassica species such as, but growth and/or infestation then it is preferred to select a target not limited to cabbage, chinese cabbage, Brussels sprouts, 65 gene which is essential for viability, growth, development or kale, rapeseed, broccoli, cauliflower, turnip, mustard or reproduction of the insect, or any gene that is involved with radish. pathogenicity or infectivity of the insect, Such that specific US 9,528,123 B2 13 14 inhibition of the target gene leads to a lethal phenotype or In the methods of the present invention, dsRNA is used to decreases or stops insect infestation. inhibit growth or to interfere with the pathogenicity or According to one non-limiting embodiment, the target infectivity of the insect. gene is such that when its expression is down-regulated or The invention thus relates to isolated double-stranded inhibited using the method of the invention, the insect is 5 RNA comprising annealed complementary Strands, one of killed, or the reproduction or growth of the insect is stopped which has a nucleotide sequence which is complementary to or retarded. This type of target genes is considered to be at least part of a target nucleotide sequence of a target gene essential for the viability of the insect and is referred to as of an insect. The target gene may be any of the target genes essential genes. Therefore, the present invention encom described herein, or a part thereof that exerts the same passes a method as described herein, wherein the target gene 10 is an essential gene. function. According to a further non-limiting embodiment, the According to one embodiment of the present invention, an target gene is such that when it is down-regulated using the isolated double-stranded RNA is provided comprising method of the invention, the infestation or infection by the annealed complementary Strands, one of which has a nucleo insect, the damage caused by the insect, and/or the ability of 15 tide sequence which is complementary to at least part of a the insect to infest or infect host organisms and/or cause nucleotide sequence of an insect target gene, wherein said such damage, is reduced. The terms “infest” and “infect” or target gene comprises a sequence which is selected from the “infestation” and “infection” are generally used interchange group comprising: ably throughout. This type of target genes is considered to be (i) sequences which are at least 75% identical to a involved in the pathogenicity or infectivity of the insect. sequence represented by any of SEQID NOs 1, 3, 5, 7, Therefore, the present invention extends to methods as 9, 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, 160-163, described herein, wherein the target gene is involved in the 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, pathogenicity or infectivity of the insect. The advantage of 225, 230, 247, 249,251,253,255, 257, 259,275 to 472, choosing the latter type of target gene is that the insect is 473, 478,483, 488, 493, 498, 503, 513,515, 517,519, blocked to infect further plants or plant parts and is inhibited 25 521,533 to 575, 576,581,586,591,596,601, 603,605, to form further generations. 607, 609, 621 to 767, 768, 773,778,783,788,793, 795, According to one embodiment, target genes are conserved 797, 799,801, 813 to 862, 863, 868,873, 878,883,888, genes or insect-specific genes. 890, 892, 894, 896, 908 to 1040, 1041, 1046, 1051, In addition, any suitable double-stranded RNA fragment 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, capable of directing RNAi or RNA-mediated gene silencing 30 1085, 1087, 1089, 1091, 1093, 1095, 1097,1099, 1101, or inhibition of an insect target gene may be used in the 1103, 1105, 1107, 1109, 1111, 1113, 1161 to 1571, methods of the invention. 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, In another embodiment, a gene is selected that is essen 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, tially involved in the growth, development, and reproduction 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, of a pest, (such as an insect). Exemplary genes include but 35 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730 to are not limited to the structural subunits of ribosomal 2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070,2075, proteins and a beta-coatamer gene. Such as the CHD3 gene. 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106,2108, Ribosomal proteins such as S4 (RpS4) and S9 (RpS9) are 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 2364, structural constituents of the ribosome involved in protein 2366, 2368, 2370, 2372, 2384 to 2460, 2461, 2466, biosynthesis and which are components of the cytosolic 40 2471, 2476 or 2481, or the complement thereof, and small ribosomal subunit, the ribosomal proteins such as L9 (ii) sequences comprising at least 17 contiguous nucleo and L19 are structural constituent of ribosome involved in tides of any of SEQID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, protein biosynthesis which is localised to the ribosome. The 19, 21, 23, 49 to 158, 159, 160-163, 168, 173, 178, 183, beta coatamer gene in C. elegans encodes a protein which is 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, a subunit of a multimeric complex that forms a membrane 45 251,253,255, 257, 259,275 to 472, 473,478,483,488, vesicle coat. Similar sequences have been found in diverse 493,498, 503,513,515,517,519,521,533 to 575,576, organisms such as Arabidopsis thaliana, Drosophila mela 581,586,591,596,601, 603, 605, 607, 609, 621 to 767, nogaster, and Saccharomyces cerevisiae. Related sequences 768, 773,778, 783, 788, 793, 795, 797, 799, 801, 813 are found in diverse organisms such as Leptinotarsa decem to 862, 863, 868, 873, 878, 883, 888, 890, 892, 894, lineata, Phaedon cochleariae, Epilachna varivestis, Antho 50 896,908 to 1040, 1041, 1046, 1051, 1056, 1061, 1071, nomus grandis, Tribolium castaneum, Myzus persicae, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, Nilaparvata lugens, Chilo suppressalis, Plutella xylostella 1091, 1093, 1095, 1097,1099, 1101, 1103, 1105, 1107, and Acheta domesticus. 1109, 1111, 1113, 1161 to 1571, 1572, 1577, 1582, Other target genes for use in the present invention may 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, include, for example, those that play important roles in 55 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, viability, growth, development, reproduction, and infectiv 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, ity. These target genes include, for example, house keeping 1698, 1700, 1702, 1704, 1730 to 2039, 2040, 2045, genes, transcription factors, and pest specific genes or lethal 2050, 2055, 2060, 2065, 2070,2075, 2080,2085, 2090, knockout mutations in Caenorhabditis or Drosophila. The 2095, 2100, 2102, 2104, 2106, 2108, 2120 to 2338, target genes for use in the present invention may also be 60 2339, 2344, 2349, 2354, 2359, 2364,2366, 2368,2370, those that are from other organisms, e.g., from insects or 2372,2384 to 2460, 2461, 2466, 2471, 2476 or 2481, arachnidae (e.g. Leptinotarsa spp., Phaedon spp., Epilachna or the complement thereof, spp., Anthonomus spp., Tribolium spp., Myzus spp., Nilapa or wherein said insect target gene is an insect orthologue of rvata spp., Chilo spp., Plutella spp., or Acheta spp.). a gene comprising at least 17 contiguous nucleotides of any Preferred target genes include those specified in Table 1A 65 of SEQID NOs 49 to 158, 275 to 472, 533 to 575, 621 to and orthologous genes from other target organisms, such as 767, 813 to 862, 908 to 1040, 1161 to 1571, 1730 to 2039, from other pest organisms. 2120 to 2338, 2384 to 2460, or the complement thereof. US 9,528,123 B2 15 16 Depending on the assay used to measure gene silencing, at least 20 or at least 21 nucleotide and still more preferably the growth inhibition can be quantified as being greater than at least 22, 23 or 24 nucleotides of the target gene. about 5%, 10%, more preferably about 20%, 25%, 33%, It is preferred that (at least part of) the double-stranded 50%. 60%, 75%, 80%, most preferably about 90%, 95%, or RNA will share 100% sequence identity with the target about 99% as compared to a pest organism that has been 5 region of the insect target gene. However, it will be appre treated with control dsRNA. ciated that 100% sequence identity over the whole length of According to another embodiment of the present inven the double stranded region is not essential for functional tion, an isolated double-stranded RNA is provided, wherein RNA inhibition. RNA sequences with insertions, deletions, at least one of said annealed complementary strands com and single point mutations relative to the target sequence prises the RNA equivalent of at least one of the nucleotide 10 sequences represented by any of SEQ ID NOS 1, 3, 5, 7, 9, have also been found to be effective for RNA inhibition. The 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, 160-163, 168, 173, terms “corresponding to’ or “complementary to” are used 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, herein interchangeable, and when these terms are used to 249,251,253,255,257, 259,275 to 472, 473,478,483,488, refer to sequence correspondence between the double 493,498, 503,513,515,517,519,521,533 to 575,576,581, 15 Stranded RNA and the target region of the target gene, they 586,591,596,601, 603, 605, 607, 609, 621 to 767, 768, 773, are to be interpreted accordingly, i.e. as not absolutely 778, 783,788,793,795, 797, 799,801, 813 to 862, 863, 868, requiring 100% sequence identity. However, the '% sequence 873, 878, 883, 888, 890, 892, 894, 896, 908 to 1040, 1041, identity between the double-stranded RNA and the target 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, region will generally be at least 80% or 85% identical, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, preferably at least 90%. 95%, 96%, or more preferably at 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161 to least 97%, 98% and still more preferably at least 99%. Two 1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, nucleic acid strands are 'substantially complementary 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, when at least 85% of their bases pair. 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, The term “complementary” as used herein relates to both 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730 to 25 DNA-DNA complementarity as to DNA-RNA complemen 2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, tarity. In analogy herewith, the term "RNA equivalent 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, substantially means that in the DNA sequence(s), the base 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, “T” may be replaced by the corresponding base “U” nor 2368, 2370, 2372,2384 to 2460, 2461, 2466, 2471, 2476 or mally present in ribonucleic acids. 2481, or wherein at least one of said annealed complemen 30 Although the dsRNA contains a sequence which corre tary Strands comprises the RNA equivalent of a fragment of sponds to the target region of the target gene it is not at least 17 basepairs in length thereof, preferably at least 18, absolutely essential for the whole of the dsRNA to corre 19, 20 or 21, more preferably at least 22, 23 or 24 basepairs spond to the sequence of the target region. For example, the in length thereof. dsRNA may contain short non-target regions flanking the If the method of the invention is used for specifically 35 target-specific sequence, provided that such sequences do controlling growth or infestation of a specific insect in or on not affect performance of the dsRNA in RNA inhibition to a host cell or host organism, it is preferred that the double a material extent. Stranded RNA does not share any significant homology with The dsRNA may contain one or more substitute bases in any host gene, or at least not with any essential gene of the order to optimise performance in RNAi. It will be apparent host. In this context, it is preferred that the double-stranded 40 to the skilled reader how to vary each of the bases of the RNA shows less than 30%, more preferably less that 20%, dsRNA in turn and test the activity of the resulting dsRNAs more preferably less than 10%, and even more preferably (e.g. in a suitable in vitro test system) in order to optimise less than 5% nucleic acid sequence identity with any gene of the performance of a given dsRNA. the host cell. 96 sequence identity should be calculated The dsRNA may further contain DNA bases, non-natural across the full length of the double-stranded RNA region. If 45 bases or non-natural backbone linkages or modifications of genomic sequence data is available for the host organism the Sugar-phosphate backbone, for example to enhance one may cross-check sequence identity with the double stability during storage or enhance resistance to degradation stranded RNA using standard bioinformatics tools. In one by nucleases. embodiment, there is no sequence identity between the It has been previously reported that the formation of short dsRNA and a host sequences over 21 contiguous nucleo 50 interfering RNAs (siRNAs) of about 21 bp is desirable for tides, meaning that in this context, it is preferred that 21 effective gene silencing. However, in applications of appli contiguous base pairs of the dsRNA do not occur in the cant it has been shown that the minimum length of dsRNA genome of the host organism. In another embodiment, there preferably is at least about 80-100 bp in order to be effi is less than about 10% or less than about 12.5% sequence ciently taken up by certain pest organisms. There are indi identity over 24 contiguous nucleotides of the dsRNA with 55 cations that in invertebrates Such as the free living nematode any nucleotide sequence from a host species. C. elegans or the plant parasitic nematode Meloidogyne The double-stranded RNA comprises annealed comple incognita, these longer fragments are more effective in gene mentary strands, one of which has a nucleotide sequence silencing, possibly due to a more efficient uptake of these which corresponds to a target nucleotide sequence of the long dsRNA by the invertebrate. target gene to be down-regulated. The other strand of the 60 It has also recently been suggested that synthetic RNA double-stranded RNA is able to base-pair with the first duplexes consisting of either 27-mer blunt or short hairpin Strand. (sh) RNAs with 29bp stems and 2-mt 3' overhangs are more The expression “target region' or “target nucleotide potent inducers of RNA interference than conventional sequence' of the target insect gene may be any Suitable 21-mer siRNAs. Thus, molecules based upon the targets region or nucleotide sequence of the gene. The target region 65 identified above and being either 27-mer blunt or short should comprise at least 17, at least 18 or at least 19 hairpin (sh) RNAs with 29-bp stems and 2-nt 3' overhangs consecutive nucleotides of the target gene, more preferably are also included within the scope of the invention. US 9,528,123 B2 17 18 Therefore, in one embodiment, the double-stranded RNA RNA constructs comprising at least two copies of said fragment (or region) will itself preferably be at least 17 bp nucleotide sequence complementary to at least part of a in length, preferably 18 or 19 bp in length, more preferably nucleotide sequence of an insect target. at least 20 bp, more preferably at least 21 bp, or at least 22 The term “multiple” in the context of the present inven bp, or at least 23 bp, or at least 24 bp, 25 bp, 26 bp or at least tion means at least two, at least three, at least four, at least 27 bp in length. The expressions “double-stranded RNA five, at least six, etc. fragment” or “double-stranded RNA region” refer to a small The expressions “a further target gene' or “at least one entity of the double-stranded RNA corresponding with (part other target gene' mean for instance a second, a third or a of) the target gene. fourth, etc. target gene. Generally, the double stranded RNA is preferably 10 DsRNA that hits more than one of the above-mentioned between about 17-1500 bp, even more preferably between targets, or a combination of different dsRNA against differ about 80-1000 bp and most preferably between about 17-27 ent of the above mentioned targets are developed and used bp or between about 80-250 bp; such as double stranded in the methods of the present invention. RNA regions of about 17 bp, 18 bp, 19 bp, 20 bp, 21 bp. 22 Accordingly the invention relates to an isolated double bp, 23 bp, 24 bp, 25bp, 27 bp, 50 bp, 80 bp, 100 bp, 150 bp, 15 Stranded RNA construct comprising at least two copies of 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 the RNA equivalent of at least one of the nucleotide bp, 600 bp, 650 bp, 700 bp, 900 bp, 100 bp, 1100 bp, 1200 sequences represented by any of SEQ ID NOS 1, 3, 5, 7, 9, bp, 1300 bp, 1400 bp or 1500 bp. 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, 160-163, 168, 173, The upper limit on the length of the double-stranded RNA 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, may be dependent on i) the requirement for the dsRNA to be 249,251,253,255,257, 259,275 to 472, 473,478,483,488, taken up by the insect and ii) the requirement for the dsRNA 493,498, 503,513,515,517,519,521,533 to 575,576,581, to be processed within the cell into fragments that direct 586,591,596,601, 603, 605, 607, 609, 621 to 767, 768,773, RNAi. The chosen length may also be influenced by the 778, 783,788,793,795, 797, 799,801, 813 to 862, 863, 868, method of synthesis of the RNA and the mode of delivery of 873, 878, 883, 888, 890, 892, 894, 896, 908 to 1040, 1041, the RNA to the cell. Preferably the double-stranded RNA to 25 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, be used in the methods of the invention will be less than 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 10,000 bp in length, more preferably 1000 bp or less, more 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161 to preferably 500 bp or less, more preferably 300 bp or less, 1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, more preferably 100 bp or less. For any given target gene 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, and insect, the optimum length of the dsRNA for effective 30 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, inhibition may be determined by experiment. 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730 to The double-stranded RNA may be fully or partially 2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, double-stranded. Partially double-stranded RNAs may 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, include short single-stranded overhangs at one or both ends 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 2364, 23.66, of the double-stranded portion, provided that the RNA is still 35 2368, 2370, 2372,2384 to 2460, 2461, 2466, 2471, 2476 or capable of being taken up by insects and directing RNAi. 2481, or at least two copies of the RNA equivalent of a The double-stranded RNA may also contain internal non fragment of at least 17 basepairs in length thereof, preferably complementary regions. at least 18, 19, 20 or 21, more preferably at least 22, 23 or The methods of the invention encompass the simultane 24 basepairs in length thereof. Preferably, said double ous or sequential provision of two or more different double 40 stranded RNA comprises the RNA equivalent of the nucleo stranded RNAS or RNA constructs to the same insect, so as tide sequence as represented in SEQID NO 159 or 160, or to achieve down-regulation or inhibition of multiple target a fragment of at least 17, preferably at least 18, 19, 20 or 21, genes or to achieve a more potent inhibition of a single target more preferably at least 22, 23 or 24 basepairs in length gene. thereof. In a further embodiment, the invention relates to an Alternatively, multiple targets are hit by the provision of 45 isolated double stranded RNA construct comprising at least one double-stranded RNA that hits multiple target two copies of the RNA equivalent of the nucleotide sequences, and a single target is more efficiently inhibited by sequence as represented by SEQ ID NO 159 or 160. the presence of more than one copy of the double stranded Accordingly, the present invention extends to methods as RNA fragment corresponding to the target gene. Thus, in described herein, wherein the dsRNA comprises annealed one embodiment of the invention, the double-stranded RNA 50 complementary strands, one of which has a nucleotide construct comprises multiple dsRNA regions, at least one sequence which is complementary to at least part of a target Strand of each dsRNA region comprising a nucleotide nucleotide sequence of an insect target gene, and which sequence that is complementary to at least part of a target comprises the RNA equivalents of at least wo nucleotide nucleotide sequence of an insect target gene. According to sequences independently chosen from each other. In one the invention, the dsRNA regions in the RNA construct may 55 embodiment, the dsRNA comprises the RNA equivalents of be complementary to the same or to different target genes at least two, preferably at least three, four or five, nucleotide and/or the dsRNA regions may be complementary to targets sequences independently chosen from the sequences repre from the same or from different insect species. sented by any of SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, The terms “hit”, “hits” and “hitting are alternative word 19, 21, 23, 49 to 158, 159, 160-163, 168, 173, 178, 183, 188, ings to indicate that at least one of the strands of the dsRNA 60 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253, is complementary to, and as Such may bind to, the target 255, 257, 259,275 to 472, 473,478,483,488, 493,498, 503, gene or nucleotide sequence. 513,515,517,519,521,533 to 575, 576,581,586,591,596, In one embodiment, the double stranded RNA region 601, 603, 605, 607, 609, 621 to 767, 768, 773,778, 783,788, comprises multiple copies of the nucleotide sequence that is 793, 795, 797, 799,801, 813 to 862, 863, 868,873, 878,883, complementary to the target gene. Alternatively, the dsRNA 65 888, 890, 892, 894, 896, 908 to 1040, 1041, 1046, 1051, hits more than one target sequence of the same target gene. 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, The invention thus encompasses isolated double stranded 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, US 9,528,123 B2 19 20 1103, 1105, 1107, 1109, 1111, 1113, 1161 to 1571, 1572, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1698, 1700, 1702, 1704, 1730 to 2039, 2040, 2045, 1694, 1696, 1698, 1700, 1702, 1704, 1730 to 2039, 2040, 2050, 2055, 2060, 2065, 2070,2075, 2080,2085, 2090, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085, 2095, 2100, 2102, 2104, 2106, 2108, 2120 to 2338, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 2364,2366, 2368,2370, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372,2384 to 2460, 2461, 2466, 2471, 2476 or 2481, 2372, 2384 to 2460, 2461, 2466, 2471, 2476 or 2481, or or the complement thereof, fragments thereof of at least 17 basepairs in length, prefer 10 or wherein said insect target gene is an insect orthologue ably at least 18, 19, 20 or 21, more preferably at least 22, 23 of a gene comprising at least 17 contiguous nucleotides of or 24 basepairs in length thereof. any of SEQID NOs 49 to 158,275 to 472, 533 to 575, 621 The at least two nucleotide sequences may be derived to 767,813 to 862, 908 to 1040, 1161 to 1571, 1730 to 2039, from the target genes herein described. According to one 2120 to 2338, 2384 to 2460, or the complement thereof. preferred embodiment the dsRNA hits at least one target 15 The dsRNA regions (or fragments) in the double stranded gene that is essential for viability, growth, development or RNA may be combined as follows: reproduction of the insect and hits at least one gene involved a) when multiple dsRNA regions targeting a single target in pathogenicity or infectivity as described hereinabove. gene are combined, they may be combined in the Alternatively, the dsRNA hits multiple genes of the same original order (ie the order in which the regions appear category, for example, the dsRNA hits at least 2 essential in the target gene) in the RNA construct, genes or at least 2 genes involved in the same cellular b) alternatively, the original order of the fragments may be function. According to a further embodiment, the dsRNA ignored so that they are scrambled and combined hits at least 2 target genes, which target genes are involved randomly or deliberately in any order into the double in a different cellular function. For example the dsRNA hits stranded RNA construct, two or more genes involved in protein synthesis (e.g. 25 c) alternatively, one single fragment may be repeated ribosome subunits), intracellular protein transport, nuclear several times, for example from 1 to 10 times, e.g. 1, 2, mRNA splicing, or involved in one of the functions 3, 4, 5, 6, 7, 8, 9 or 10 times, in the dsRNA construct, described in Table 1A. O Preferably, the present invention extends to methods as d) the dsRNA regions (targeting a single or different target described herein, wherein said insect target gene comprises 30 genes) may be combined in the sense or antisense a sequence which is which is selected from the group orientation. comprising: In addition, the target gene(s) to be combined may be (i) sequences which are at least 75% identical to a chosen from one or more of the following categories of sequence represented by any of SEQID NOs 1, 3, 5, 7, genes: 9, 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, 160-163, 35 e) "essential” genes or “pathogenicity genes' as described 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, above encompass genes that are vital for one or more 225, 230, 247, 249,251,253,255, 257, 259,275 to 472, target insects and result in a lethal or severe (e.g. 473, 478,483,488, 493, 498, 503, 513,515, 517,519, feeding, reproduction, growth) phenotype when 521,533 to 575, 576,581,586,591,596,601, 603, 605, silenced. The choice of a strong lethal target gene 607, 609, 621 to 767, 768,773,778,783,788, 793, 795, 40 results in a potent RNAi effect. In the RNA constructs 797, 799,801, 813 to 862,863, 868,873, 878,883, 888, of the invention, multiple dsRNA regions targeting the 890, 892, 894, 896, 908 to 1040, 1041, 1046, 1051, same or different (very effective) lethal genes can be 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, combined to further increase the potency, efficacy or 1085, 1087, 1089, 1091, 1093, 1095, 1097,1099, 1101, speed of the RNAi effect in insect control. 1103, 1105, 1107, 1109, 1111, 1113, 1161 to 1571, 45 “weak” genes encompass target genes with a particu 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, larly interesting function in one of the cellular path 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, ways described herein, but which result in a weak 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, phenotypic effect when silenced independently. In the 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730 to RNA constructs of the invention, multiple dsRNA 2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070,2075, 50 regions targeting a single or different weak gene(s) may 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106,2108, be combined to obtain a stronger RNAi effect. 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 2364, g) “insect specific' genes encompass genes that have no 2366, 2368, 2370, 2372, 2384 to 2460, 2461, 2466, Substantial homologous counterpart in non-insect 2471, 2476 or 2481, or the complement thereof, and organisms as can be determined by bioinformatics (ii) sequences comprising at least 17 contiguous nucleo 55 homology searches, for example by BLAST searches. tides of any of SEQID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, The choice of an insect specific target gene results in a 19, 21, 23, 49 to 158, 159, 160-163, 168, 173, 178, 183, species specific RNAi effect, with no effect or no 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, Substantial (adverse) effect in non-target organisms. 251,253,255, 257, 259,275 to 472, 473,478,483,488, h)"conserved genes' encompass genes that are conserved 493,498, 503,513,515,517,519,521,533 to 575, 576, 60 (at the amino acid level) between the target organism 581,586,591,596,601, 603, 605, 607, 609, 621 to 767, and non-target organism(s). To reduce possible effects 768, 773,778, 783, 788, 793, 795, 797, 799, 801, 813 on non-target species, such effective but conserved to 862, 863, 868, 873, 878, 883, 888, 890, 892, 894, genes are analysed and target sequences from the 896, 908 to 1040, 1041, 1046, 1051, 1056, 1061, 1071, variable regions of these conserved genes are chosen to 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 65 be targeted by the dsRNA regions in the RNA con 1091, 1093, 1095, 1097,1099, 1101, 1103, 1105, 1107, struct. Here, conservation is assessed at the level of the 1109, 1111, 1113, 1161 to 1571, 1572, 1577, 1582, nucleic acid sequence. Such variable regions thus US 9,528,123 B2 21 22 encompass the least conserved sections, at the level of from another, e.g. the additional, sequence of interest, which the nucleic acid sequence, of the conserved target preferably provides some additional function to the RNA gene(s). COnStruct. i) “conserved pathway” genes encompass genes that are In one particular embodiment of the invention, the involved in the same biological pathway or cellular 5 dsRNA constructs of the present invention are provided with process, or encompass genes that have the same func an aptamer to facilitate uptake of the dsRNA by the insect. tionality in different insect species resulting in a spe The aptamer is designed to bind a substance which is taken cific and potent RNAi effect and more efficient insect up by the insect. Such substances may be from an insect or control; plant origin. One specific example of an aptamer, is an j) alternatively, the RNA constructs according to the 10 aptamer that binds to a transmembrane protein, for example present invention target multiple genes from different a transmembrane protein of an insect. Alternatively, the biological pathways, resulting in a broad cellular RNAi aptamer may bind a (plant) metabolite or nutrient which is effect and more efficient insect control. taken up by the insect. According to the invention, all double stranded RNA Alternatively, the linkers are self-cleaving in the endo regions comprise at least one strand that is complementary 15 Somes. This may be advantageous when the constructs of the to at least part or a portion of the nucleotide sequence of any present invention are taken up by the insect via endocytosis of the target genes herein described. However, provided one or transcytosis, and are therefore compartmentalized in the of the double stranded RNA regions comprises at least one endoSomes of the insect species. The endoSomes may have Strand that is complementary to a portion of the nucleotide a low pH environment, leading to cleavage of the linker. sequence of any one of the target genes herein described, the The above mentioned linkers that are self-cleaving in other double stranded RNA regions may comprise at least hydrophobic conditions are particularly useful in dsRNA one strand that is complementary to a portion of any other constructs of the present invention when used to be trans insect target gene (including known target genes). ferred from one cell to another via the transit in a cell wall, According to yet another embodiment of the present for example when crossing the cell wall of an insect pest invention there is provided an isolated double stranded RNA 25 organism. or RNA construct as herein described, further comprising at An intron may also be used as a linker. An "intron’ as least one additional sequence and optionally a linker. In one used herein may be any non-coding RNA sequence of a embodiment, the additional sequence is chosen from the messenger RNA. Particular suitable intron sequences for the group comprising (i) a sequence facilitating large-scale constructs of the present invention are (1) U-rich (35-45%); production of the dsRNA construct; (ii) a sequence effecting 30 (2) have an average length of 100 bp (varying between about an increase or decrease in the stability of the dsRNA; (iii) a 50 and about 500 bp) which base pairs may be randomly sequence allowing the binding of proteins or other mol chosen or may be based on known intron sequences; (3) start ecules to facilitate uptake of the RNA construct by insects: at the 5' end with -AG:GT- or -CG:GT and/or (4) have at (iv) a sequence which is an aptamer that binds to a receptor their 3' end -AG:GC- or -AG:AA. or to a molecule on the Surface or in the cytoplasm of an 35 A non-complementary RNA sequence, ranging from insect to facilitate uptake, endocytosis and/or transcytosis by about 1 base pair to about 10,000 base pairs, may also be the insect; or (V) additional sequences to catalyze processing used as a linker. of dsRNA regions. In one embodiment, the linker is a Without wishing to be bound by any particular theory or conditionally self-cleaving RNA sequence, preferably a pH mechanism, it is thought that long double-stranded RNAs sensitive linker or a hydrophobic sensitive linker. In one 40 are taken up by the insect from their immediate environ embodiment, the linker is an intron. ment. Double-stranded RNAs taken up into the gut and In one embodiment, the multiple dsRNA regions of the transferred to the gut epithelial cells are then processed double-stranded RNA construct are connected by one or within the cell into short double-stranded RNAs, called more linkers. In another embodiment, the linker is present at small interfering RNAs (siRNAs), by the action of an a site in the RNA construct, separating the dsRNA regions 45 endogenous endonuclease. The resulting siRNAs then medi from another region of interest. Different linker types for the ate RNAi via formation of a multi-component RNase com dsRNA constructs are provided by the present invention. plex termed the RISC or RNA interfering silencing complex. In another embodiment, the multiple dsRNA regions of In order to achieve down-regulation of a target gene the double-stranded RNA construct are connected without within an insect cell the double-stranded RNA added to the linkers. 50 exterior of the cell wall may be any dsRNA or dsRNA In a particular embodiment of the invention, the linkers construct that can be taken up into the cell and then may be used to disconnect Smaller dsRNA regions in the pest processed within the cell into siRNAs, which then mediate organism. Advantageously, in this situation the linker RNAi, or the RNA added to the exterior of the cell could sequence may promote division of a long dsRNA into itself be an siRNA that can be taken up into the cell and Smaller dsRNA regions under particular circumstances, 55 thereby direct RNAi. resulting in the release of separate dsRNA regions under siRNAs are generally short double-stranded RNAs having these circumstances and leading to more efficient gene a length in the range of from 19 to 25 base pairs, or from 20 silencing by these smaller dsRNA regions. Examples of to 24 base pairs. In preferred embodiments siRNAs having suitable conditionally self-cleaving linkers are RNA 19, 20, 21, 22, 23, 24 or 25 base pairs, and in particular 21 sequences that are self-cleaving at high pH conditions. 60 or 22 base pairs, corresponding to the target gene to be Suitable examples of such RNA sequences are described by down-regulated may be used. However, the invention is not Borda et al. (Nucleic Acids Res. 2003 May 15:31 (10):2595 intended to be limited to the use of such siRNAs. 600), which document is incorporated herein by reference. siRNAS may include single-stranded overhangs at one or This sequence originates from the catalytic core of the both ends, flanking the double-stranded portion. In a par hammerhead ribozyme HH16. 65 ticularly preferred embodiment the siRNA may contain 3 In another aspect of the invention, a linker is located at a overhanging nucleotides, preferably two 3' overhanging site in the RNA construct, separating the dsRNA regions thymidines (dTdT) or uridines (UU). 3'TT or UU overhangs US 9,528,123 B2 23 24 may be included in the siRNA if the sequence of the target as orthologues. e.g. the C-globin genes in mouse and human gene immediately upstream of the sequence included in are orthologues. (ii) paralogues are homologous genes in double-stranded part of the dsRNA is AA. This allows the within a single species. e.g. the C- and B-globin genes in TT or UU overhang in the siRNA to hybridise to the target mouse are paralogues gene. Although a 3' TT or UU overhang may also be Preferred homologues are genes comprising a sequence included at the other end of the siRNA it is not essential for which is at least about 85% or 87.5%, still more preferably the target sequence downstream of the sequence included in about 90%, still more preferably at least about 95% and most double-stranded part of the siRNA to have AA. In this preferably at least about 99% identical to a sequence context, siRNAs which are RNA/DNA chimeras are also selected from the group of sequences represented by SEQID contemplated. These chimeras include, for example, the 10 NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, siRNAs comprising a double-stranded RNA with 3' over 160-163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, hangs of DNA bases (e.g. dTdT), as discussed above, and 220, 225, 230, 247, 249,251,253,255, 257, 259,275 to 472, also double-stranded RNAs which are polynucleotides in 473, 478,483, 488, 493, 498, 503, 513,515,517,519, 521, which one or more of the RNA bases or ribonucleotides, or 533 to 575, 576,581,586,591,596,601, 603, 605, 607, 609, even all of the ribonucleotides on an entire strand, are 15 621 to 767, 768,773,778,783,788,793, 795, 797,799,801, replaced with DNA bases or deoxynucleotides. 813 to 862, 863, 868,873, 878,883, 888,890, 892, 894, 896, The dsRNA may be formed from two separate (sense and 908 to 1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, antisense) RNA strands that are annealed together by (non 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, covalent) basepairing. Alternatively, the dsRNA may have a 1093, 1095, 1097,1099, 1101, 1103, 1105,1107, 1109, 1111, foldback stem-loop or hairpin structure, wherein the two 1113, 1161 to 1571, 1572, 1577, 1582, 1587, 1592, 1597, annealed strands of the dsRNA are covalently linked. In this 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, embodiment the sense and antisense stands of the dsRNA 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, are formed from different regions of single polynucleotide 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, molecule that is partially self-complementary. RNAs having 1704, 1730 to 2039, 2040, 2045, 2050, 2055, 2060, 2065, this structure are convenient if the dsRNA is to be synthe 25 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, sised by expression in vivo, for example in a host cell or 2106, 2108, 2120 to 2338, 2339, 2344, 2349, 2354, 2359, organism as discussed below, or by in vitro transcription. 2364, 2366, 2368, 2370, 2372,2384 to 2460, 2461, 2466, The precise nature and sequence of the “loop' linking the 2471,2476 or 2481, or the complement thereof. Methods for two RNA strands is generally not material to the invention, determining sequence identity are routine in the art and except that it should not impair the ability of the double 30 include use of the Blast software and EMBOSS software stranded part of the molecule to mediate RNAi. The features (The European Molecular Biology Open Software Suite of “hairpin” or “stem-loop” RNAs for use in RNAi are (2000), Rice, P. Longden, I. and Bleasby, A. Trends in generally known in the art (see for example WO99/53050, Genetics 16, (6) pp. 276-277). The term “identity” as used in the name of CSIRO, the contents of which are incorpo herein refers to the relationship between sequences at the rated herein by reference). In other embodiments of the 35 nucleotide level. The expression “9% identical' is determined invention, the loop structure may comprise linker sequences by comparing optimally aligned sequences, e.g. two or or additional sequences as described above. more, over a comparison window wherein the portion of the Another aspect of the present invention are target nucleo sequence in the comparison window may comprise inser tide sequences of the insect target genes herein disclosed. tions or deletions as compared to the reference sequence for Such target nucleotide sequences are particularly important 40 optimal alignment of the sequences. The reference sequence to design the dsRNA constructs according to the present does not comprise insertions or deletions. The reference invention. Such target nucleotide sequences are preferably at window is chosen from between at least 10 contiguous least 17, preferably at least 18, 19, 20 or 21, more preferably nucleotides to about 50, about 100 or to about 150 nucleo at least 22, 23 or 24 nucleotides in length. Non-limiting tides, preferably between about 50 and 150 nucleotides. “/6 examples of preferred target nucleotide sequences are given 45 identity” is then calculated by determining the number of in the examples. nucleotides that are identical between the sequences in the According to one embodiment, the present invention window, dividing the number of identical nucleotides by the provides an isolated nucleotide sequence encoding a double number of nucleotides in the window and multiplying by stranded RNA or double stranded RNA construct as 100. described herein. 50 Other homologues are genes which are alleles of a gene According to a more specific embodiment, the present comprising a sequence as represented by any of SEQ ID invention relates to an isolated nucleic acid sequence con NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, sisting of a sequence represented by any of SEQID NOS 49 160-163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, to 158, 275 to 472,533 to 575, 621 to 767,813 to 862, 908 220, 225, 230, 247, 249,251,253,255, 257, 259,275 to 472, to 1040, 1161 to 1571, 1730 to 2039, 2120 to 2338, 2384 to 55 473, 478,483, 488, 493, 498, 503, 513,515,517,519, 521, 2460, or a fragment of at least 17 preferably at least 18, 19. 533 to 575, 576,581,586,591,596,601, 603, 605, 607, 609, 20 or 21, more preferably at least 22, 23 or 24 nucleotides 621 to 767, 768,773,778,783,788,793, 795, 797,799,801, thereof. 813 to 862, 863, 868,873, 878,883, 888,890, 892, 894, 896, A person skilled in the art will recognize that homologues 908 to 1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, of these target genes can be found and that these homologues 60 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, are also useful in the methods of the present invention. 1093, 1095, 1097,1099, 1101, 1103, 1105,1107, 1109, 1111, Protein, or nucleotide sequences are likely to be homolo 1113, 1161 to 1571, 1572, 1577, 1582, 1587, 1592, 1597, gous if they show a “significant' level of sequence similarity 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, or more preferably sequence identity. Truely homologous 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, sequences are related by divergence from a common ances 65 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, tor gene. Sequence homologues can be of two types: (i) 1704, 1730 to 2039, 2040, 2045, 2050, 2055, 2060, 2065, where homologues exist in different species they are known 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, US 9,528,123 B2 25 26 2106, 2108, 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 1093, 1095, 1097,1099, 1101, 1103, 1105,1107, 1109, 1111, 2364, 2366, 2368, 2370, 2372,2384 to 2460, 2461, 2466, 1113, 1161 to 1571, 1572, 1577, 1582, 1587, 1592, 1597, 2471,2476 or 2481. Further preferred homologues are genes 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, comprising at least one single nucleotide polymorphism 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, (SNIP) compared to a gene comprising a sequence as 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, represented by any of SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 1704, 1730 to 2039, 2040, 2045, 2050, 2055, 2060, 2065, 17, 19, 21, 23, 49 to 158, 159, 160-163, 168, 173, 178, 183, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 2106, 2108, 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 253,255, 257, 259,275 to 472, 473,478,483,488, 493,498, 2364, 2366, 2368, 2370, 2372,2384 to 2460, 2461, 2466, 503, 513,515,517,519,521,533 to 575, 576,581,586,591, 10 2471, 2476 or 248. By way of example, nematode ortho 596,601, 603,605, 607, 609, 621 to 767, 768, 773,778, 783, logues may comprise a nucleotide sequence as represented 788, 793, 795, 797,799,801, 813 to 862, 863, 868,873, 878, in any of SEQID NOs 124 to 135, 435 to 446,563 to 564, 883, 888,890, 892, 894, 896,908 to 1040, 1041, 1046, 1051, 739 to 751, 853, 854, 1011 to 1025, 1438 to 1473, 1988 to 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 2001, 2291 to 2298, 2439 or 2440, or a fragment of at least 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 15 17, 18, 19, 20 or 21 nucleotides thereof. According to 1103, 1105, 1107, 1109, 1111, 1113, 1161 to 1571, 1572, another aspect, the invention thus encompasses any of the 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, methods described herein for controlling nematode growth 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662, in an organism, or for preventing nematode infestation of an 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, organism Susceptible to nemataode infection, comprising 1694, 1696, 1698, 1700, 1702, 1704, 1730 to 2039, 2040, contacting nematode cells with a double-stranded RNA, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085, wherein the double-stranded RNA comprises annealed 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120 to 2338, complementary strands, one of which has a nucleotide 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, sequence which is complementary to at least part of the 2372,2384 to 2460, 2461, 2466, 2471, 2476 or 2481. nucleotide sequence of a target gene comprising a fragment According to another embodiment, the invention encom 25 of at least 17, 18, 19, 20 or 21 nucleotides of any of the passes target genes which are insect orthologues of a gene sequences as represented in SEQID NOs 124 to 135, 435 to comprising a nucleotide sequence as represented in any of 446,563 to 564, 739 to 751, 853, 854, 1011 to 1025, 1438 SEQID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49 to to 1473, 1988 to 2001, 2291 to 2298, 2439 or 2440, whereby 158, 159, 160-163, 168, 173, 178, 183, 188, 193, 198, 203, the double-stranded RNA is taken up by the nematode and 208, 215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259, 30 thereby controls growth or prevents infestation. The inven 275 to 472, 473,478,483,488, 493,498, 503,513,515,517, tion also relates to nematode-resistant transgenic plants 519,521,533 to 575, 576,581,586,591,596,601, 603, 605, comprising a fragment of at least 17, 18, 19, 20 or 21 607, 609, 621 to 767, 768,773,778,783,788, 793,795, 797, nucleotides of any of the sequences as represented in SEQ 799,801, 813 to 862, 863, 868,873, 878,883, 888,890, 892, ID NOs 124 to 135,435 to 446,563 to 564, 739 to 751, 853, 894, 896, 908 to 1040, 1041, 1046, 1051, 1056, 1061, 1071, 35 854, 1011 to 1025, 1438 to 1473, 1988 to 2001, 2291 to 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 2298, 2439 or 2440. A non-limiting list of nematode ortho 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, logues genes or sequences comprising at least a fragment of 1109, 1111, 1113, 1161 to 1571, 1572, 1577, 1582, 1587, 17 bp of one of the sequences of the invention, is given in 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, Tables 5. 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 40 According to another embodiment, the invention encom 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, passes target genes which are fungal orthologues of a gene 1700, 1702, 1704, 1730 to 2039, 2040, 2045, 2050, 2055, comprising a nucleotide sequence as represented in any of 1. 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, 160-163, 2102, 2104, 2106, 2108, 2120 to 2338, 2339, 2344, 2349, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 2354, 2359, 2364, 2366, 2368, 2370, 2372,2384 to 2460, 45 230, 247, 249,251,253,255, 257, 259,275 to 472, 473,478, 2461, 2466, 2471, 2476 or 2481. By way of example, 483,488, 493,498, 503, 513,515,517,519,521,533 to 575, orthologues may comprise a nucleotide sequence as repre 576,581,586,591,596,601, 603, 605, 607, 609, 621 to 767, sented in any of SEQID NOs 49 to 123, 275 to 434, 533 to 768,773,778,783,788,793,795, 797, 799,801, 813 to 862, 562, 621 to 738, 813 to 852, 908 to 1010, 1161 to 1437, 1730 863, 868, 873, 878, 883, 888, 890, 892, 894, 896, 908 to to 1987, 2120 to 2290, and 2384 to 2438, or a fragment 50 1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, thereof of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, nucleotides. A non-limiting list of insect or arachnida ortho 1095, 1097,1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, logues genes or sequences comprising at least a fragment of 1161 to 1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 17 bp of one of the sequences of the invention, is given in 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, Tables 4. 55 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, According to another embodiment, the invention encom 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, passes target genes which are nematode orthologues of a 1730 to 2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, gene comprising a nucleotide sequence as represented in any 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, of 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, 2108, 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 2364, 160-163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 60 2366, 2368, 2370, 2372,2384 to 2460, 2461, 2466, 2471, 220, 225, 230, 247, 249,251,253,255, 257, 259,275 to 472, 2476 or 2481. By way of example, fungal orthologues may 473, 478,483,488, 493, 498, 503, 513,515,517,519, 521, comprise a nucleotide sequence as represented in any of 533 to 575, 576,581,586,591,596,601, 603, 605, 607, 609, SEQID NOs 136 to 158,447 to 472,565 to 575, 752 to 767, 621 to 767, 768,773,778,783,788,793, 795, 797,799,801, 855 to 862, 1026 to 1040, 1475 to 1571, 2002 to 2039, 2299 813 to 862, 863, 868,873, 878, 883, 888,890, 892, 894, 896, 65 to 2338, 2441 to 2460, or a fragment of at least 17, 18, 19, 908 to 1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides thereof. Accord 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, ing to another aspect, the invention thus encompasses any of US 9,528,123 B2 27 28 the methods described herein for controlling fungal growth In this embodiment the insect can be any insect, but is on a cell or an organism, or for preventing fungal infestation preferably plant pathogenic insect. Preferred plant patho of a cell or an organism Susceptible to fungal infection, genic insects include, but are not limited to, those listed comprising contacting fungal cells with a double-stranded above. RNA, wherein the double-stranded RNA comprises A plant to be used in the methods of the invention, or a annealed complementary Strands, one of which has a nucleo transgenic plant according to the invention encompasses any tide sequence which is complementary to at least part of the plant, but is preferably a plant that is susceptible to infes nucleotide sequence of a target gene comprising a fragment tation by a plant pathogenic insect. of at least 17, 18, 19, 20 or 21 nucleotides of any of the Accordingly, the present invention extends to methods as 10 described herein wherein the plant is chosen from the sequences as represented in SEQID NOS 136 to 158, 447 to following group of plants (or crops): alfalfa, apple, apricot, 472,565 to 575, 752 to 767, 855 to 862, 1026 to 1040, 1475 artichoke, asparagus, avocado, banana, barley, beans, beet, to 1571, 2002 to 2039, 2299 to 2338,2441 to 2460, whereby blackberry, blueberry, broccoli, brussel sprouts, cabbage, the double-stranded RNA is taken up by the fungus and canola, carrot, cassava, cauliflower, a cereal, celery, cherry, thereby controls growth or prevents infestation. The inven 15 citrus, clemintine, coffee, corn, cotton, cucumber, eggplant, tion also relates to fungal-resistant transgenic plants com endive, eucalyptus, figes, grape, grapefruit, groundnuts, prising a fragment of at least 17, 18, 19, 20 or 21 of any of ground cherry, kiwifruit, lettuce, leek, lemon, lime, pine, the sequences as represented in SEQID NOS 136 to 158,447 maize, mango, melon, millet, mushroom, nut aot, okra, to 472, 565 to 575, 752 to 767, 855 to 862, 1026 to 1040, onion, orange, an ornamental plant or flower or tree, papaya, 1475 to 1571, 2002 to 2039, 2299 to 2338, 2441 to 2460. A parsley, pea, peach, peanut, peat, pepper, persimmon, pine non-limiting list of fungal orthologues genes or sequences apple, plantain, plum, pomegranate, potato, pumpkin, radic comprising at least a fragment of 17 bp of one of the chio, radish, rapeseed, raspberry, rice, rye, Sorghum, soy, sequences of the invention, is given in Tables 6. Soybean, spinach, Strawberry, Sugarbeet, Sugargcane, Sun In one preferred embodiment of the invention the dsRNA flower, Sweet potato, tangerine, tea, tobacco, tomato, a vine, may be expressed by (e.g. transcribed within) a host cell or 25 watermelon, wheat, yams and Zucchini. host organism, the host cell or organism being an organism In one embodiment the present invention extends to susceptible or vulnerable to infestation by an insect. In this methods as described herein, wherein the plant is potato and embodiment RNAi-mediated gene silencing of one or more the target gene is a gene from an insect selected from the target genes in the insect may be used as a mechanism to group consisting of Leptinotarsa spp. (e.g. L. decemlineata control growth of the insect in or on the host organism 30 (Colorado potato beetle), L. juncta (false potato beetle), or and/or to prevent or reduce insect infestation of the host L. texana (Texan false potato beetle)); Lema spp. (e.g. L. organism. Thus, expression of the double-stranded RNA trilineata (three-lined potato beetle)): Epitrix spp. (e.g. E. within cells of the host organism may confer resistance to a cucumeris (potato flea beetle) or E. tuberis (tuber flea particular insect or to a class of insects. In case the dsRNA beetle)); Epicauta spp. (e.g. E. vittata (striped blister hits more than one insect target gene, expression of the 35 beetle)); Phaedon spp. (e.g. P. cochleariae (mustard leaf double-stranded RNA within cells of the host organism may beetle)); Empoasca spp. (e.g. E. fabae (potato leafhopper)); confer resistance to more than one insect or more than one Myzus spp. (e.g. M. persicae (green peach aphid)); Para class of insects. trioza spp. (e.g. P. Cockerelli (potato psyllid)); Ostrinia spp. In a preferred embodiment the host organism is a plant (e.g. O. nubilalis (European corn borer)); Conoderus spp. and the insect is a plant pathogenic insect. In this embodi 40 (e.g. C. falli (Southern potato wireworm), or C. vespertinus ment the insect is contacted with the double-stranded RNA (tobacco wireworm)); and Phthorimaea spp. (e.g. P. Oper by expressing the double-stranded RNA in a plant or plant culella (potato tuberworm)); in another embodiment the cell that is infested with or susceptible to infestation with the present invention extends to methods as described herein, plant pathogenic insect. wherein the plant is tomato and the target gene is a gene In this context the term “plant' encompasses any plant 45 from an insect selected from the group consisting of Mac material that it is desired to treat to prevent or reduce insect rosiphum spp. (e.g. M. euphorbiae (potato aphid)); Myzus growth and/or insect infestation. This includes, inter alia, spp. (e.g. M. persicae (green peach aphid)); Trialeurodes whole plants, seedlings, propagation or reproductive mate spp. (e.g. T. vaporariorum (greenhouse whitefly), or T. rial Such as seeds, cuttings, grafts, explants, etc. and also abutilonia (banded-winged whitefly)); Bemisia spp. (e.g. B. plant cell and tissue cultures. The plant material should 50 argentifolii (silverleaf whitefly)); Frankliniella spp. (e.g. F. express, or have the capability to express, dsRNA corre occidentalis (western flower thrips)); Leptinotarsa spp. (e.g. sponding to one or more target genes of the insect. L. decemlineata (Colorado potato beetle), L. juncta (false Therefore, in a further aspect the invention provides a potato beetle), or L. texana (Texan false potato beetle)); plant, preferably a transgenic plant, or propagation or repro Epitrix spp. (e.g. E. hirtipennis (flea beetle)); Lygus spp. ductive material for a (transgenic) plant, or a plant cell 55 (e.g. L. lineolaris (tarnished plant bug), or L. hesperus culture expressing or capable of expressing at least one (western tarnished plant bug)); Euschistus spp. (e.g. E. double-stranded RNA, wherein the double-stranded RNA conspresus (consperse Stinkbug)); Nezara spp. (e.g. N. comprises annealed complementary Strands, one of which viridula (Southern green Stinkbug)); Thyanta spp. (e.g. T. has a nucleotide sequence which is complementary to at pallidovirens (redshouldered Stinkbug)); Phthorimaea spp. least part of a target nucleotide sequence of a target gene of 60 (e.g. P. operculella (potato tuberworm)); Helicoverpa spp. an insect, such that the double-stranded RNA is taken up by (e.g. H. Zea (tomato fruitworm); Keiferia spp. (e.g. K. an insect upon plant-insect interaction, said double stranded lycopersicella (tomato pinworm)); Spodoptera spp. (e.g. S. RNA being capable of inhibiting the target gene or down exigua (beet armyworm), or S. praefica (western yellow regulating expression of the target gene by RNA interfer striped armyworm)); Limonius spp. (wireworms); Agrotis ence. The target gene may be any of the target genes herein 65 spp. (e.g. A. ipsilon (black cutworm)); Manduca spp. (e.g. described, for instance a target gene that is essential for the M. sexta (tobacco hornworm), or M. quinquemaculata (to viability, growth, development or reproduction of the insect. mato hornworm)); Liriomyza spp. (e.g. L. sativae, L. trifoli US 9,528,123 B2 29 30 or L. huidobrensis (leafminer)); and Paratrioza spp. (e.g. P stem borer), C. auricilius (gold-fringed stem borer), or C. cockerelli (tomato psyllid)); In another embodiment the polychrysus (dark-headed stem borer)); Chilotraea spp. (e.g. present invention extends to methods as described herein, C. polychrysa (rice stalk borer)); Sesamia spp. (e.g. S. wherein the plant is corn and the target gene is a gene from inferens (pink rice borer)); Tryporyza spp. (e.g. T. innotata an insect selected from the group consisting of Diabrotica (white rice borer)); Tryporyza spp. (e.g.T. incertulas (yellow spp. (e.g. D. virgifera virgifera (western corn rootworm), D. rice borer)). Cnaphalocrocis spp. (e.g. C. medinalis (rice barberi (northern corn rootworm), D. undecimpunctata leafroller)); Agromyza spp. (e.g. A. Oryzae (leafminer)); howardi (southern corn rootworm), D. virgifera zeae (Mexi Diatraea spp. (e.g. D. Saccharalis (Sugarcane borer)); Nar can corn rootworm); D. balteata (banded cucumber beetle)); naga spp. (e.g. N. aenescens (green rice caterpillar)); Xan Ostrinia spp. (e.g. O. nubilalis (European corn borer)); 10 thodes spp. (e.g. X. transverse (green caterpillar)); Spodop Agrotis spp. (e.g. A. ipsilon (black cutworm)); Helicoverpa tera spp. (e.g. S. frugiperda (fall armyworm)); Mythinna spp. (e.g. H. Zea (corn earworm)); Spodoptera spp. (e.g. S. spp. (e.g. Mythmna (Pseudaletia) seperata (armyworm)); frugiperda (fall armyworm)); Diatraea spp. (e.g. D. gran Helicoverpa spp. (e.g. H. Zea (corn earworm)); Colaspis diosella (Southwestern corn borer), or D. Saccharalis (Sug spp. (e.g. C. brunnea (grape colaspis)): Lissorhoptrus spp. arcane borer)); Elasmopalpus spp. (e.g. E. lignosellus (lesser 15 (e.g. L. Oryzophilus (rice water weevil)); Echinocnemus spp. cornstalk borer); Melanotus spp. (wireworms); Cyclo (e.g. E. squamos (rice plant weevil)); Diclodispa spp. (e.g. cephala spp. (e.g. C. borealis (northern masked chafer)); D. armigera (rice hispa)); Oulema spp. (e.g. O. Oryzae (leaf Cyclocephala spp. (e.g. C. immaculate (Southern masked beetle); Sitophilus spp. (e.g. S. oryzae (rice weevil)); Pachy chafer)); Popillia spp. (e.g. P. japonica (Japanese beetle)); diplosis spp. (e.g. P. Oryzae (rice gall midge)); Hydrellia spp. Chaetocnema spp. (e.g. C. pullicaria (corn flea beetle)); (e.g. H. griseola (Small rice leafminer)); Chlorops spp. (e.g. Sphenophorus spp. (e.g. S. maidis (maize billbug)); Rho C. oryzae (stem maggot)); and Hydrellia spp. (e.g. H. Sasakii palosiphum spp. (e.g. R. maidis (corn leaf aphid)); Anura (rice stem maggot)); phis spp. (e.g. A. maidiradicis (corn root aphid)); Blissus Transgenic plants according to the invention extend to all spp. (e.g. B. leucopterus leucopterus (chinch bug)); Mel plant species specifically described above being resistant to anoplus spp. (e.g. M. femurrubrum (redlegged grasshopper), 25 the respective insect species as specifically described above. M. Sanguinipes (migratory grasshopper)); Hylenya spp. Preferred transgenic plants (or reproductive or propagation (e.g. H. platura (seedcorn maggot)); Agromyza spp. (e.g. A. material for a transgenic plant, or a cultured transgenic plant parvicornis (corn blot leafminer)); Anaphothrips spp. (e.g. cell) are plants (or reproductive or propagation material for A. Obscrurus (grass thrips)); Solenopsis spp. (e.g. S. milesta a transgenic plant, or a cultured transgenic plant cell) (thief ant)); and Tetranychus spp. (e.g. T. urticae (twospotted 30 wherein said plant comprises a nucleic acid sequence which spider mite)); in another embodiment the present invention is selected from the group comprising: extends to methods as described herein, wherein the plant is (i) sequences which are at least 75% identical to a cotton and the target gene is a gene from an insect selected sequence represented by any of SEQID NOs 1, 3, 5, 7, from the group consisting of Helicoverpa spp. (e.g. H. Zea 9, 11, 13, 15, 17, 19, 21, 23, 49 to 158, 159, 160-163, (cotton bollworm)); Pectinophora spp. (e.g. P. gossypiella 35 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, (pink bollworm)); Helicoverpa spp. (e.g. H. armigera 225, 230, 247, 249,251,253,255, 257, 259,275 to 472, (American bollworm)); Earias spp. (e.g. E. vittella (spotted 473, 478,483, 488, 493, 498, 503, 513,515, 517,519, bollworm)); Heliothis spp. (e.g. H. virescens (tobacco bud 521,533 to 575, 576,581,586,591,596,601, 603,605, worm)); Spodoptera spp. (e.g. S. exigua (beet armyworm)); 607, 609, 621 to 767, 768, 773,778,783,788,793, 795, Anthonomus spp. (e.g. A. grandis (boll weevil)); Pseudato 40 797, 799,801, 813 to 862, 863, 868,873, 878,883,888, moscelis spp. (e.g. P. seriatus (cotton fleahopper)); Trialeu 890, 892, 894, 896, 908 to 1040, 1041, 1046, 1051, rodes spp. (e.g. T. abutiloneus (banded-winged whitefly) T. 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, vaporariorum (greenhouse whitefly)); Bemisia spp. (e.g. B. 1085, 1087, 1089, 1091, 1093, 1095, 1097,1099, 1101, argentifolii (silverleaf whitefly): Aphis spp. (e.g. A. gossypii 1103, 1105, 1107, 1109, 1111, 1113, 1161 to 1571, (cotton aphid)); Lygus spp. (e.g. L. lineolaris (tarnished 45 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, plant bug) or L. hesperus (western tarnished plant bug)); 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, Euschistus spp. (e.g. E. conspersus (consperse Stink bug)); 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, Chlorochroa spp. (e.g. C. sayi (Say Stinkbug)); Nezara spp. 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730 to (e.g. N. viridula (green Stinkbug)); Thrips spp. (e.g. T. tabaci 2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070,2075, (onion thrips)); Franklinkiella spp. (e.g. F. fisca (tobacco 50 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106,2108, thrips), or F. Occidentalis (western flower thrips)); Melano 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 2364, plus spp. (e.g. M. femurrubrum (redlegged grasshopper), or 2366, 2368, 2370, 2372, 2384 to 2460, 2461, 2466, M. differentialis (differential grasshopper)); and Tetranychus 2471, 2476 or 2481, or the complement thereof, and spp. (e.g. T. cinnabarinus (carmine spider mite), or T. urticae (ii) sequences comprising at least 17 contiguous nucleo (two spotted spider mite)); in another embodiment the pres 55 tides of any of SEQID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, ent invention extends to methods as described herein, 19, 21, 23, 49 to 158, 159, 160-163, 168, 173, 178, 183, wherein the plant is rice and the target gene is a gene from 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, an insect selected from the group consisting of Nilaparvata 251,253,255, 257, 259,275 to 472, 473,478,483,488, spp. (e.g. N. lugens (brown planthopper)); Laodelphax spp. 493,498, 503,513,515,517,519,521,533 to 575,576, (e.g. L. striatellus (Small brown planthopper)); Nephotettix 60 581,586,591,596,601, 603, 605, 607, 609, 621 to 767, spp. (e.g. N. virescens or N. cincticeps (green leafhopper), or 768, 773,778, 783, 788, 793, 795, 797, 799, 801, 813 N. nigropictus (rice leafhopper)); Sogatella spp. (e.g. S. to 862, 863, 868, 873, 878, 883, 888, 890, 892, 894, fircifera (white-backed planthopper)); Blissus spp. (e.g. B. 896,908 to 1040, 1041, 1046, 1051, 1056, 1061, 1071, leucopterus leucopterus (chinch bug)); Scotinophora spp. 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, (e.g. S. vermidulate (rice blackbug)); Acrosternum spp. (e.g. 65 1091, 1093, 1095, 1097,1099, 1101, 1103, 1105, 1107, A. hilare (green Stink bug)); Parnara spp. (e.g. P. guttata 1109, 1111, 1113, 1161 to 1571, 1572, 1577, 1582, (rice skipper)); Chilo spp. (e.g. C. suppressalis (rice striped 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, US 9,528,123 B2 31 32 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, “operably linked as used herein refers to a functional 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, linkage between the promoter sequence and the gene of 1698, 1700, 1702, 1704, 1730 to 2039, 2040, 2045, interest, such that the promoter sequence is able to initiate 2050, 2055, 2060, 2065, 2070,2075, 2080, 2085, 2090, transcription of the gene of interest. 2095, 2100, 2102, 2104, 2106, 2108, 2120 to 2338, By way of example, the transgene nucleotide sequence 2339, 2344, 2349, 2354, 2359, 2364,2366, 2368,2370, encoding the double-stranded RNA could be placed under 2372,2384 to 2460, 2461, 2466, 2471, 2476 or 2481, the control of an inducible or growth or developmental or the complement thereof, stage-specific promoter which permits transcription of the or wherein said nucleic acid is an insect orthologue of a dsRNA to be turned on, by the addition of the inducer for an gene comprising at least 17 contiguous nucleotides of 10 inducible promoter or when the particular stage of growth or any of SEQ ID NOs 49 to 158,275 to 472,533 to 575, development is reached. 621 to 767,813 to 862,908 to 1040, 1161 to 1571, 1730 Alternatively, the transgene encoding the double-stranded to 2039, 2120 to 2338, 2384 to 2460, or the comple RNA is placed under the control of a strong constitutive ment thereof. promoter Such as any selected from the group comprising The present invention also encompasses plants (or repro 15 the CaMV35S promoter, doubled CaMV35S promoter, ductive or propagation material for a transgenic plant, or a ubiquitin promoter, actin promoter, rubisco promoter, GOS2 cultured transgenic plant cell) which express or are capable promoter, Figwort mosaic viruse (FMV) 34S promoter, of expressing at least one of the nucleotides of the invention, cassava vein mosaic virus (CsVMV) promoter (Verdaguer for instance at least one of the nucleotide sequences repre B. etal, Plant Mol Biol. 199837(6):1055-67). sented in any of SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, Alternatively, the transgene encoding the double-stranded 19, 21, 23, 49 to 158, 159, 160-163, 168, 173, 178, 183, 188, RNA is placed under the control of a tissue specific promoter 193, 198,203,208,215, 220, 225, 230, 240 to 247, 249, 251, Such as any selected from the group comprising root specific 253,255, 257, 259,275 to 472, 473,478,483,488, 493,498, promoters of genes encoding PsMTA Class III chitinase, 503, 508 to 513,515, 517,519, 521, 533 to 575, 576, 581, photosynthetic tissue-specific promoters such as promoters 586,591,596,601, 603, 605, 607, 609, 621 to 767, 768, 773, 25 of cab1 and cab2, rbcS, gap A, gapB and ST-LS1 proteins, 778, 783,788,793,795, 797, 799,801, 813 to 862, 863, 868, JAS promoters, chalcone synthase promoter and promoter of 873, 878, 883, 888, 890, 892, 894, 896, 908 to 1040, 1041, RJ39 from strawberry. 1046, 1051, 1056, 1061, 1066 to 1071, 1073, 1075, 1077, In another embodiment, the transgene encoding the 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, double-stranded RNA is placed under the control of an 1097,1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161 30 insect-induced promoter, for instance the potato proteinase to 1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, inhibitor II (PinII) promoter (Duan X et al. Nat Biotechnol. 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1996, 14(4):494-8)); or a wounding, induced promoter, for 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, instance the jasmonates and ethylene induced promoters, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730 to PDF1.2 promoter (Manners J M et al., Plant Mol Biol. 1998, 2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 35 38(6):1071-80); or under a defense related promoter, for 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, instance the Salicylic acid induced promoters and plant 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, pathogenesis related protein (PR protein) promoters (PR1 2368, 2370, 2372,2384 to 2460, 2461, 2466, 2471, 2476, promoter (Cornelissen B J et al., Nucleic Acids Res. 1987, 2481 or 2486, or the complement thereof, or comprising a 15(17):6799-811; COMT promoter (Toquin V et al, Plant fragment thereof comprising at least 17, preferably at least 40 Mol Biol. 2003, 52(3):495-509). 18, 19, 20 or 21, more preferably at least 22, 23 or 24 Furthermore, when using the methods of the present nucleotides. invention for developing transgenic plants resistant against The plant may be provided in a form wherein it is actively insects, it might be beneficial to place the nucleic acid expressing (transcribing) the double-stranded RNA in one or encoding the double-stranded RNA according to the present more cells, cell types or tissues. Alternatively, the plant may 45 invention under the control of a tissue-specific promoter. In be “capable of expressing, meaning that it is transformed order to improve the transfer of the dsRNA from the plant with a transgene which encodes the desired dsRNA but that cell to the pest, the plants could preferably express the the transgene is not active in the plant when (and in the form dsRNA in a plant part that is first accessed or damaged by in which) the plant is Supplied. the plant pest. In case of plant pathogenic insects, preferred Therefore, according to another embodiment, a recombi 50 tissues to express the dsRNA are the leaves, stems, roots, nant DNA construct is provided comprising the nucleotide and seeds. Therefore, in the methods of the present inven sequence encoding the dsRNA or dsRNA construct accord tion, a plant tissue-preferred promoter may be used. Such as ing to the present invention operably linked to at least one a leaf-specific promoter, a stem-specific promoter, a phloem regulatory sequence. Preferably, the regulatory sequence is specific promoter, a xylem-specific promoter, a root-specific selected from the group comprising constitutive promoters 55 promoter, or a seed-specific promoter (Sucrose transporter or tissue specific promoters as described below. gene AtSUC promoter (Baud Set al., Plant J. 2005, 43(6): The target gene may be any target gene herein described. 824-36), wheat high molecular weight glutenin gene pro Preferably the regulatory element is a regulatory element moter (Robert L S et al., Plant Cell. 1989, 1(6):569-78.)). that is active in a plant cell. More preferably, the regulatory Suitable examples of a root specific promoter are PsMTA element is originating from a plant. The term “regulatory 60 (Fordam-Skelton, A. P. et al., 1997 Plant Molecular Biology sequence' is to be taken in a broad context and refers to a 34: 659-668.) and the Class III Chitinase promoter. regulatory nucleic acid capable of effecting expression of the Examples of leaf- and stem-specific or photosynthetic tis sequences to which it is operably linked. Sue-specific promoters that are also photoactivated are pro Encompassed by the aforementioned term are promoters moters of two chlorophyll binding proteins (cab1 and cab2) and nucleic acids or synthetic fusion molecules or deriva 65 from sugar beet (Stahl D. J., et al., 2004 BMC Biotechnol tives thereof which activate or enhance expression of a ogy 2004 4:31), ribulose-bisphosphate carboxylase nucleic acid, so called activators or enhancers. The term (Rubisco), encoded by rbcS (Nomura M. et al., 2000 Plant US 9,528,123 B2 33 34 Mol. Biol. 44:99-106), A (gapA) and B (gapB) subunits of material, propagation material or cell culture material which chloroplast glyceraldehyde-3-phosphate dehydrogenase does not actively express the dsRNA but has the capability (Conley T. R. et al. 1994 Mol. Cell Biol. 19:2525-33; Kwon to do so. H. B. et al. 1994 Plant Physiol. 105:357-67), promoter of the Accordingly, the present invention encompasses a plant Solanum tuberosum gene encoding the leaf and stem specific (e.g. a rice plant), or a seed (e.g. a rice seed), or a cell (e.g. (ST-LS1) protein (Zaidi M. A. et al., 2005 Transgenic Res. a bacterial or plant cell), comprising at least one double 14:289-98), stem-regulated, defense-inducible genes, such stranded RNA or at least one double-stranded RNA con as JAS promoters (patent publication no. 2005.0034.192/US struct as described herein: or at least one nucleotide A1). An example of a flower-specific promoter is for sequence or at least one recombinant DNA construct as 10 descrobed herein; or at least one plant cell as described instance, the chalcone synthase promoter (Faktor O. et al. herein. The present invention also encompasses a plant (e.g. 1996 Plant Mol. Biol. 32: 849) and an example of a an alfalfa, apple, apricot, artichoke, asparagus, avocado, fruit-specific promoter is for instance RJ39 from strawberry banana, barley, beans, beet, blackberry, blueberry, broccoli, (WO 98.31812). brussel sprouts, cabbage, canola, carrot, cassava, cauli In yet other embodiments of the present invention, other 15 flower, a cereal, celery, cherry, citrus, clemintine, coffee, promoters useful for the expression of dsRNA are used and corn, cotton, cucumber, eggplant, endive, eucalyptus, figes, include, but are not limited to, promoters from an RNA PolI. grape, grapefruit, groundnuts, ground cherry, kiwifruit, let an RNA PolII, an RNA PolIII, T7 RNA polymerase or SP6 tuce, leek, lemon, lime, pine, maize, mango, melon, millet, RNA polymerase. These promoters are typically used for in mushroom, nutaot, okra, onion, orange, an ornamental plant vitro-production of dsRNA, which dsRNA is then included or flower or tree, papaya, parsley, pea, peach, peanut, peat, in an antiinsecticidal agent, for example, in an anti-insecti pepper, persimmon, pineapple, plantain, plum, pomegran cidal liquid, spray or powder. ate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, Therefore, the present invention also encompasses a rice, rye, Sorghum, Soy, Soybean, spinach, Strawberry, Sug method for generating any of the double-stranded RNA or arbeet, Sugargcane, Sunflower, Sweet potato, tangerine, tea, RNA constructs of the invention. This method comprises the 25 tobacco, tomato, a vine, watermelon, wheat, yams or Zuc steps of chini plant; preferably a potato, eggplant, tomato, pepper, a. contacting an isolated nucleic acid or a recombinant tobacco, ground cherry, rice corn or cotton plant), or a seed DNA construct of the invention with cell-free compo or tuber (e.g. an alfalfa, apple, apricot, artichoke, asparagus, nents; or avocado, banana, barley, beans, beet, blackberry, blueberry, b. introducing (e.g. by transformation, transfection or 30 broccoli, brussel Sprouts, cabbage, canola, carrot, cassava, injection) an isolated nucleic acid or a recombinant cauliflower, a cereal, celery, cherry, citrus, clemintine, cof DNA construct of the invention in a cell, fee, corn, cotton, cucumber, eggplant, endive, eucalyptus, under conditions that allow transcription of said nucleic figes, grape, grapefruit, groundnuts, ground cherry, kiwi acid or recombinant DNA construct to produce the dsRNA fruit, lettuce, leek, lemon, lime, pine, maize, mango, melon, or RNA construct. 35 millet, mushroom, nutaot, okra, onion, orange, an ornamen Optionally, one or more transcription termination tal plant or flower or tree, papaya, parsley, pea, peach, sequences may also be incorporated in the recombinant peanut, peat, pepper, persimmon, pineapple, plantain, plum, construct of the invention. The term “transcription termina pomegranate, potato, pumpkin, radicchio, radish, rapeseed, tion sequence' encompasses a control sequence at the end of raspberry, rice, rye, Sorghum, Soy, soybean, spinach, Straw a transcriptional unit, which signals 3' processing and poly 40 berry, Sugarbeet, Sugargcane, Sunflower, Sweet potato, tan adenylation of a primary transcript and termination of tran gerine, tea, tobacco, tomato, a vine, watermelon, wheat, Scription. Additional regulatory elements, such as transcrip yams or Zucchini plant; preferably a potato, eggplant, tional or translational enhancers, may be incorporated in the tomato, pepper, tobacco, ground cherry, rice, corn or cotton expression construct. seed or tuber), or a cell (e.g. a bacterial or plant cell), The recombinant constructs of the invention may further 45 comprising at least one double-stranded RNA or at least one include an origin of replication which is required for main double-stranded RNA construct as described herein: or at tenance and/or replication in a specific cell type. One least one nucleotide sequence or at least one recombinant example is when an expression construct is required to be DNA construct as descrobed herein. Preferably, these plants maintained in a bacterial cell as an episomal genetic element or seeds or cells comprise a recombinant construct wherein (e.g. plasmid or cosmid molecule) in a cell. Preferred origins 50 the nucleotide sequence encoding the dsRNA or dsRNA of replication include, but are not limited to, fl-ori and colE1 construct according to the present invention is operably O1. linked to at least one regulatory element as described above. The recombinant construct may optionally comprise a The plant may be provided in a form wherein it is actively selectable marker gene. As used herein, the term “selectable expressing (transcribing) the RNA molecule in one or more marker gene' includes any gene, which confers a phenotype 55 cells, cell types or tissues. Alternatively, the plant may be on a cell in which it is expressed to facilitate the identifi “capable of expressing, meaning that it is transformed with cation and/or selection of cells, which are transfected or a transgene which encodes the desired RNA molecule but transformed, with an expression construct of the invention. that the transgene is not active in the plant when (and in the Examples of suitable selectable markers include resistance form in which) the plant is supplied. genes against amplicillin (Ampr), tetracycline (Tcr), 60 In one particular embodiment, there is provided a recom kanamycin (Kanr), phosphinothricin, and chloramphenicol binant (expression) construct for expression of an RNA (CAT) gene. Other Suitable marker genes provide a meta molecule in a plant or in a plant cell comprising at least one bolic trait, for example mana. Visual marker genes may also regulatory sequence operably linked to a nucleic acid mol be used and include for example beta-glucuronidase (GUS), ecule comprising at least 14, 15, 16, 17, 18, 19, 20, 21, 22 luciferase and Green Fluorescent Protein (GFP). 65 etc. nucleotides, up to all of the nucleotides of the sequence Plants that have been stably transformed with a transgene set forth as SEQID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, encoding the dsRNA may be supplied as seed, reproductive 23, 49 to 158, 159, 160-163, 168, 173, 178, 183, 188, 193, US 9,528,123 B2 35 36 198,203,208,215, 220, 225, 230, 240 to 247, 249,251, 253, Bacillus thuringiensis insecticidal protein is selected from 255, 257, 259,275 to 472, 473,478,483,488, 493,498, 503, the group consisting of a Cry 1, a Cry3, a TIC851, a 508 to 513,515,517,519, 521, 533 to 575, 576, 581,586, CryET170, a Cry22, a binary insecticidal protein CryET33 591,596,601, 603, 605, 607, 609, 621 to 767, 768, 773,778, and CryET34, a binary insecticidal protein CryET80 and 783,788, 793, 795, 797, 799,801, 813 to 862, 863, 868,873, CryET76, a binary insecticidal protein TIC100 and TIC101, 878, 883, 888,890, 892, 894, 896,908 to 1040, 1041, 1046, and a binary insecticidal protein PS149B1. 1051, 1056, 1061, 1066 to 1071, 1073, 1075, 1077, 1079, In a further embodiment, the invention rela 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, tes to a composition for controlling insect growth and/or 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161 to preventing or reducing insect infestation, comprising at 1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 10 least a plant part, plant cell, plant tissue or seed 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, comprising at least one double-stranded RNA, wherein 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, said double-stranded RNA comprises annealed comple 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730 to mentary Strands, one of which has a nucleotide 2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, sequence which is complementary to at least part of a 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 15 nucleotide sequence of an insect target gene. Option 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, ally, the composition further comprises at least one 2368, 2370, 2372,2384 to 2460, 2461, 2466, 2471, 2476, Suitable carrier, excipient or diluent. The target gene 2481 or 2486, or comprising at least 14, 15, 16, 17, 18, 19. may be any target gene described herein. Preferably the 20, 21, 22 etc. up to all nucleotides of the sequence of an insect target gene is essential for the viability, growth, orthologous nucleic acid molecule from a different target development or reproduction of the insect. species. Many vectors are available for this purpose, and In another aspect the invention relates to a composition as selection of the appropriate vector will depend mainly on the described above, wherein the insect target gene comprises a size of the nucleic acid to be inserted into the vector and the sequence which is at least 75%, preferably at least 80%, particular host cell to be transformed with the vector. 85%, 90%, more preferably at least 95%, 98% or 99% General techniques for expression of exogenous double 25 identical to a sequence selected from the group of sequences stranded RNA in plants for the purposes of RNAi are known represented by any of SEQID NOs 1, 3, 5, 7, 9, 11, 13, 15, in the art (see Baulcombe D, 2004, Nature. 431(7006):356 17, 19, 21, 23, 49 to 158, 159, 160-163, 168, 173, 178, 183, 63. RNA silencing in plants, the contents of which are 188, 193, 198,203,208,215, 220, 225, 230, 240 to 247, 249, incorporated herein by reference). More particularly, meth 251,253,255, 257, 259,275 to 472, 473,478,483,488, 493, ods for expression of double-stranded RNA in plants for the 30 498, 503, 508 to 513,515, 517,519, 521, 533 to 575, 576, purposes of down-regulating gene expression in plant pests 581,586,591,596,601, 603, 605, 607, 609, 621 to 767, 768, such as nematodes or insects are also known in the art. 773,778,783,788,793, 795, 797, 799,801, 813 to 862, 863, Similar methods can be applied in an analogous manner in 868, 873, 878, 883, 888, 890, 892, 894, 896, 908 to 1040, order to express double-stranded RNA in plants for the 1041, 1046, 1051, 1056, 1061, 1066 to 1071, 1073, 1075, purposes of down-regulating expression of a target gene in 35 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, a plant pathogenic insect. In order to achieve this effect it is 1095, 1097,1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, necessary only for the plant to express (transcribe) the 1161 to 1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, double-stranded RNA in a part of the plant which will come 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, into direct contact with the insect, such that the double 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, stranded RNA can be taken up by the insect. Depending on 40 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, the nature of the insect and its relationship with the host 1730 to 2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, plant, expression of the dsRNA could occur within a cell or 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, tissue of a plant within which the insect is also present 2108, 2120 to 2338, 2339, 2344, 2349, 2354, 2359, 2364, during its life cycle, or the RNA may be secreted into a space 2366, 2368, 2370, 2372,2384 to 2460, 2461, 2466, 2471, between cells, such as the apoplast, that is occupied by the 45 2476,2481 or 2486, or the complement thereof, or wherein insect during its life cycle. Furthermore, the dsRNA may be said insect target gene is an insect orthologue of a gene located in the plant cell, for example in the cytosol, or in the comprising at least 17 contiguous nucleotides of any of SEQ plant cell organelles Such as a chloroplast, mitochondrion, ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49 to 158, vacuole or endoplastic reticulum. 159, 160-163, 168, 173, 178, 183, 188, 193, 198, 203, 208, Alternatively, the dsRNA may be secreted by the plant 50 215, 220, 225, 230, 240 to 247, 249,251,253,255, 257, 259, cell and by the plant to the exterior of the plant. As such, the 275 to 472, 473, 478, 483, 488, 493, 498, 503, 508 to 513, dsRNA may form a protective layer on the surface of the 515,517,519,521,533 to 575,576,581,586,591,596,601, plant. 603, 605, 607, 609, 621 to 767, 768,773,778, 783,788,793, In a further aspect, the invention also provides combina 795, 797, 799,801, 813 to 862, 863, 868,873, 878,883,888, tions of methods and compositions for preventing or pro 55 890, 892, 894, 896, 908 to 1040, 1041, 1046, 1051, 1056, tecting plants from pest infestation. For instance, one means 1061, 1066 to 1071, 1073, 1075, 1077, 1079, 1081, 1083, provides using the plant transgenic approach combining 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, methods using expression of dsRNA molecules and methods 1103, 1105, 1107, 1109, 1111, 1113, 1161 to 1571, 1572, using expression of Such Bt insecticidal proteins. 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, Therefore the invention also relates to a method or a plant 60 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662, cell or plant described herein, wherein said plant cell or plant 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, expressing said RNA molecule comprises or expresses a 1694, 1696, 1698, 1700, 1702, 1704, 1730 to 2039, 2040, pesticidal agent selected from the group consisting of a 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085, patatin, a Bacillus thuringiensis insecticidal protein, a Xeno 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120 to 2338, rhabdus insecticidal protein, a Photorhabdus insecticidal 65 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, protein, a Bacillus laterosporous insecticidal protein, and a 2372,2384 to 2460, 2461, 2466, 2471, 2476,2481 or 2486, Bacillus sphearicus insecticidal protein. Preferably said or the complement thereof. US 9,528,123 B2 37 38 According to a still further embodiment, the present moving these insects were not able to right themselves invention extends to a method for increasing plant yield once placed on their backs. Target LD002 (SEQ ID NO comprising introducing in a plant any of the nucleotide 168); Target LD010 (SEQID NO 188); Target LD014 (SEQ sequences or recombinant DNA constructs as herein ID NO 198); Target LD016 (SEQ ID NO 220); glp dsRNA described in an expressible format. Plants encompassed by 5 (SEQ ID NO 235). this method are as described earlier. FIG. 7-LD. Mortality and growth/developmental delay of The invention will be further understood with reference to larval survivors of the Colorado potato beetle, Leptinotarsa the following non-limiting examples. decemlineata, on transgenic potato plants. Seven CPB L1 larvae were fed on transgenic potato siblings harbouring BRIEF DESCRIPTION OF FIGURES AND 10 LD002 construct (O), empty vector (A), or wild type line V TABLES plants () for seven days. Mortality is expressed in percent age and average larval weight in mg. FIG. 1-LD: Survival of L. decemlineata on artificial diet FIG. 1-PC: Effects of ingested target dsRNAs on survival treated with dsRNA. Insects of the second larval stage were and growth of P. cochleariae larvae. Neonate larvae were fed diet treated with 50 ul of topically-applied solution of 15 fed oilseed rape leaf discs treated with 25 ul of topically dsRNA (targets orgfp control). Diet was replaced with fresh applied solution of 0.1 ug/ul dsRNA (targets orgfp control). diet containing topically-applied dsRNA after 7 days. The After 2 days, the insects were transferred onto fresh dsRNA number of Surviving insects were assessed at days 2, 5, 7, 8, treated leaf discs. At day 4, larvae from one replicate for 9, & 13. The percentage of surviving larvae was calculated every treatment were collected and placed in a Petri dish relative to day 0 (start of assay). Target LD006: (SEQID NO containing fresh untreated oilseed rape foliage. The insects 178); Target LD007 (SEQID NO 183); Target LD010 (SEQ were assessed at days 2, 4, 7, 9 & 11. (a) Survival of E. ID NO 188); Target LD011 (SEQ ID NO 193); Target varivestis larvae on oilseed rape leaf discs treated with LD014 (SEQ ID NO 198); gfp dsRNA (SEQ ID NO 235). dsRNA. The percentage of surviving larvae was calculated FIG. 2-LD: Survival of L. decemlineata on artificial diet relative to day 0 (start of assay). (b) Average weights of P treated with dsRNA. Insects of the second larval stage were 25 cochleariae larvae on oilseed rape leaf discs treated with fed diet treated with 50 ul of topically-applied solution of dsRNA. Insects from each replicate were weighed together dsRNA (targets orgfp control). Diet was replaced with fresh and the average weight per determined. Error bars diet only after 7 days. The number of surviving insects was represent standard deviations. Target 1: SEQ ID NO 473; assessed at days 2, 5, 6, 7, 8, 9, 12, & 14. The percentage of target 3: SEQID NO 478; target 5: SEQID NO 483; target surviving larvae was calculated relative to day 0 (start of 30 10: SEQID NO 488; target 14: SEQID NO 493; target 16: assay). Target LD001 (SEQ ID NO 163): Target LD002 SEQ ID NO 498; target 27: SEQ ID NO 503; gifp dsRNA: (SEQ ID NO 168); Target LD003 (SEQID NO 173); Target SEQ ID NO 235. LD015 (SEQID NO 215); Target LD016 (SEQID NO 220): FIG. 2-PC: Survival of P. cochleariae on oilseed rape leaf gfp dsRNA (SEQ ID NO 235). discs treated with different concentrations of dsRNA of (a) FIG. 3-LD: Average weight of L. decemlineata larvae on 35 target PC010 and (b) target PC027. Neonate larvae were potato leaf discs treated with dsRNA. Insects of the second placed on leaf discs treated with 25 ul of topically-applied larval stage were fed leaf discs treated with 20 ul of a solution of dsRNA. Insects were transferred to fresh treated topically-applied solution (10 ng/ul) of dsRNA (target leaf discs at day 2. At day 4 for target PC010 and day 5 for LD002 or gfp). After two days the insects were transferred target PC027, the insects were transferred to untreated on to untreated leaves every day. 40 leaves. The number of Surviving insects were assessed at FIG. 4-LD: Survival of L. decemlineata on artificial diet days 2, 4, 7, 8, 9 & 11 for PC010 and 2, 5, 8, 9 & 12 for treated with shorter versions of target LD014 dsRNA and PC027. The percentage of surviving larvae was calculated concatemer dsRNA. Insects of the second larval stage were relative to day 0 (start of assay). fed diet treated with 50 ul of topically-applied solution of FIG. 1-EV: Survival of E. varivestis larvae on bean leaf dsRNA (gfp or targets). The number of surviving insects 45 discs treated with dsRNA. Neonate larvae were fed bean leaf were assessed at days 3, 4, 5, 6, & 7. The percentage of discs treated with 25 ul of topically-applied solution of 1 surviving larvae were calculated relative to day 0 (start of ug/ul dsRNA (targets or gfp control). After 2 days, the assay). insects were transferred onto fresh dsRNA-treated leaf discs. FIG. 5-LD: Survival of L. decemlineata larvae on artifi At day 4, larvae from one treatment were collected and cial diet treated with different concentrations of dsRNA of 50 placed in a plastic box containing fresh untreated bean target LD002 (a), target LD007 (b), target LD010 (c), target foliage. The insects were assessed for mortality at days 2, 4, LD011 (d), target LD014 (e), target LD015 (f), LD016 (g) 6, 8 & 10. The percentage of surviving larvae was calculated and target LD027 (h). Insects of the second larval stage were relative to day 0 (start of assay). Target 5: SEQID NO 576: fed diet treated with 50 ul of topically-applied solution of target 10: SEQ ID NO 586; target 15: SEQ ID NO 591; dsRNA. Diet was replaced with fresh diet containing topi- 55 target 16: SEQ ID NO 596; gfp dsRNA: SEQ ID NO 235. cally-applied dsRNA after 7 days. The number of surviving FIG. 2-EV: Effects of ingested target dsRNAs on survival insects were assessed at regular intervals. The percentage of of E. varivestis adults and resistance to Snap bean foliar surviving larvae were calculated relative to day 0 (start of insect damage. (a) Survival of E. varivestis adults on bean assay). leaf treated with dsRNA. Adults were fed bean leaf discs FIG. 6-LD. Survival of L. decemlineata adults on potato 60 treated with 75 ul of topically-applied solution of 0.1 ug/ul leaf discs treated with dsRNA. Young adult insects were fed dsRNA (targets or gfp control). After 24 hours, the insects double-stranded-RNA-treated leaf discs for the first two were transferred onto fresh dsRNA-treated leaf discs. After days and were then placed on untreated potato foliage. The a further 24 hours, adults from one treatment were collected number of Surviving insects were assessed regularly; mobile and placed in a plastic box containing potted fresh untreated insects were recorded as insects which were alive and 65 whole bean plants. The insects were assessed for mortality appeared to move normally; moribund insects were recorded at days 4, 5, 6, 7, 8, & 11. The percentage of Surviving adults as insects which were alive but appeared sick and slow was calculated relative to day 0 (start of assay). Target 10: US 9,528,123 B2 39 40 SEQID NO 586; target 15: SEQID NO 591; target 16: SEQ cell culture of OD1 A13301-105 (DE3) of the C. elegans ID NO 596; gfp dsRNA: SEQID NO 235. (b) Resistance to dsRNA library carrying each a vector with a C. elegans bean foliar damage caused by adults of the E. varivestis by genomic fragment for expression of the dsRNA. To each dsRNA. Whole plants containing insects from one treatment well, 4 of the synchronized L1 worms were added and were (see (a)) were checked visually for foliar damage on day 9. incubated at 25° C. for at least 4 to 5 days. These experi (i) target 10; (ii) target 15; (iii) target 16; (iv) gfp dsRNA, (v) ments were performed in quadruplicate. In the screen 6 untreated. controls were used: FIG. 1-TC: Survival of T. castaneum larvae on artificial pGN29-negative control, wild type diet treated with dsRNA of target 14. Neonate larvae were pGZ1 unc-22=twitcher phenotype fed diet based on a flour/milk mix with 1 mg dsRNA target 10 pGZ18- synthase-embryonic lethal 14. Control was water (without dsRNA) in diet. Four pGZ25-pos-1=embryonic lethal replicates of 10 first larvae per replicate were per formed for each treatment. The insects were assessed for pGZ59–bli-4D-acute lethal Survival as average percentage means at days 6, 17, 31, 45 ACC-acetyl co-enzym A carboxylase-acute lethal and 60. The percentage of Surviving larvae was calculated 15 After 5 days, the phenotype of the C. elegans nuc-1 relative to day 0 (start of assay). Error bars represent (e1392) worms fed with the producing dsRNA were standard deviations. Target TC014: SEQ ID NO 878. compared to the phenotype of worms fed with the empty FIG. 1-MP. Effect of ingested target 27 dsRNA on the vector (pGN29) and the other controls. The worms that were Survival of Myzus persicae nymphs. First instars were fed with the dsRNA were screened for lethality (acute or placed in feeding chambers containing 50 ul of liquid diet larval) lethality for the parent (Po) generation, (embryonic) with 2 ug/ul dsRNA (target 27 or gfp dsRNA control). Per lethality for the first filial (F1) generation, or for growth treatment, 5 feeding chambers were set up with 10 instars in retardation of Po as follows: (i) Acute lethality of Po: L1s each feeding chamber. Number of survivors were assessed at have not developed and are dead, this phenotype never gives 8 days post start of bioassay. Error bars represent standard progeny and the well looks quite empty; (ii) (Larval) lethal deviations. Target MP027: SEQ ID NO 1061; gfp dsRNA: 25 ity of Po: Po died in a later stage than L1, this phenotype also SEQ ID NO 235. never gives progeny. Dead larvae or dead adult worms are FIG. 1-NL: Survival of Nilaparvata lugens on liquid found in the wells; (iii) Lethality for F1: L1s have devel artificial diet treated with dsRNA. Nymphs of the first to oped until adult stage and are still alive. This phenotype has second larval stage were fed diet supplemented with 2 no progeny. This can be due to sterility, embryonic lethality mg/ml Solution of dsRNA targets in separate bioassays: (a) 30 (dead eggs on the bottom of well), embryonic arrest or larval NL002, NL003, NL005, NL010; (b) NL009, NL016; (c) arrest (eventually ends up being lethal): (iv) Arrested in NL014, NL018; (d) NL013, NL015, NL021. Insect survival growth and growth retardation/delay: Compared to a well on targets were compared to diet only and diet with gfp with normal development and normal # of progeny. dsRNA control at same concentration. Diet was replaced For the target sequences presented in Table 1A, it was with fresh diet containing dsRNA every two days. The 35 concluded that dsRNA mediated silencing of the C. elegans number of Surviving insects were assessed every day target gene in nematodes, such as C. elegans, had a fatal FIG. 2-NL: Survival of Nilaparvata lugens on liquid effect on the growth and viability of the worm. artificial diet treated with different concentrations of target Subsequent to the above dsRNA silencing experiment, a dsRNA NL002. Nymphs of the first to second larval stage more detailed phenotyping experiment was conducted in C. were fed diet supplemented with 1, 0.2, 0.08, and 0.04 40 elegans in a high throughput format on 24 well plates. The mg/ml (final concentration) of NL002. Diet was replaced dsRNA library produced in bacterial strain AB301-105 with fresh diet containing dsRNA every two days. The (DE3), as described above, was fed to C. elegans nuc-1 numbers of Surviving insects were assessed every day. (e1392) worms on 24 well plates as follows: nuc-1 eggs were transferred to a separate plate and allowed to hatch EXAMPLES 45 simultaneously at 20 C for synchronization of the L1 gen eration. Subsequently 100 of the synchronized L1 worms Example 1 were soaked in a mixture of 500 uLS-complete fed medium, comprising 5 ug/mL cholesterol. 4 LL/mL PEG and 1 mM Silencing C. elegans Target Genes in C. elegans in IPTG, and 500 uL of bacterial cell culture of OD1 High Throughput Screening 50 AB301-105 (DE3) of the C. elegans dsRNA library carrying each a vector with a C. elegans genomic fragment for A C. elegans genome wide library was prepared in the expression of the dsRNA. The soaked L1 worms were rolled pGN9A vector (WO 01/88121) between two identical for 2 hours at 25 C. T7-promoters and terminators, driving its expression in the After centrifugation and removal of 950 uL of the super sense and antisense direction upon expression of the T7 55 natant, 5 LIL of the remaining and resuspended pellet (com polymerase, which was induced by IPTG. prising about 10 to 15 worms) was transferred in the middle This library was transformed into the bacterial strain of each well of a 24 well plate, filled with a layer of agar LB A13301-105 (DE3) in 96 well plate format. For the genome broth. The inoculated plate was incubated at 25° C. for 2 wide screening, these bacterial cells were fed to the nuclease days. At the adult stage, 1 adult worm was singled and deficient C. elegans nuc-1 (e1392) strain. 60 incubated at 25°C. for 2 days for inspection of its progeny. Feeding the dsRNA produced in the bacterial strain The other adult worms are inspected in situ on the original A13301-105 (DE3), to C. elegans nuc-1 (e1392) worms, 24 well plate. These experiments were performed in qua was performed in a 96 well plate format as follows: nuc-1 druplicate. eggs were transferred to a separate plate and allowed to This detailed phenotypic screen was repeated with a hatch simultaneously at 20°C. for synchronization of the L1 65 second batch of worms, the only difference being that the generation. 96 well plates were filled with 100 uL liquid worms of the second batch were incubated at 20 C for 3 growth medium comprising IPTG and with 10 uL bacterial days. US 9,528,123 B2 41 42 The phenotype of the worms fed with C. elegans dsRNA The sequences of the degenerate primers used for ampli was compared to the phenotype of C. elegans nuc-1 (e1392) fication of each of the genes are given in Table 2-LD, which worms fed with the empty vector. displays Leptintarsa decemlineata target genes including Based on this experiment, it was concluded that silencing primer sequences and cDNA sequences obtained. These the C. elegans target genes as represented in Table 1A had 5 primers were used in respective PCR reactions with the a fatal effect on the growth and viability of the worm and that following conditions: 10 minutes at 95°C., followed by 40 the target gene is essential to the viability of nematodes. cycles of 30 seconds at 95° C., 1 minute at 55° C. and 1 Therefore these genes are good target genes to control (kill minute at 72° C., followed by 10 minutes at 72° C. The or prevent from growing) nematodes via dsRNA mediated resulting PCR fragments were analyzed on agarose gel. 10 purified (QIAquick Gel Extraction kit, Cat. Nr. 28706, gene silencing. Accordingly, the present invention encom Qiagen), cloned into the pCR8/GW/topo vector (Cat. Nr. passes the use of nematode orthologues of the above C. K2500 20, Invitrogen), and sequenced. The sequences of the elegans target gene, to control nematode infestation, Such as resulting PCR products are represented by the respective nematode infestation of plants. SEQ ID NOs as given in Table 2-LD and are referred to as 15 the partial sequences. The corresponding partial amino acid Example 2 sequence are represented by the respective SEQID NOs as given in Table 3-LD, where the start of the reading frame is Identification of D. melanogaster Ortholoques indicated in brackets. B. dsRNA Production of the Leptinotarsa decemlineata As described above in Example 1, numerous C. elegans Genes lethal sequences were identified and can be used for iden dsRNA was synthesized in milligram amounts using the tifying orthologues in other species and genera. For commercially available kit T7 RibomaxTM Express RNAi example, the C. elegans lethal sequences can be used to System (Cat. Nr. P1700, Promega). First two separate single identify orthologous D. melanogasters sequences. That is, 5' T7 RNA polymerase promoter templates were generated each C. elegans sequence can be queried against a public 25 in two separate PCR reactions, each reaction containing the database, such as GenBank, for orthologous sequences in D. target sequence in a different orientation relative to the T7 melanogaster. Potential D. melanogaster orthologues were promoter. selected that share a high degree of sequence homology (E For each of the target genes, the sense T7 template was value preferably less than or equal to 1 E-30) and the generated using specific T7 forward and specific reverse sequences are blast reciprocal best hits, the latter means that 30 primers. The sequences of the respective primers for ampli the sequences from different organisms (e.g. C. elegans and fying the sense template for each of the target genes are D. melanogaster) are each other’s top blast hits. For given in Table 8-LD. The conditions in the PCR reactions example, sequence C from C. elegans is compared against were as follows: 4 minutes at 95°C., followed by 35 cycles sequences in D. melanogaster using BLAST. If sequence C of 30 seconds at 95°C., 30 seconds at 55° C. and 1 minute has the D. melanogaster sequence D as best hit and when D 35 at 72°C., followed by 10 minutes at 72°C. The anti-sense is compared to all the sequences of C. elegans, also turns out T7 template was generated using specific forward and to be sequence C, then D and C are reciprocal best hits. This specific T7 reverse primers in a PCR reaction with the same criterium is often used to define orthology, meaning similar conditions as described above. The sequences of the respec sequences of different species, having similar function. The tive primers for amplifying the anti-sense template for each D. melanogaster sequence identifiers are represented in 40 of the target genes are given in Table 8-LD. The resulting Table 1A. PCR products were analyzed on agarose gel and purified by PCR purification kit (Qiaquick PCR Purification Kit, Cat. Example 3 Nr. 28106, Qiagen) and NaClO precipitation. The generated T7 forward and reverse templates were mixed to be tran Leptinotarsa decemlineata (Colorado Potato Beetle) 45 scribed and the resulting RNA strands were annealed, DNase and RNase treated, and purified by sodium acetate, A. Cloning Partial Gene Sequences from Leptinotarsa following the manufacturers instructions. The sense Strand decemlineata of the resulting dsRNA for each of the target genes is given High quality, intact RNA was isolated from 4 different in Table 8-LD. Table 8-LD displays sequences for preparing larval stages of Leptinotarsa decemlineata (Colorado potato 50 ds RNA fragments of Leptinotarsa decemlineata target beetle; source: Jeroen van Schaik, Entocare CV Biologische sequences and concatemer sequences, including primer Gewashescherming, Postbus 162, 6700 AD Wageningen, the Sequences. Netherlands) using TRIZol Reagent (Cat. Nr. 15596-026/ C. Cloning Leptinotarsa decemlineata Genes into Plant 15596-018, Invitrogen, Rockville, Md., USA) following the Vector pK7GWIWG2D(II) manufacturer's instructions. Genomic DNA present in the 55 Since the mechanism of RNA interference operates RNA preparation was removed by DNase treatment follow through dsRNA fragments, the target nucleotide sequences ing the manufacturers instructions (Cat. Nr. 1700, Pro of the target genes, as selected above, were cloned in mega). cDNA was generated using a commercially available anti-sense and sense orientation, separated by the intron kit (SuperScript TM III Reverse Transcriptase, Cat. Nr. CmR-intron, whereby CmR is the chloramphenicol resis 18080044. Invitrogen, Rockville, Md., USA) following the 60 tance marker, to form a dsRNA hairpin construct. These manufacturers instructions. hairpin constructs were generated using the LR recombina To isolate cDNA sequences comprising a portion of the tion reaction between an att-containing entry clone (see LD001, LD002, LD003, LD006, LD007, LD010, LD011, Example 1) and an attR-containing destination vector LD014, LD015, LD016, LC018 and LD027 genes, a series (pK7GWIWG2D(II)). The plant vector pK7GWIWG2D of PCR reactions with degenerate primers were performed 65 (II) was obtained from the VIB/Plant Systems Biology with using Amplitaq Gold (Cat. Nr. N8080240, Applied Biosys a Material Transfer Agreement. LR recombination reaction tems) following the manufacturers instructions. was performed by using LR ClonaseTM II enzyme mix (Cat. US 9,528,123 B2 43 44 Nr. 11791-020. Invitrogen) following the manufacturers TABLE A-continued instructions. These cloning experiments resulted in a hairpin construct for each of the LD002, LD006, LD007, LD010, Ingredients for Artificial diet LD011, LD014 and LD016 genes, having either the pro Ingredients Volume for 1 L moter-sense-intron-CmR-intron-antisense orientation, or 5 fructose 20 g promoter—antisense-intron-CmR-intron-sense orientation, Wesson salt mixture 4 g and wherein the promoter is the plant operable 35S pro tomato fruit powder 12.5 g. moter. The binary vector pK7GWIWG2D(II) with the 35S potato leaf powder 25 g promoter is suitable for transformation into A. tumefaciens. b-sitosterol 1 g 10 sorbic acid 0.8 g. For LD002 and LD010, a double digest with restriction methyl paraben 0.8 g. enzymes BsoBI & Pvul was done on LD002 cloned into Vanderzant vitamin mix 12 g pCR8/GW/topo (see Example 3A). For LD006, LD007, neomycin Sulfate 0.2 g LD011, LD014, LD016 and LD027, a digest with restriction aureomycin 0.130 g rifampicin 0.130 g enzyme BsoBI was done on LD006 cloned into pCR8/GW/ chloramphenicol 0.130 g topo (see Example 3A). The band containing the gene of 15 nystatin 0.050 g interest flanked by the attl sites using Qiaquick Gel Extrac soybean oil 2 ml tion Kit (Cat. Nr. 28706, Qiagen) was purified. An amount wheat germ oil 2 ml of 150 ng of purified fragment and 150 ng pK7GWIWG2D (II) was added together with the LR clonase II enzyme and Fifty ul of a solution of dsRNA at a concentration of 1 incubated for at least 1 h at 25° C. After proteinase K mg/ml was applied topically onto the solid artificial diet in solution treatment (10 min at 37° C.), the whole recombi nation mix was transformed into Top 10 chemically com the wells of the multiwell plate. The diet was dried in a petent cells. Positive clones were selected by restriction laminair flow cabin. Per treatment, twenty-four Colorado digest analysis. The complete sequence of the hairpin con potato beetle larvae (2" stage), with two insects per well, struct for: 25 were tested. The plates were stored in the insect rearing LD002 (antisense-intron-CmR-intron-sense) is set forth chamber at 25+2° C., 60% relative humidity, with a 16:8 in SEQ ID NO 240; hours light:dark photoperiod. The beetles were assessed as LD006 (sense-intron-CmR-intron-antisense) is set forth live or dead every 1, 2 or 3 days. After seven days, for targets in SEQ ID NO 241: LD006, LD007, LD010, LD011, and LD014, the diet was LD007 sense-intron-CmR-intron-antisense) is set forth in 30 replaced with fresh diet with topically applied dsRNA at the SEQ ID NO 242: same concentration (1 mg/ml); for targets LD001, LD002, LD010 (antisense-intron-CmR-intron-sense) is set forth LD003, LD015, and LD016, the diet was replaced with fresh in SEQ ID NO 243: diet only. The dsRNA targets were compared to diet only or LD011 (sense-intron-CmR-intron-antisense) is set forth diet with topically applied dsRNA corresponding to a frag in SEQ ID NO 244: 35 ment of the GFP (green fluorescent protein) coding sequence LD014 (sense-intron-CmR-intron-antisense) is set forth (SEQ ID NO 235). in SEQ ID NO 245; Feeding artificial diet containing intact naked dsRNAs to LD016 (antisense-intron-CmR-intron-sense) is set forth L. decemlineata larvae resulted in significant increases in in SEQ ID NO 246; larval mortalities as indicated in two separate bioassays LD027 (sense-intron-CmR-intron-antisense) is set forth 40 (FIGS. 1LD-2LD). in SEQ ID NO 2486. All dsRNAs tested resulted ultimately in 100% mortality Table 9-LD provides complete sequences for each hairpin after 7 to 14 days. Diet with or without GFP dsRNA COnStruct. Sustained the insects throughout the bioassays with very D. Screening dsRNA Targets Using Artificial Diet for little or no mortality. Activity Against Leptinotarsa decemlineata 45 Typically, in all assays observed, CPB second-stage lar Artificial diet for the Colorado potato beetle was prepared vae fed normally on diet with or without dsRNA for 2 days as follows (adapted from Gelman et al., 2001, J. Ins. Sc., vol. and molted to the third larval stage. At this new larval stage 1, no. 7, 1-10): water and agar were autoclaved, and the the CPB were observed to reduce significantly or stop remaining ingredients (shown in Table Abelow) were added altogether their feeding, with an increase in mortality as a when the temperature dropped to 55° C. At this temperature, 50 result. the ingredients were mixed well before the diet was ali E. Bioassay of dsRNA Targets Using Potato Leaf Discs quoted into 24-well plates (Nunc) with a quantity of 1 ml of for Activity Against the Leptinotarsa decemlineata diet per well. The artificial diet was allowed to solidify by An alternative bioassay method was employed using cooling at room temperature. Diet was stored at 4°C. for up potato leaf material rather than artificial diet as food source to three weeks. 55 for CPB. Discs of approximately 1.1 cm in diameter (or 0.95 cm) were cut out off leaves of 2 to 3-week old potato plants TABLE A using a suitably-sized cork borer. Treated leaf discs were prepared by applying 20 Jul of a 10 ng/ul solution of target Ingredients for Artificial diet LD002 dsRNA or control gfp dsRNA on the adaxial leaf 60 surface. The leaf discs were allowed to dry and placed Ingredients Volume for 1 L individually in 24 wells of a 24-well multiplate (Nunc). A Water 768 mil single second-larval stage CPB was placed into each well, agar 14 g rolled oats 40 g which was then covered with tissue paper and a multiwell Torula yeast 60 g plastic lid. The plate containing the insects and leaf discs lactalbumin hydrolysate 30 g 65 were kept in an insect chamber at 28°C. with a photoperiod casein 10 g of 16 h light/8 h dark. The insects were allowed to feed on the leaf discs for 2 days after which the insects were US 9,528,123 B2 45 46 transferred to a new plate containing fresh treated leaf discs. on agarose gel and purified by PCR purification kit (Qia Thereafter, the insects were transferred to a plate containing quick PCR Purification Kit, Cat. Nr. 28106, Qiagen) and untreated leaf discs every day until day 7. Insect mortality NaClO precipitation. The generated T7 forward and reverse and weight scores were recorded. templates were mixed to be transcribed and the resulting Feeding potato leaf discs with Surface-applied intact RNA strands were annealed, Dnase and Rnase treated, and naked dsRNA of target LD002 to L. decemlineata larvae purified by sodium acetate, following the manufacturers resulted in a significant increase in larval mortalities (i.e. at instructions. The sense strand of the resulting dsRNA is day 7 all insects were dead; 100% mortality) whereas herein represented by SEQ ID NO 203. control gfp dsRNA had no effect on CPB survival. Target For LD014 F2, the sense T7 template was generated LD002 dsRNA severely affected the growth of the larvae 10 after 2 to 3 days whereas the larvae fed with gfp dsRNA at using the specific T7 forward primer oGBM161 and the the same concentration developed as normal (FIG. 3-LD). specific reverse primer oGBM166 (represented herein as F. Screening Shorter Versions of dsRNAs Using Artificial SEQ ID NO 209 and SEQ ID NO 210, respectively) in a Diet for Activity Against Leptinotarsa decemlineata PCR reaction with the following conditions: 4 minutes at This example exemplifies the finding that shorter (60 or 15 95° C., followed by 35 cycles of 30 seconds at 95°C., 30 100 bp) dsRNA fragments on their own or as concatemer seconds at 55° C. and 1 minute at 72° C., followed by 10 constructs are sufficient in causing toxicity towards the minutes at 72°C. The anti-sense T7 template was generated Colorado potato beetle. using the specific forward primer oGBM165 and the specific LD014, a target known to induce lethality in Colorado T7 reverse primer oGBM162 (represented herein as SEQ ID potato beetle, was selected for this example. This gene NO 211 and SEQ ID NO 212, respectively) in a PCR encodes a V-ATPase subunit E (SEQ ID NO 15). reaction with the same conditions as described above. The A 100 base pair fragment, LD014 F1, at position 195-294 resulting PCR products were analyzed on agarose gel and on SEQ ID NO 15 (SEQ ID NO 159) and a 60 base pair purified by PCR purification kit (Qiaquick PCR Purification fragment, LD014 F2, at position 235-294 on SEQID NO 15 Kit, Cat. Nr. 28106, Qiagen) and NaClO precipitation. The (SEQ ID NO 160) were further selected. See also Table 25 generated T7 forward and reverse templates were mixed to 7-LD. be transcribed and the resulting RNA strands were annealed, Two concatemers of 300 base pairs, LD014 C1 and Dnase and Rnase treated, and purified by sodium acetate, LD014 C2, were designed (SEQ ID NO 161 and SEQ ID following the manufacturers instructions. The sense Strand NO 162). LD014 C1 contained 3 repeats of the 100 base of the resulting dsRNA is herein represented by SEQID NO pair fragment described above (SEQ ID NO 159) and 30 208. LD014 C2 contained 5 repeats of the 60 base pair fragment Also for the concatemers, separate single 5' T7 RNA described above (SEQ ID NO 160). See also Table 7-LD. polymerase promoter templates were generated in two sepa The fragments LD014 F1 and LD014 F2 were synthe rate PCR reactions, each reaction containing the target sized as sense and antisense primers. These primers were sequence in a different orientation relative to the T7 pro annealed to create the double strands DNA molecules prior 35 moter. The recombinant plasmids p3 and p4 containing to cloning. Xbal and Xmal restrictions sites were included LD014 C1 & LD014 02 were linearised with Xbal or at the 5' and 3' ends of the primers, respectively, to facilitate Xmal, the two linear fragments for each construct purified the cloning. and used as template for the in vitro transcription assay, The concatemers were made as 300 base pairs synthetic using the T7 promoters flanking the cloning sites. Double genes. Xbal and Xmal restrictions sites were included at the 40 Stranded RNA was prepared by in vitro transcription using 5' and 3' ends of the synthetic DNA fragments, respectively, the T7 RiboMAXTM Express RNAi System (Promega). The to facilite the cloning. sense strands of the resulting dsRNA for LD014 C1 and The 4 DNA molecules, i.e. the 2 single units (LD014 F1 LD014 C2 are herein represented by SEQ ID NO 213 and & LD014 F2) and the 2 concatemers (LD014 C1 & 2114, respectively. LD014 C2), were digested with Xbal and Xmal and sub 45 Shorter sequences of target LD014 and concatemers were cloned in pRluescriptiISK+ linearised by Xbal and Xmal able to induce lethality in Leptinotarsa decemlineata, as digests, resulting in recombinant plasmids p1, p.2, p3, & p4. shown in FIG. 4-LD. respectively. G. Screening dsRNAs at Different Concentrations. Using Double-stranded RNA production: dsRNA was synthe Artificial Diet for Activity Against Leptinotarsa decemlin sized using the commercially available kit T7 RibomaxTM 50 eata Express RNAi System (Cat. Nr. P1700, Promega). First two Fifty ul of a solution of dsRNA at serial ten-fold concen separate single 5' T7 RNA polymerase promoter templates trations from 1 ug/ul (for target LD027 from 0.1 g/ul) down were generated in two separate PCR reactions, each reaction to 0.01 ng/ul was applied topically onto the solid artificial containing the target sequence in a different orientation diet in the wells of a 24-well plate (Nunc). The diet was relative to the T7 promoter. For LD014 F1, the sense T7 55 dried in a laminair flow cabin. Per treatment, twenty-four template was generated using the specific T7 forward primer Colorado potato beetle larvae (2" stage), with two insects oGBM159 and the specific reverse primer oGBM164 (rep per well, were tested. The plates were stored in the insect resented herein as SEQ ID NO 204 and SEQ ID NO 205, rearing chamber at 25+2°C., 60% relative humidity, with a respectively) in a PCR reaction with the following condi 16:8 hours light:dark photoperiod. The beetles were tions: 4 minutes at 95° C., followed by 35 cycles of 30 60 assessed as live or dead at regular intervals up to day 14. seconds at 95°C., 30 seconds at 55° C. and 1 minute at 72° After seven days, the diet was replaced with fresh diet with C., followed by 10 minutes at 72° C. The anti-sense T7 topically applied dsRNA at the same concentrations. The template was generated using the specific forward primer dsRNA targets were compared to diet only. oGBM163 and the specific T7 reverse primer oGBM160 Feeding artificial diet containing intact naked dsRNAs of (represented herein as SEQID NO 206 and SEQID NO 207, 65 different targets to L. decemlineata larvae resulted in high respectively) in a PCR reaction with the same conditions as larval mortalities at concentrations as low as between 0.1 described above. The resulting PCR products were analyzed and 10 ng dsRNA/ul as shown in FIG. 5-LD. US 9,528,123 B2 47 48 H. Adults are Extremely Susceptible to Orally Ingested the susceptible diploid Solanum tuberosum 6487-9 were dsRNA Corresponding to Target Genes. used as starting material for transformation. The example provided below highlights the finding that In vitro derived explants were inoculated with Agrobac adult insects (and not only insects of the larval stage) are terium tumifaciens C58C Rif containing the hairpin con extremely susceptible to orally ingested dsRNA correspond- 5 structs. After three days co-cultivation the explants were put ing to target genes. onto a selective medium containing 100 mg/l Kanamycin Four targets were chosen for this experiment: targets 2, and 300 mg/l Timentin. After 6 weeks post-transformation 10, 14 and 16 (SEQ ID NO 168, 188, 198 and 220, the first putative shoots were removed and rooted on selec respectively). GFP fragment dsRNA (SEQ ID NO 235) was tive medium. Shoots originating from different explants used as a control. Young adults (2 to 3 days old) were picked 10 were treated as independent events, shoots originating from at random from our laboratory-reared culture with no bias the same callus were termed siblings until their clonal status can be verified by Southerns, and nodal cuttings of a towards insect gender. Ten adults were chosen per treatment. shoot were referred to as 'clones. The adults were prestarved for at least 6 hours before the The transgenic status of the rooting shoots was checked onset of the treatment. On the first day of treatment, each 15 either by GFP fluorescence or by plus/minus PCR for the adult was fed four potato leaf discs (diameter 1.5 cm) which target sequence. Positive shoots were then clonally propa were pretreated with a topical application of 25 ul of 0.1 gated in tissue culture to ensure enough replicates were ug/ul target dsRNA (synthesized as described in Example available for the Colorado potato beetle assay with the first 3A; topical application as described in Example 3E) per plants being available to test fourteen weeks post transfor disc. Each adult was confined to a small petridish (diameter mation. 3 cm) in order to make Sure that all insects have ingested Bioassay equal amounts of food and thus received equal doses of Transgenic potato plants were grown to the 8-12 unfolded dsRNA. The following day, each adult was again fed four leaf stage in a plant growth room chamber with the follow treated leaf discs as described above. On the third day, all ten ing conditions: 23+2° C., 60% relative humidity, 16:8 hour adults per treatment were collected and placed together in a 25 light:dark photoperiod. The plants were caged by placing a cage consisting of a plastic box (dimensions 30 cmx20 500 ml bottle upside down over the plant with the neck of cmx15 cm) with a fine nylon mesh built into the lid to the bottle firmly placed in the soil in a pot and base cut open provide good aeration. Inside the box, Some moistened filter and covered with a fine nylon mesh to permit aeration, paper was placed in the base. Some (untreated) potato reduce condensation inside and prevent larval escape. foliage was placed on top of the paper to maintain the adults 30 In this bioassay, seven neonate CPB larvae were placed on during the experiment. From day 5, regular assessments the foliage of each transgenic potato plant. Six transgenic were carried out to count the number of dead, alive (mobile) potato siblings per transformation event (i.e. plants derived and moribund insects. For insect moribundity, adults were from one callus) of the hairpin construct LD002 (comprising laid on their backs to check whether they could right SEQ ID NO 240) (labeled as pGBNB001/28A to F) and themselves within several minutes; an insect was considered 35 empty vector (labeled as pK7GWIWG2D(II)/11A to F), and moribund only if it was not able to turn onto its front. two wild type plants were tested. Temperature, humidity and Clear specific toxic effects of double-stranded RNA cor lighting conditions were the same as described above. At day responding to different targets towards adults of the Colo 7 (7 days after the start of the bioassay), the number of rado potato beetle, Leptinotarsa decemlineata, were dem Survivors were counted and the average weight of larval onstrated in this experiment (FIG. 6-LD). Double-stranded 40 Survivors from each plant recorded. Data was analysed using RNA corresponding to a gfp fragment showed no toxicity the Spotfire R DecisionSite.R. 9.0 software (Version towards CPB adults on the day of the final assessment (day 17.1.779) from Spotfire Inc. 19). This experiment clearly showed that the survival of In this experiment, all larvae of the Colorado potato beetle CPB adults was severely reduced only after a few days of on two sibling plants (labeled as pGENB001/28A and exposure to dsRNA when delivered orally. For example, for 45 pGBNB001/28F), harbouring hairpin construct LD002, gen target 10, on day 5, 5 out of 10 adults were moribund (sick erated from a single transformation event, were dead on day and slow moving); on day 6, 4 out of 10 adults were dead 7 (FIG. 7-LD). Feeding damage by CPB larvae on these two with three of the survivors moribund; on day 9 all adults plants was very low when compared to the empty vector were observed dead. transgenic plants or wild type line V plants. As a consequence of this experiment, the application of 50 target double-stranded RNAS against insect pests may be Example 4 broadened to include the two life stages of an insect pest (i.e. larvae and adults) which could cause extensive crop damage, Phaedon cochleariae (Mustard Leaf Beetle) as is the case with the Colorado potato beetle. I. Laboratory Trials to Test Transgenic Potato Plants 55 A. Cloning of a Partial Sequence of the Phaedon cochle Against Larvae of the Colorado Potato Beetle, Leptinotarsa ariae (Mustard Leaf Beetle) PC001, PC003, PC005, PC010, decemlineata PC014, PC016 and PC027 Genes Via Family PCR The example provided below is an exemplification of the High quality, intact RNA was isolated from the third finding that transgenic potato plants expressing CPB-gene larval stage of Phaedon cochleariae (mustard leaf beetle: specific hairpin RNAs adversely affected Colorado potato 60 source: Dr. Caroline Muller, Julius-von-Sachs-Institute for beetles. Biosciences, Chemical Ecology Group, University of Wuer Potato Transformation zburg, Julius-von-Sachs-Platz 3, D-97082 Wuerzburg, Ger Stably transformed potato plants were obtained using an many) using TRIZol Reagent (Cat. Nr. 15596-026/15596 adapted protocol received through Julie Gilbert at the NSF 018, Invitrogen, Rockville, Md., USA) following the Potato Genome Project. Stem intemode explants of potato 65 manufacturers instructions. Genomic DNA present in the Line V (obtained from the Laboratory of Plant Breeding at RNA preparation was removed by DNase (Cat. Nr. 1700, PRI Wageningen, the Netherlands) which was derived from Promega) treatment following the manufacturers instruc US 9,528,123 B2 49 50 tions. cDNA was generated using a commercially available anti-sense and sense orientation, separated by the intron kit (SuperScript TM III Reverse Transcriptase, Cat. Nr. CmR-intron, whereby CmR is the chloramphenicol resis 18080044. Invitrogen, Rockville, Md., USA) following the tance marker, to form a dsRNA hairpin construct. These manufacturers instructions. hairpin constructs were generated using the LR recombina To isolate cDNA sequences comprising a portion of the tion reaction between an att-containing entry clone (see PC001, PC003, PC005, PC010, PC014, PC016 and PC027 Example 4A) and an attR-containing destination vector genes, a series of PCR reactions with degenerate primers (pK7GWIWG2D(II)). The plant vector pK7GWIWG2D were performed using Amplitaq Gold (Cat. Nr. N8080240, (II) was obtained from the VIB/Plant Systems Biology with Applied Biosystems) following the manafacturers instruc a Material Transfer Agreement. LR recombination reaction tions. 10 was performed by using LR ClonaseTM II enzyme mix (Cat. The sequences of the degenerate primers used for ampli Nr. 11791-020. Invitrogen) following the manufacturers fication of each of the genes are given in Table 2-PC. These instructions. These cloning experiments resulted in a hairpin primers were used in respective PCR reactions with the construct for each of the PC001, PC010, PC014, PC016 and following conditions: 10 minutes at 95°C., followed by 40 PC027 genes, having the promoter sense-intron-CmR-in cycles of 30 seconds at 95° C., 1 minute at 55° C. and 1 15 tron-antisense orientation, and wherein the promoter is the minute at 72° C., followed by 10 minutes at 72° C. The plant operable 35S promoter. The binary vector resulting PCR fragments were analyzed on agarose gel. pK7GWIWG2D(II) with the 35S promoter is suitable for purified (QIAquick Gel Extraction kit, Cat. Nr. 28706, transformation into A. tumefaciens. Qiagen), cloned into the pCR4/TOPO vector (Cat. Nr. Restriction enzyme digests were carried out on pCR8/ K4530-20, Invitrogen) and sequenced. The sequences of the GW/TOPO plasmids containing the different targets (see resulting PCR products are represented by the respective Example 4B): for PC001, a double digest with BsoBI & SEQ ID NOs as given in Table 2-PC and are referred to as Pvul; for PC010, a double digest with Pvul & PvulI; for the partial sequences. PC014, a triple digest with HincII, Pvul & XhoI; for PC016, The corresponding partial amino acid sequence are rep a single digest with Apal I; for PC027, a double digest with resented by the respective SEQ ID NOs as given in Table 25 Aval & DrdI. The band containing the gene of interest 3-PC. Table 3-PC provides amino acid sequences of cDNA flanked by the attl sites using Qiaquick Gel Extraction Kit clones, and the start of the reading frame is indicated in (Cat. Nr. 28706, Qiagen) was purified. An amount of 150 ng brackets. of purified fragment and 150 ng pK7GWIWG2D(II) was B. dsRNA Production of the Phaedon cochleariae Genes added together with the LR clonase II enzyme and incubated dsRNA was synthesized in milligram amounts using the 30 for at least 1 h at 25° C. After proteinase K solution commercially available kit T7 RibomaxTM Express RNAi treatment (10 min at 37° C.), the whole recombination mix System (Cat. Nr. P1700, Promega). First two separate single was transformed into Top 10 chemically competent cells. 5' T7 RNA polymerase promoter templates were generated Positive clones were selected by restriction digest analyses. in two separate PCR reactions, each reaction containing the The complete sequence of the hairpin construct for: target sequence in a different orientation relative to the T7 35 PC001 (sense-intron-CmR-intron-antisense) is repre promoter. sented in SEQ ID NO 508; For each of the target genes, the sense T7 template was PC010 (sense-intron-CmR-intron-antisense) is repre generated using specific T7 forward and specific reverse sented in SEQ ID NO 509; primers. The sequences of the respective primers for ampli PC014 (sense-intron-CmR-intron-antisense) is repre fying the sense template for each of the target genes are 40 sented in SEQ ID NO 510; given in Table 8-PC. Table 8-PC provides details for pre PC016 (sense-intron-CmR-intron-antisense) is repre paring ds RNA fragments of Phaedon cochleariae target sented in SEQ ID NO 511; sequences, including primer sequences. PC027 (sense-intron-CmR-intron-antisense) is repre The conditions in the PCR reactions were as follows: 1 sented in SEQ ID NO 512: minute at 95°C., followed by 20 cycles of 30 seconds at 95° 45 Table 9-PC provides sequences for each hairpin construct. C., 30 seconds at 60° C. and 1 minute at 72°C., followed by D. Laboratory Trials to Test dsRNA Targets. Using Oil 15 cycles of 30 seconds at 95°C., 30 seconds at 50° C. and seed Rape Leaf Discs for Activity Against Phaedon cochle 1 minute at 72° C. followed by 10 minutes at 72° C. The ariae Larvae anti-sense T7 template was generated using specific forward The example provided below is an exemplification of the and specific T7 reverse primers in a PCR reaction with the 50 finding that the mustard leaf beetle (MLB) larvae are sus same conditions as described above. The sequences of the ceptible to orally ingested dsRNA corresponding to own respective primers for amplifying the anti-sense template for target genes. each of the target genes are given in Table 8-PC. The To test the different double-stranded RNA samples against resulting PCR products were analyzed on agarose gel and MLB larvae, a leaf disc assay was employed using oilseed purified by PCR purification kit (Qiaquick PCR Purification 55 rape (Brassica napus variety SW Oban; source: Nick Kit, Cat. Nr. 28106, Qiagen) and NaClO precipitation. The Balaam, Sw Seed Ltd., 49 North Road, Abington, Cam generated T7 forward and reverse templates were mixed to bridge, CB1 6AS, UK) leaf material as food source. The be transcribed and the resulting RNA strands were annealed, insect cultures were maintained on the same variety of DNase and RNase treated, and purified by sodium acetate, oilseed rape in the insect chamber at 25+2° C. and 60+5% following the manufacturers instructions. The sense Strand 60 relative humidity with a photoperiod of 16 h light/8 h dark. of the resulting dsRNA for each of the target genes is given Discs of approximately 1.1 cm in diameter (or 0.95 cm) in Table 8-PC. were cut out off leaves of 4- to 6-week old rape plants using C. Recombination of the Phaedon cochleariae (Mustard a suitably-sized cork borer. Double-stranded RNA samples Leaf Beetle) Genes into the Plant Vector pK7GWIWG2D(II) were diluted to 0.1 lug/ul in Milli-Q water containing 0.05% Since the mechanism of RNA interference operates 65 Triton X-100. Treated leaf discs were prepared by applying through dsRNA fragments, the target nucleotide sequences 25ul of the diluted solution of target PC001, PC003, PC005, of the target genes, as selected above, were cloned in PC010, PC014, PC016, PC027 dsRNA and control gfp US 9,528,123 B2 51 52 dsRNA or 0.05% Triton X-100 on the adaxial leaf Surface. F. Laboratory Trials of Myzus periscae (Green Peach The leaf discs were left to dry and placed individually in Aphid) Infestation on Transgenic Arabidopsis thaliana each of the 24 wells of a 24-well multiplate containing 1 ml Plants of gellified 2% agar which helps to prevent the leaf disc from Generation of Transgenic Plants drying out. Two neonate MLB larvae were placed into each 5 Arabidopsis thaliana plants were transformed using the well of the plate, which was then covered with a multiwell floral dip method (Clough and Bent (1998) Plant Journal plastic lid. The plate (one treatment containing 48 insects) 16:735-743). Aerial parts of the plants were incubated for a was divided into 4 replicates of 12 insects per replicate (each few seconds in a solution containing 5% Sucrose, resus row). The plate containing the insects and leaf discs were pended Agrobacterium tumefaciens strain C58C1 Rif cells kept in an insect chamber at 25+2° C. and 60+5% relative 10 from an overnight culture and 0.03% of the surfactant Silwet humidity with a photoperiod of 16 h light/8 h dark. The L-77. After inoculation, plants were covered for 16 hours insects were fed leaf discs for 2 days after which they were with a transparent plastic to maintain humidity. To increase transferred to a new plate containing freshly treated leaf the transformation efficiency, the procedure was repeated discs. Thereafter, 4 days after the start of the bioassay, the after one week. Watering was stopped as seeds matured and insects from each replicate were collected and transferred to 15 dry seeds were harvested and cold-treated for two days. a Petri dish containing untreated fresh oilseed rape leaves. After sterilization, seeds were plated on a kanamycin-con Larval mortality and average weight were recorded at days taining growth medium for selection of transformed plants. 2, 4 7, 9 and 11. The selected plants are transferred to soil for optimal T2 P. cochleariae larvae fed on intact naked target dsRNA seed production. treated oilseed rape leaves resulted in significant increases in Bioassay larval mortalities for all targets tested, as indicated in FIG. Transgenic Arabidopsis thaliana plants are selected by 1(a). Tested double-stranded RNA for target PC010 led to allowing the segregating T2 seeds to germinate on appro 100% larval mortality at day 9 and for target PCO27 at day priate selection medium. When the roots of these transgenics 11. For all other targets, significantly high mortality values are well-established they are then transferred to fresh arti were reached at day 11 when compared to control gfp 25 ficial growth medium or soil and allowed to grow under dsRNA, 0.05% Trition X-100 alone or untreated leaf only: optimal conditions. Whole transgenic plants are tested (average value in percentage-confidence interval with alpha against nymphs of the green peach aphid (Myzus persicae) 0.05) PC001 (94.4+8.2); PC003 (86.1+4.1): PC005 to show (1) a significant resistance to plant damage by the (83.3+7.8); PC014 (63.9+20.6); PC016 (75.0+16.8); gfp feeding nymph, (2) increased nymphal mortality, and/or (3) dsRNA (11.1+8.2): 0.05% Triton X-100 (19.4+10.5); leaf 30 decreased weight of nymphal Survivors (or any other aber only (8.3+10.5). rant insect development). Larval survivors were assessed based on their average weight. For all targets tested, the mustard leaf beetle larvae Example 5 had significantly reduced average weights after day 4 of the bioassay; insects fed control gfp dsRNA or 0.05% Triton 35 Epilachna varivetis (Mexican Bean Beetle) X-100 alone developed normally, as for the larvae on leaf only (FIG. 1(b)-PC). A. Cloning Epilachna varivetis Partial Gene Sequences E. Laboratory Trials to Screen dsRNAs at Different Con High quality, intact RNA was isolated from 4 different centrations. Using Oilseed Rape Leaf Discs for Activity larval stages of Epilachna varivetis (Mexican bean beetle: Against Phaedon cochleariae Larvae 40 Source: Thomas Dorsey, Supervising Entomologist, New Twenty-five ul of a solution of dsRNA from target PC010 Department of Agriculture, Division of Plant Indus or PC027 at serial ten-fold concentrations from 0.1 lug/ul try, Bureau of , Phillip Alampi Ben down to 0.1 ng/ul was applied topically onto the oilseed rape eficial Insect Laboratory, PO Box 330, Trenton, N.J. 08625 leaf disc, as described in Example 4D above. As a negative 0330, USA) using TRIZol Reagent (Cat. Nr. 15596-026/ control, 0.05% Triton X-100 only was administered to the 45 15596-018, Invitrogen, Rockville, Md., USA) following the leaf disc. Per treatment, twenty-four mustard leaf beetle manufacturers instructions. Genomic DNA present in the neonate larvae, with two insects per well, were tested. The RNA preparation was removed by DNase treatment follow plates were stored in the insect rearing chamber at 25+2°C., ing the manafacturer's instructions (Cat. Nr. 1700, Pro 60+5% relative humidity, with a 16:8 hours light:dark pho mega). cDNA was generated using a commercially available toperiod. At day 2, the larvae were transferred on to a new 50 kit (SuperScript TM III Reverse Transcriptase, Cat. Nr. plate containing fresh dsRNA-treated leaf discs. At day 4 for 18080044. Invitrogen, Rockville, Md., USA) following the target PC010 and day 5 for target PC027, insects from each manufacturers instructions. replicate were transferred to a Petridish containing abundant To isolate cDNA sequences comprising a portion of the untreated leaf material. The beetles were assessed as live or EV005, EVO09, EVO10, EVO15 and EV016 genes, a series dead on days 2, 4, 7, 8, 9, and 11 for target PC010, and 2. 55 of PCR reactions with degenerate primers were performed 5, 8, 9 and 12 for target PC027. using Amplitaq Gold (Cat. Nr. N8080240, Applied Biosys Feeding oilseed rape leaf discs containing intact naked tems) following the manufacturers instructions. dsRNAs of the two different targets, PC010 and PC027, to The sequences of the degenerate primers used for ampli P. cochleariae larvae resulted in high mortalities at concen fication of each of the genes are given in Table 2-EV, which trations down to as low as 1 ng dsRNA/ul solution, as shown 60 displays Epilachna varivetis target genes including primer in FIGS. 2 (a) and (b). Average mortality values in sequences and cDNA sequences obtained. These primers percentage-confidence interval with alpha 0.05 for different were used in respective PCR reactions with the following concentrations of dsRNA for target PC010 at day 11, Oug/ul: conditions: for EVO05 and EV009, 10 minutes at 95°C., 8.3+9.4; 0.1 ug/ul: 100; 0.01 g/ul: 79.2+20.6; 0.001 ug/ul: followed by 40 cycles of 30 seconds at 95°C., 1 minute at 58.3-9.4; 0.0001 ug/ul: 12.5+15.6; and for target PC027 at 65 50° C. and 1 minute 30 seconds at 72° C., followed by 7 day 12, Oug/ul: 8.3-9.4; 0.1 lug/ul: 95.8+8.2: 0.01 lug/ul: minutes at 72°C.; for EVO14, 10 minutes at 95°C., followed 95.8+8.2: 0.001 ug/ul: 83.3+13.3; 0.0001 ug/ul: 12.5+8.2. by 40 cycles of 30 seconds at 95°C., 1 minute at 53° C. and US 9,528,123 B2 53 54 1 minute at 72° C., followed by 7 minutes at 72° C.; for vector pK7GWIWG2D(II) with the 35S promoter is suitable EV010 and EV016, 10 minutes at 95°C., followed by 40 for transformation into A. tumefaciens. cycles of 30 seconds at 95° C. 1 minute at 54° C. and 1 Restriction enzyme digests were carried out on pCR8/ minute 40 seconds at 72°C., followed by 7 minutes at 72° GW/TOPO plasmids containing the different targets (see C. The resulting PCR fragments were analyzed on agarose Example B). The band containing the gene of interest gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706, flanked by the attl sites using Qiaquick Gel Extraction Kit Qiagen), cloned into the pCR4/TOPO vector (Cat. Nr. (Cat. Nr. 28706, Qiagen) is purified. An amount of 150 ng K4530-20, Invitrogen), and sequenced. The sequences of the of purified fragment and 150 ng pK7GWIWG2D(II) is resulting PCR products are represented by the respective added together with the LR clonase II enzyme and incubated 10 for at least 1 h at 25° C. After proteinase K solution SEQ ID NOs as given in Table 2-EV and are referred to as treatment (10 min at 37° C.), the whole recombination mix the partial sequences. The corresponding partial amino acid is transformed into Top 10 chemically competent cells. sequences are represented by the respective SEQID NOs as Positive clones are selected by restriction digest analyses. given in Table 3-EV, where the start of the reading frame is D. Laboratory Trials to Test dsRNA Targets Using Bean indicated in brackets. 15 Leaf Discs for Activity Against Epilachna varivetis Larvae B. dsRNA Production of the Epilachna varivetis Genes The example provided below is an exemplification of the dsRNA was synthesized in milligram amounts using the finding that the Mexican bean beetle (MBB) larvae are commercially available kit T7 RibomaxTM Express RNAi Susceptible to orally ingested dsRNA corresponding to own System (Cat. Nr. P1700, Promega). First two separate single target genes. 5' T7 RNA polymerase promoter templates were generated To test the different double-stranded RNA samples against in two separate PCR reactions, each reaction containing the MBB larvae, a leaf disc assay was employed using Snap target sequence in a different orientation relative to the T7 bean (Phaseolus vulgaris variety Montano; source: Aveve N promoter. V. Belgium) leaf material as food source. The same variety For each of the target genes, the sense T7 template was of beans was used to maintain insect cultures in the insect generated using specific T7 forward and specific reverse 25 chamber at 25+2° C. and 60+5% relative humidity with a primers. The sequences of the respective primers for ampli photoperiod of 16 h light/8 h dark. Discs of approximately fying the sense template for each of the target genes are 1.1 cm in diameter (or 0.95 cm) were cut out off leaves of given in Table 8-EV. 1- to 2-week old bean plants using a suitably-sized cork The conditions in the PCR reactions were as follows: 1 borer. Double-stranded RNA samples were diluted to 1 lug/ul minute at 95°C., followed by 20 cycles of 30 seconds at 95° 30 in Milli-Q water containing 0.05% Triton X-100. Treated C., 30 seconds at 60° C. and 1 minute at 72°C., followed by leaf discs were prepared by applying 25 ul of the diluted 15 cycles of 30 seconds at 95°C., 30 seconds at 50° C. and solution of target Ev005, Ev010, Ev015, Ev016 dsRNA and 1 minute at 72° C. followed by 10 minutes at 72° C. The control gfp dsRNA or 0.05% Triton X-100 on the adaxial anti-sense T7 template was generated using specific forward leaf surface. The leaf discs were left to dry and placed and specific T7 reverse primers in a PCR reaction with the 35 individually in each of the 24 wells of a 24-well multiplate same conditions as described above. The sequences of the containing 1 ml of gellified 2% agar which helps to prevent respective primers for amplifying the anti-sense template for the leaf disc from drying out. A single neonate MBB larva each of the target genes are given in Table 8-EV. The was placed into each well of a plate, which was then covered resulting PCR products were analyzed on agarose gel and with a multiwell plastic lid. The plate was divided into 3 purified by PCR purification kit (Qiaquick PCR Purification 40 replicates of 8 insects per replicate (row). The plate con Kit, Cat. Nr. 28106, Qiagen) and NaClO precipitation. The taining the insects and leaf discs were kept in an insect generated T7 forward and reverse templates were mixed to chamber at 25+2° C. and 60+5% relative humidity with a be transcribed and the resulting RNA strands were annealed, photoperiod of 16 h light/8 h dark. The insects were fed on DNase and RNase treated, and purified by sodium acetate, the leaf discs for 2 days after which the insects were following the manufacturers instructions. The sense Strand 45 transferred to a new plate containing freshly treated leaf of the resulting dsRNA for each of the target genes is given discs. Thereafter, 4 days after the start of the bioassay, the in Table 8-EV. insects were transferred to a petriplate containing untreated C. Recombination of the Epilachna varivetis Genes into fresh bean leaves every day until day 10. Insect mortality the Plant Vector pK7GWIWG2D(II) was recorded at day 2 and every other day thereafter. Since the mechanism of RNA interference operates 50 Feeding Snap bean leaves containing Surface-applied through dsRNA fragments, the target nucleotide sequences intact naked target dsRNAs to E. varivestis larvae resulted in of the target genes, as selected above, are cloned in anti significant increases in larval mortalities, as indicated in sense and sense orientation, separated by the intron-CmR FIG. 1. Tested double-stranded RNAs of targets Ev010, intron, whereby CmR is the chloramphenicol resistance Ev015, & Ev016 led to 100% mortality after 8 days, whereas marker, to form a dsRNA hairpin construct. These hairpin 55 dsRNA of target Ev005 took 10 days to kill all larvae. The constructs are generated using the LR recombination reac majority of the insects fed on treated leaf discs containing tion between an attL-containing entry clone (see Example control gfp dsRNA or only the surfactant Triton X-100 were 5A) and an attR-containing destination vector sustained throughout the bioassay (FIG. 1-EV). (=pK7GWIWG2D(II)). The plant vector pK7GWIWG2D E. Laboratory Trials to Test dsRNA Targets Using Bean (II) is obtained from the VIB/Plant Systems Biology with a 60 Leaf Discs for Activity Against Epilachna varivestis Adults Material Transfer Agreement. LR recombination reaction is The example provided below is an exemplification of the performed by using LR ClonaseTM II enzyme mix (Cat. Nr. finding that the Mexican bean beetle adults are susceptible 11791-020, Invitrogen) following the manufacturers to orally ingested dsRNA corresponding to own target genes. instructions. These cloning experiments result in a hairpin In a similar bioassay set-up as for Mexican bean beetle construct for each of the target genes, having the promoter— 65 larvae, adult MBBs were tested against double-stranded sense-intron-CmR-intron-antisense orientation, and wherein RNAs topically-applied to bean leaf discs. Test dsRNA from the promoter is the plant operable 35S promoter. The binary each target Ev010, Ev015 and Ev016 was diluted in 0.05% US 9,528,123 B2 55 56 Triton X-100 to a final concentration of 0.1 g/ul. Bean leaf kit, Cat. Nr. 28706, Qiagen), cloned into the pCR8/GW/ discs were treated by topical application of 30 ul of the test TOPO vector (Cat. Nr. K2500-20, Invitrogen) and solution onto each disc. The discs were allowed to dry sequenced. The sequences of the resulting PCR products are completely before placing each on a slice of gellified 2% represented by the respective SEQID NOs as given in Table agar in each well of a 24-well multiwell plate. Three-day-old 2-AG and are referred to as the partial sequences. The adults were collected from the culture cages and fed nothing corresponding partial amino acid sequence are represented for 7-8 hours prior to placing one adult to each well of the by the respective SEQ ID NOs as given in Table 3-AG. bioassay plate (thus 24 adults per treatment). The plates B. dsRNA Production of the Anthonomus grandis (Cotton were kept in the insect rearing chamber (under the same Boll Weevil) Genes conditions as for MBB larvae for 24 hours) after which the 10 dsRNA was synthesized in milligram amounts using the adults were transferred to a new plate containing fresh commercially available kit T7 RibomaxTM Express RNAi dsRNA-treated leaf discs. After a further 24 hours, the adults System (Cat. Nr. P1700, Promega). First two separate single from each treatment were collected and placed in a plastic 5' T7 RNA polymerase promoter templates were generated box with dimensions 30 cmx15 cmx10 cm containing two in two separate PCR reactions, each reaction containing the potted and untreated 3-week-old bean plants. Insect mortal 15 target sequence in a different orientation relative to the T7 ity was assessed from day 4 until day 11. promoter. All three target dsRNAs (Ev010, Ev015 and Ev016) For each of the target genes, the sense T7 template was ingested by adults of Epillachna varivestis resulted in sig generated using specific T7 forward and specific reverse nificant increases in mortality from day 4 (4 days post primers. The sequences of the respective primers for ampli bioassay start), as shown in FIG. 2-EV(a). From day 5, fying the sense template for each of the target genes are dramatic changes in feeding patterns were observed between given in Table 8-AG. A touchdown PCR was performed as insects fed initially with target-dsRNA-treated bean leaf follows: 1 minute at 95° C., followed by 20 cycles of 30 discs and those that were fed discs containing control gfp seconds at 95°C., 30 seconds at 60° C. with a decrease in dsRNA or surfactant Triton X-100. Reductions in foliar temperature of 0.5° C. per cycle and 1 minute at 72° C. damage by MBB adults of untreated bean plants were 25 followed by 15 cycles of 30 seconds at 95°C., 30 seconds clearly visible for all three targets when compared to gfp at 50° C. and 1 minute at 72°C., followed by 10 minutes at dsRNA and surfactant only controls, albeit at varying levels: 72° C. The anti-sense T7 template was generated using insects fed target 15 caused the least damage to bean foliage specific forward and specific T7 reverse primers in a PCR (FIG. 2-EV(b)). reaction with the same conditions as described above. The 30 sequences of the respective primers for amplifying the Example 6 anti-sense template for each of the target genes are given in Table 8-AG. The resulting PCR products were analyzed on Anthonomus grandis (Cotton Boll Weevil) agarose gel and purified by PCR purification kit (Qiaquick PCR Purification Kit, Cat. Nr. 28106, Qiagen) and NaClO. A. Cloning Anthonomus grandis Partial Sequences 35 precipitation. The generated T7 forward and reverse tem High quality, intact RNA was isolated from the 3 instars plates were mixed to be transcribed and the resulting RNA of Anthonomus grandis (cotton boll weevil; source: Dr. Gary strands were annealed, DNase and RNase treated, and Benzon, Benzon Research Inc., 7 Kuhn Drive, Carlisle, Pa. purified by sodium acetate, following the manufacturers 17013, USA) using TRIZol Reagent (Cat. Nr. 15596-026/ instructions. The sense strand of the resulting dsRNA for 15596-018, Invitrogen, Rockville, Md., USA) following the 40 each of the target genes is given in Table 8-AG. manufacturer's instructions. Genomic DNA present in the C. Recombination of Anthonomus grandis Genes into the RNA preparation was removed by DNase treatment follow Plant Vector pK7GWIWG2D(II) ing the manafacturer's instructions (Cat. Nr. 1700, Pro Since the mechanism of RNA interference operates mega). cDNA was generated using a commercially available through dsRNA fragments, the target nucleotide sequences kit (SuperScriptTM III 45 of the target genes, as selected above, are cloned in anti Reverse Transcriptase, Cat. Nr. 18080044, Invitrogen, sense and sense orientation, separated by the intron-CmR Rockville, Md., USA) following the manufacturers instruc intron, whereby CmR is the chloramphenicol resistance tions. marker, to form a dsRNA hairpin construct. These hairpin To isolate cDNA sequences comprising a portion of the constructs are generated using the LR recombination reac AG001, AG005, AG010, AG014 and AG016 genes, a series 50 tion between an attL-containing entry clone (see Example of PCR reactions with degenerate primers were performed 6A) and an attR-containing destination vector using Amplitaq Gold (Cat. Nr. N8080240, Applied Biosys (pK7GWIWG2D(II)). The plant vector pK7GWIWG2D tems) following the manafacturers instructions. (II) is obtained from the VIB/Plant Systems Biology with a The sequences of the degenerate primers used for ampli Material Transfer Agreement. LR recombination reaction is fication of each of the genes are given in Table 2-AG. These 55 performed by using LR ClonaseTM II enzyme mix (Cat. Nr. primers were used in respective PCR reactions with the 11791-020, Invitrogen) following the manufacturers following conditions: for AG001, AG005 and AG016, 10 instructions. These cloning experiments result in a hairpin minutes at 95°C., followed by 40 cycles of 30 seconds at construct for each of the target genes, having the promoter— 95° C., 1 minute at 50° C. and 1 minute and 30 seconds at sense-intron-CmR-intron-antisense orientation, and wherein 72° C., followed by 7 minutes at 72° C.; for AG010, 10 60 the promoter is the plant operable 35S promoter. The binary minutes at 95°C., followed by 40 cycles of 30 seconds at vector pK7GWIWG2D(II) with the 35S promoter is suitable 95°C., 1 minute at 54° C. and 2 minutes and 30 seconds at for transformation into A. tumefaciens. 72° C., followed by 7 minutes at 72° C.; for AG014, 10 Restriction enzyme digests were carried out on pCR8/ minutes at 95°C., followed by 40 cycles of 30 seconds at GW/TOPO plasmids containing the different targets (see 95°C., 1 minute at 55° C. and 1 minute at 72°C., followed 65 Example 6B). The band containing the gene of interest by 7 minutes at 72°C. The resulting PCR fragments were flanked by the attl sites using Qiaquick Gel Extraction Kit analyzed on agarose gel, purified (QIAquick Gel Extraction (Cat. Nr. 28706, Qiagen) is purified. An amount of 150 ng US 9,528,123 B2 57 58 of purified fragment and 150 ng pK7GWIWG2D(II) is The sequences of the degenerate primers used for ampli added together with the LR clonase II enzyme and incubated fication of each of the genes are given in Table 2-TC. These for at least 1 h at 25° C. After proteinase K solution primers were used in respective PCR reactions with the treatment (10 min at 37° C.), the whole recombination mix following conditions: 10 minutes at 95°C., followed by 40 is transformed into Top 10 chemically competent cells. 5 cycles of 30 seconds at 95° C., 1 minute at 50° C. and 1 Positive clones are selected by restriction digest analyses. minute and 30 seconds at 72°C., followed by 7 minutes at D. Laboratory Trials to Test Escherichia coli Expressing 72° C. (TC001, TC014, TC015); 10 minutes at 95° C., dsRNA Targets Against Anthonomus grandis followed by 40 cycles of 30 seconds at 95°C., 1 minute at Plant-Based Bioassays 54° C. and 2 minutes and 30 seconds at 72°C., followed by Whole plants are sprayed with suspensions of chemically 10 7 minutes at 72°C. (TC010); 10 minutes at 95°C., followed by 40 cycles of 30 seconds at 95°C., 1 minute at 53° C. and induced bacteria expressing dsRNA prior to feeding the 1 minute at 72°C., followed by 7 minutes at 72°C. (TC002). plants to CBW. The are grown from in a plant growth room The resulting PCR fragments were analyzed on agarose gel. chamber. The plants are caged by placing a 500 ml plastic purified (QIAquick Gel Extraction kit, Cat. Nr. 28706, bottle upside down over the plant with the neck of the bottle 15 Qiagen), cloned into the pCR8/GW/TOPO vector (Cat. Nr. firmly placed in the Soil in a pot and the base cut open and K2500-20, Invitrogen), and sequenced. The sequences of the covered with a fine nylon mesh to permit aeration, reduce resulting PCR products are represented by the respective condensation inside and prevent insect escape. CBW are SEQ ID NOs as given in Table 2-TC and are referred to as placed on each treated plant in the cage. Plants are treated the partial sequences. The corresponding partial amino acid with a suspension of E. coli AB301-105(DE3) harboring the sequences are represented by the respective SEQID NOs as pGXXX0XX plasmids or pGN29 plasmid. Different quan given in Table 3-TC. tities of bacteria are applied to the plants: for instance 66, 22. B. dsRNA Production of the Tribolium castaneum Genes and 7 units, where one unit is defined as 10 bacterial cells dsRNA was synthesized in milligram amounts using the in 1 ml of a bacterial Suspension at optical density value of commercially available kit T7 RibomaxTM Express RNAi 1 at 600 nm wavelength. In each case, a total volume of 25 System (Cat. Nr. P1700, Promega). First two separate single between 1 and 10 mls sprayed on the plant with the aid of 5' T7 RNA polymerase promoter templates were generated a vaporizer. One plant is used per treatment in this trial. The in two separate PCR reactions, each reaction containing the number of survivors are counted and the weight of each target sequence in a different orientation relative to the T7 survivor recorded. promoter. Spraying plants with a suspension of E. coli bacterial 30 For each of the target genes, the sense T7 template was strain AB301-105(DE3) expressing target dsRNA from generated using specific T7 forward and specific reverse pGXXX0XX lead to a dramatic increase in insect mortality primers. The sequences of the respective primers for ampli when compared to pGN29 control. These experiments show fying the sense template for each of the target genes are that double-stranded RNA corresponding to an insect gene given in Table 8-TC. The conditions in the PCR reactions target sequence produced in either wild-type or RNaseIII 35 were as follows: 1 minute at 95°C., followed by 20 cycles deficient bacterial expression systems is toxic towards the of 30 seconds at 95° C., 30 seconds at 60° C. (-0.5° insect in terms of Substantial increases in insect mortality C./cycle) and 1 minute at 72° C., followed by 15 cycles of and growth/development delay for larval survivors. It is also 30 seconds at 95°C., 30 seconds at 50° C. and 1 minute at clear from these experiments that an exemplification is 72°C., followed by 10 minutes at 72°C. The anti-sense T7 provided for the effective protection of plants/crops from 40 template was generated using specific forward and specific insect damage by the use of a spray of a formulation T7 reverse primers in a PCR reaction with the same condi consisting of bacteria expressing double-stranded RNA cor tions as described above. The sequences of the respective responding to an insect gene target. primers for amplifying the anti-sense template for each of the target genes are given in Table 8-TC. The resulting PCR Example 7 45 products were analyzed on agarose gel and purified by PCR purification kit (Qiaquick PCR Purification Kit, Cat. Nr. Tribolium castaneum (Red Flour Beetle) 28106, Qiagen) and NaClO precipitation. The generated T7 forward and reverse templates were mixed to be transcribed A. Cloning Tribolium castaneum Partial Sequences and the resulting RNA strands were annealed, DNase and High quality, intact RNA was isolated from all the dif 50 RNase treated, and purified by sodium acetate, following the ferent insect stages of Tribolium castaneum (red flour beetle: manufacturers instructions. The sense strand of the result Source: Dr. Lara Senior, Insect Investigations Ltd., Capital ing dsRNA for each of the target genes is given in Table Business Park, Wentloog, Cardiff, CF3 2PX, Wales, UK) 8-TC. using TRIZol Reagent (Cat. Nr. 15596-026/15596-018, C. Recombination of Tribolium castaneum Genes into the Invitrogen, Rockville, Md., USA) following the manufac 55 Plant Vector pK7GWIWG2D(II) turer's instructions. Genomic DNA present in the RNA Since the mechanism of RNA interference operates preparation was removed by DNase treatment following the through dsRNA fragments, the target nucleotide sequences manafacturers instructions (Cat. Nr. 1700, Promega). of the target genes, as selected above, are cloned in anti cDNA was generated using a commercially available kit sense and sense orientation, separated by the intron-CmR (SuperScriptTM III Reverse Transcriptase, Cat. Nr. 60 intron, whereby CmR is the chloramphenicol resistance 18080044. Invitrogen, Rockville, Md., USA) following the marker, to form a dsRNA hairpin construct. These hairpin manufacturers instructions. constructs are generated using the LR recombination reac To isolate cDNA sequences comprising a portion of the tion between an attL-containing entry clone (see Example TC001, TC002, TC010, TC014 and TC015 genes, a series of 7A) and an attR-containing destination vector PCR reactions with degenerate primers were performed 65 (pK7GWIWG2D(II)). The plant vector pK7GWIWG2D using Amplitaq Gold (Cat. Nr. N8080240, Applied Biosys (II) is obtained from the VIB/Plant Systems Biology with a tems) following the manafacturers instructions. Material Transfer Agreement. LR recombination reaction is US 9,528,123 B2 59 60 performed by using LR ClonaseTM II enzyme mix (Cat. Nr. Rockville, Md., USA) following the manufacturer's instruc 11791-020, Invitrogen) following the manufacturers tions. Genomic DNA present in the RNA preparation was instructions. These cloning experiments result in a hairpin removed by DNase treatment following the manafacturers construct for each of the target genes, having the promoter— instructions (Cat. Nr. 1700, Promega). cDNA was generated sense-intron-CmR-intron-antisense orientation, and wherein using a commercially available kit (SuperScriptTM III the promoter is the plant operable 35S promoter. The binary Reverse Transcriptase, Cat. Nr. 18080044, Invitrogen, Rock vector pK7GWIWG2D(II) with the 35S promoter is suitable ville, Md., USA) following the manufacturer's instructions. for transformation into A. tumefaciens. To isolate cDNA sequences comprising a portion of the Restriction enzyme digests were carried out on pCR8/ MP001, MP002, MP010, MP016 and MP027 genes, a series GW/TOPO plasmids containing the different targets (see 10 of PCR reactions with degenerate primers were performed Example 7B). The band containing the gene of interest using Amplitaq Gold (Cat. Nr. N8080240, Applied Biosys flanked by the attl sites using Qiaquick Gel Extraction Kit tems) following the manafacturers instructions. (Cat. Nr. 28706, Qiagen) is purified. An amount of 150 ng The sequences of the degenerate primers used for ampli of purified fragment and 150 ng pK7GWIWG2D(II) is fication of each of the genes are given in Table 2-MP. These added together with the LR clonase II enzyme and incubated 15 primers were used in respective PCR reactions with the for at least 1 h at 25° C. After proteinase K solution following conditions: for MP001, MP002 and MP016, 10 treatment (10 min at 37° C.), the whole recombination mix minutes at 95°C., followed by 40 cycles of 30 seconds at is transformed into Top 10 chemically competent cells. 95° C., 1 minute at 50° C. and 1 minute 30 seconds at 72° Positive clones are selected by restriction digest analyses. C., followed by 7 minutes at 72° C.; for MP027, a touch D. Laboratory Trials to Test dsRNA Targets. Using Arti down program was used: 10 minutes at 95°C., followed by ficial Diet for Activity Against Tribolium castaneum Larvae 10 cycles of 30 seconds at 95°C., 40 seconds at 60° C. with The example provided below is an exemplification of the a decrease in temperature of 1° C. per cycle and 1 minute 10 finding that the red flour beetle (RFB) larvae are susceptible seconds at 72° C., followed by 30 cycles of 30 seconds at to orally ingested dsRNA corresponding to own target genes. 95°C., 40 seconds at 50° C. and 1 minute 10 seconds at 72° Red flour beetles, Tribolium Castaneum, were maintained 25 C., followed by 7 minutes at 72° C.; for MP010, 10 minutes at Insect Investigations Ltd. (origin: Imperial College of at 95°C., followed by 40 cycles of 30 seconds at 95°C., 1 Science, Technology and Medicine, Silwood Park, Berk minute at 54° C. and 3 minutes at 72° C., followed by 7 shire, UK). Insects were cultured according to company minutes at 72° C. The resulting PCR fragments were ana SOP/251/01. Briefly, the beetles were housed in plastic jars lyzed on agarose gel, purified (QIAquick Gel Extraction kit, or tanks. These have an open top to allow ventilation. A 30 Cat. Nr. 28706, Qiagen), cloned into the pCR8/GW/TOPO piece of netting was fitted over the top and secured with an vector (Cat. Nr. K2500-20, Invitrogen), and sequenced. The elastic band to prevent escape. The larval rearing medium sequences of the resulting PCR products are represented by (flour) was placed in the container where the beetles can the respective SEQID NOs as given in Table 2-MP and are breed. The stored product beetle colonies were maintained in referred to as the partial sequences. The corresponding a controlled temperature room at 25+3°C. with a 16:8 hour 35 partial amino acid sequences are represented by the respec light:dark cycle. tive SEQ ID NOs as given in Table 3-MP. Double-stranded RNA from target TC014 (with sequence B. dsRNA Production of Myzus persicae Genes corresponding to SEQID NO 799) was incorporated into dsRNA was synthesized in milligram amounts using the a mixture of flour and milk powder (wholemeal flour: commercially available kit T7 RibomaxTM Express RNAi powdered milk in the ratio 4:1) and left to dry overnight. 40 System (Cat. Nr. P1700, Promega). First two separate single Each replicate was prepared separately: 100 ul of a 10 ug/ul 5' T7 RNA polymerase promoter templates were generated dsRNA solution (1 mg dsRNA) was added to 0.1 g flour/ in two separate PCR reactions, each reaction containing the milk mixture. The dried mixture was ground to a fine target sequence in a different orientation relative to the T7 powder. Insects were maintained within Petri dishes (55 mm promoter. diameter), lined with a double layer of filter paper. The 45 For each of the target genes, the sense T7 template was treated diet was placed between the two filter paper layers. generated using specific T7 forward and specific reverse Ten first instar, mixed sex larvae were placed in each dish primers. The sequences of the respective primers for ampli (replicate). Four replicates were performed for each treat fying the sense template for each of the target genes are ment. Control was Milli-Q water. Assessments (number of given in Table 8-MP. A touchdown PCR was performed as Survivors) were made on a regular basis. During the trial, the 50 follows: 1 minute at 95° C., followed by 20 cycles of 30 test conditions were 25-33°C. and 20-25% relative humid seconds at 95°C., 30 seconds at 55° C. (for MP001, MP002, ity, with a 12:12 hour light:dark photoperiod. MP016, MP027 and gfp) or 30 seconds at 50° C. (for Survival of larvae of T. Castaneum over time on artificial MP010) with a decrease in temperature of 0.5° C. per cycle diet treated with target TC014 dsRNA was significantly and 1 minute at 72°C., followed by 15 cycles of 30 seconds reduced when compared to diet only control, as shown in 55 at 95° C., 30 seconds at 45° C. and 1 minute at 72° C. FIG 1-TC. followed by 10 minutes at 72°C. The anti-sense T7 template was generated using specific forward and specific T7 reverse Example 8 primers in a PCR reaction with the same conditions as described above. The sequences of the respective primers for Myzus persicae (Green Peach Aphid) 60 amplifying the anti-sense template for each of the target genes are given in Table 8-MP. The resulting PCR products A. Cloning Myzus persicae Partial Sequences were analyzed on agarose gel and purified by PCR purifi High quality, intact RNA was isolated from nymphs of cation kit (Qiaquick PCR Purification Kit, Cat. Nr. 28106, Myzus persicae (green peach aphid; source: Dr. Rachel Qiagen) and NaClO precipitation. The generated T7 for Down, Insect & Pathogen Interactions, Central Science 65 ward and reverse templates were mixed to be transcribed Laboratory, Sand Hutton, York, YO41 1LZ, UK) using and the resulting RNA strands were annealed, DNase and TRIZol Reagent (Cat. Nr. 15596-026/15596-018, Invitrogen, RNase treated, and purified by sodium acetate, following the US 9,528,123 B2 61 62 manufacturers instructions. The sense strand of the result The amino acids were dissolved in 30 ml Milli-Q H2O ing dsRNA for each of the target genes is given in Table except for tyrosine which was first dissolved in a few drops 8-MP. of 1 M HCl before adding to the amino acid mix. The C. Recombination of Myzus persicae Genes into the Plant Vitamin mix component of the diet was prepared as a 5x Vector pK7GWIWG2D(II) concentrate stock as follows: in mg/L, amino benzoic acid Since the mechanism of RNA interference operates 100, ascorbic acid 1000, biotin 1, calcium panthothenate 50, through dsRNA fragments, the target nucleotide sequences choline chloride 500, folic acid 10, myoinositol 420, nico of the target genes, as selected above, were cloned in tinic acid 100, pyridoxine hydrochloride 25, riboflavin 5, anti-sense and sense orientation, separated by the intron thiamine hydrochloride 25. The riboflavin was dissolved in CmR-intron, whereby CmR is the chloramphenicol resis 10 1 ml H2O at 50° C. and then added to the vitamin mix stock. tance marker, to form a dsRNA hairpin construct. These The vitamin mix was aliquoted in 20 ml per aliquot and hairpin constructs were generated using the LR recombina stored at -20°C. One aliquot of vitamin mix was added to tion reaction between an att-containing entry clone (see the amino acid solution. Sucrose and MgSO4.7HO was Example 8A) and an attR-containing destination vector added with the following amounts to the mix: 20 g and 242 (=pK7GWIWG2D(II)). The plant vector pK7GWIWG2D 15 mg, respectively. Trace metal stock solution was prepared as (II) was obtained from the VIB/Plant Systems Biology with follows: in mg/100 ml, CuSO4.5H2O4.7, FeC6HO 44.5, a Material Transfer Agreement. LR recombination reaction MnC14H2O 6.5, NaCl 25.4, ZnCl 8.3. Ten ml of the trace was performed by using LR ClonaseTM II enzyme mix (Cat. metal solution and 250 mg KHPO was added to the diet Nr. 11791-020. Invitrogen) following the manufacturers and Milli-Q water was added to a final liquid diet volume of instructions. These cloning experiments resulted in a hairpin 100 ml. The pH of the diet was adjusted to 7 with 1 M KOH construct for each of the MP001, MP002, MP010, MPO16 solution. The liquid diet was filter-sterilised through an 0.22 and MP026 genes, having the promoter-sense-intron um filter disc (Millipore). CmR-intron-antisense orientation and wherein the promoter Green peach aphids (Myzus persicae; source: Dr. Rachel is the plant operable 35S promoter. The binary vector Down, Insect & Pathogen Interactions, Central Science pK7GWIWG2D(II) with the 35S promoter is suitable for 25 Laboratory, Sand Hutton, York, YO41 1LZ, UK) were transformation into A. tumefaciens. reared on 4- to 6-week-old oilseed rape (Brassica napus A digest with restriction enzyme Alwa41 was done for all variety SW Oban; source: Nick Balaam, Sw Seed Ltd., 49 the targets cloned into pCR8/GW/topo (see Example 8B). North Road, Abington, Cambridge, CB1 6AS, UK) in alu The band containing the gene of interest flanked by the atti, minium-framed cages containing 70 um mesh in a controlled sites using Qiaquick Gel Extraction Kit (Cat. Nr. 28706, 30 environment chamber with the following conditions: 23+2 Qiagen) was purified. An amount of 150 ng of purified C. and 60+5% relative humidity, with a 16:8 hours light:dark fragment and 150 ng pK7GWIWG2D(II) was added photoperiod. together with the LR clonase II enzyme and incubated for at One day prior to the start of the bioassay, adults were least 1 h at 25°C. After proteinase K solution treatment (10 collected from the rearing cages and placed on fresh min at 37° C), the whole recombination mix was trans 35 detached oilseed rape leaves in a Petridish and left overnight formed into Top 10 chemically competent cells. Positive in the insect chamber. The following day, first-instar nymphs clones were selected by restriction digest analysis. The were picked and transferred to feeding chambers. A feeding complete sequence of the hairpin construct for: chamber comprised of 10 first instar nymphs placed in a MP001 (sense-intron-CmR-intron-antisense) is repre small Petri dish (with diameter 3 cm) covered with a single sented in SEQ ID NO 1066: 40 layer of thinly stretched parafilm Monto which 50 ul of diet MP002 (sense-intron-CmR-intron-antisense) is repre was added. The chamber was sealed with a second layer of sented in SEQ ID NO 1067: parafilm and incubated under the same conditions as the MP010 (sense-intron-CmR-intron-antisense) is repre adult cultures. Diet with dsRNA was refreshed every other sented in SEQ ID NO 1068: day and the insects survival assessed on day 8 i.e. 8" day MPO16 (sense-intron-CmR-intron-antisense) is repre 45 post bioassay start. Per treatment, 5 bioassay feeding cham sented in SEQ ID NO 1069; bers (replicates) were set up simultaneously. Test and control MP027 (sense-intron-CmR-intron-antisense) is repre (gfp) dsRNA solutions were incorporated into the diet to a sented in SEQ ID NO 1070. final concentration of 2 g/ul. The feeding chambers were Table 9-MP provides complete sequences for each hairpin kept at 23+2° C. and 60+5% relative humidity, with a 16:8 COnStruct. 50 hours light:dark photoperiod. A Mann-Whitney test was D. Laboratory Trials to Test dsRNA Targets Using Liquid determined by GraphPad Prism version 4 to establish Artificial Diet for Activity Against Myzus persicae whether the medians do differ significantly between target Liquid artificial diet for the green peach aphid, Myzus 27 (MP027) and gfp dsRNA. persicae, was prepared based on the diet Suitable for pea In the bioassay, feeding liquid artificial diet Supplemented aphids (Acyrthosiphon pisum), as described by Febvay et al. 55 with intact naked dsRNA from target 27 (SEQID NO 1061) (1988) Influence of the amino acid balance on the improve to nymphs of Myzus persicae using a feeding chamber, ment of an artificial diet for a biotype of Acyrthosiphon resulted in a significant increase in mortality, as shown in pisum (Homoptera: Aphididae). Can. J. Zool. 66: 2449 FIG. 1. Average percentage Survivors for target 27, gfp 2453, but with some modifications. The amino acids com dsRNA and diet only treatment were 2, 34 and 82, respec ponent of the diet was prepared as follows: in mg/100 ml, 60 tively. Comparison of target 027 with gfp dsRNA groups alanine 178.71, beta-alanine 6.22, arginine 244.9, aspara using the Mann-Whitney test resulted in an one-tailed gine 298.55, aspartic acid 88.25, cysteine 29.59, glutamic P-value of 0.004 which indicates that the median of target acid 149.36, glutamine 445.61, glycine 166.56, histidine 027 is significantly different (P<0.05) from the expected 136.02, isoleucine 164.75, leucine 231.56, lysine hydrochlo larger median of gfp dsRNA. The green peach aphids on the ride 351.09, methionine 72.35, ornithine (HCl) 9.41, phe 65 liquid diet with incorporated target 27 dsRNA were notice nylalanine 293, proline 129.33, serine 124.28, threonine ably smaller than those that were fed on diet only or with gfp 127.16, tryptophane 42.75, tyrosine 38.63, L-valine 190.85. dsRNA control (data not presented). US 9,528,123 B2 63 64 E. Laboratory Trials of Myzus periscae (Green Peach 72° C.; for NL006: 10 minutes at 95° C., followed by 40 Aphid) Infestation on Transgenic Arabidopsis thaliana cycles of 30 seconds at 95° C., 1 minute at 55° C. and 3 Plants minute 30 seconds at 72°C., followed by 10 minutes at 72° Generation of Transgenic Plants C.; for NL007: 10 minutes at 95°C., followed by 40 cycles Arabidopsis thaliana plants were transformed using the of 30 seconds at 95°C., 1 minute at 54° C. and 1 minute 15 floral dip method (Clough and Bent (1998) Plant Journal seconds at 72° C., followed by 10 minutes at 72° C.; for 16:735-743). Aerial parts of the plants were incubated for a NL008 & NL014: 10 minutes at 95° C., followed by 40 few seconds in a solution containing 5% sucrose, resus cycles of 30 seconds at 95°C., 1 minute at 53° C. and 1 pended Agrobacterium tumefaciens strain C58C1 Rif cells minute at 72° C., followed by 10 minutes at 72° C.; for from an overnight culture and 0.03% of the surfactant Silwet 10 NL009, NL011, NL012 & NL019; 10 minutes at 95° C., L-77. After inoculation, plants were covered for 16 hours followed by 40 cycles of 30 seconds at 95°C., 1 minute at with a transparent plastic to maintain humidity. To increase 55° C. and 1 minute at 72° C., followed by 10 minutes at 72° the transformation efficiency, the procedure was repeated C.; for NL010: 10 minutes at 95°C., followed by 40 cycles after one week. Watering was stopped as seeds matured and of 30 seconds at 95°C., 1 minute at 54° C. and 2 minute 30 dry seeds were harvested and cold-treated for two days. 15 seconds at 72° C., followed by 10 minutes at 72° C.; for After sterilization, seeds were plated on a kanamycin-con NL013: 10 minutes at 95°C., followed by 40 cycles of 30 taining growth medium for selection of transformed plants. seconds at 95° C., 1 minute at 54° C. and 1 minute 10 The selected plants are transferred to soil for optimal T2 seconds at 72° C., followed by 10 minutes at 72° C.; for seed production. NL015 & NL016: 10 minutes at 95° C., followed by 40 Bioassay cycles of 30 seconds at 95°C., 1 minute at 54° C. and 1 Transgenic Arabidopsis thaliana plants are selected by minute 40 seconds at 72°C., followed by 10 minutes at 72° allowing the segregating T2 seeds to germinate on appro C.; for NL018: 10 minutes at 95°C., followed by 40 cycles priate selection medium. When the roots of these transgenics of 30 seconds at 95°C., 1 minute at 54° C. and 1 minute 35 are well-established they are then transferred to fresh arti seconds at 72° C., followed by 10 minutes at 72° C.; for ficial growth medium or soil and allowed to grow under 25 NL021, NL022 & NL027: 10 minutes at 95°C., followed by optimal conditions. Whole transgenic plants are tested 40 cycles of 30 seconds at 95°C., 1 minute at 54° C. and 1 against nymphs of the green peach aphid (Myzus persicae) minute 45 seconds at 72°C., followed by 10 minutes at 72° to show (1) a significant resistance to plant damage by the C. The resulting PCR fragments were analyzed on agarose feeding nymph, (2) increased nymphal mortality, and/or (3) gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706, decreased weight of nymphal Survivors (or any other aber 30 Qiagen), cloned into the pCR8/GW/topo vector (Cat. Nr. rant insect development). K2500 20, Invitrogen), and sequenced. The sequences of the resulting PCR products are represented by the respective Example 9 SEQ ID NOs as given in Table 2-NL and are referred to as the partial sequences. The corresponding partial amino acid Nilaparvata lugens (Brown Plant Hopper) 35 sequences are represented by the respective SEQID NOs as given in Table 3-NL. A. Cloning Nilaparvata lugens Partial Sequences B. Cloning of a Partial Sequence of the Nilaparvata From high quality total RNA of Nilaparvata lugens lugens NL023 Gene Via EST Sequence (source: Dr. J. A. Gatehouse, Dept. Biological Sciences, From high quality total RNA of Nilaparvata lugens Durham University, UK) cDNA was generated using a 40 (source: Dr. J. A. Gatehouse, Dept. Biological Sciences, commercially available kit (SuperScriptTM III Reverse Tran Durham University, UK) cDNA was generated using a scriptase, Cat N. 18080044. Invitrogen, Rockville, Md., commercially available kit (SuperScriptTM III Reverse Tran USA) following the manufacturer's protocol. scriptase, Cat N. 18080044. Invitrogen, Rockville, Md., To isolate cDNA sequences comprising a portion of the USA) following the manufacturer's protocol. Nilaparvata lugens NL001, NL002, NL003, NL004, 45 A partial clNA sequence, NL023, was amplified from NL005, NL006, NL007, NL008, NL009, NL010, NL011, Nilaparvata lugens cDNA which corresponded to a Nilapa NL012, NL013, NL014, NL015, NL016, NLO18, NL019, rvata lugens EST sequence in the public database Genbank NL021, NL022, and NL027 genes, a series of PCR reactions with accession number CAH65679.2. To isolate cDNA with degenerate primers were performed using Amplitaq sequences comprising a portion of the NL023 gene, a series Gold (Cat N. N8080240; Applied Biosystems) following 50 of PCR reactions with EST based specific primers were the manufacturer's protocol. performed using PerfectShot TM EXTaq (Cat N. RR005A, The sequences of the degenerate primers used for ampli Takara Bio Inc.) following the manafacturer's protocol. fication of each of the genes are given in Table 2-NL. These For NL023, the specific primers oGBKW002 and primers were used in respective PCR reactions with the oGBKW003 (represented herein as SEQ ID NO 1157 and following conditions: for NL001: 5 minutes at 95° C. 55 SEQ ID NO 1158, respectively) were used in two indepen followed by 40 cycles of 30 seconds at 95°C., 1 minute at dent PCR reactions with the following conditions: 3 minutes 55° C. and 1 minute at 72° C., followed by 10 minutes at 72° at 95°C., followed by 30 cycles of 30 seconds at 95°C., 30 C.: for NL002: 3 minutes at 95°C., followed by 40 cycles seconds at 56° C. and 2 minutes at 72°C., followed by 10 of 30 seconds at 95°C., 1 minute at 55° C. and 1 minute at minutes at 72°C. The resulting PCR products were analyzed 72° C., followed by 10 minutes at 72° C.; for NL003: 3 60 on agarose gel, purified (QIAquick R. Gel Extraction Kit; minutes at 95°C., followed by 40 cycles of 30 seconds at Cat. N. 28706, Qiagen), cloned into the pCR4-TOPO 95°C., 1 minute at 61° C. and 1 minute at 72°C., followed vector (Cat N. K4575-40, Invitrogen) and sequenced. The by 10 minutes at 72° C.; for NL004: 10 minutes at 95°C., consensus sequence resulting from the sequencing of both followed by 40 cycles of 30 seconds at 95°C., 1 minute at PCR products is herein represented by SEQID NO 1111 and 51° C. and 1 minute at 72° C.; for NL005: 10 minutes at 95° 65 is referred to as the partial sequence of the NL023 gene. The C., followed by 40 cycles of 30 seconds at 95°C., 1 minute corresponding partial amino acid sequence is herein repre at 54° C. and 1 minute at 72°C., followed by 10 minutes at sented as SEQ ID NO 1112. US 9,528,123 B2 65 66 C. dsRNA Production of Nilaparvata lugens Genes the following conditions: 4 minutes at 95°C., followed by dsRNA was synthesized in milligram amounts using the 35 cycles of 30 seconds at 95°C., 30 seconds at 55° C. and commercially available kit T7 RibomaxTM Express RNAi 1 minute at 72° C., followed by 10 minutes at 72° C. The System (Cat. Nr. P1700, Promega). First two separate single anti-sense T7 template was generated using the specific FW 5' T7 RNA polymerase promoter templates were generated 5 primer oGAU181 and the specific T7 RV primer oGAU184 in two separate PCR reactions, each reaction containing the (represented herein as SEQID NO 238 and SEQID NO 239, target sequence in a different orientation relative to the T7 respectively) in a PCR reaction with the same conditions as promoter. described above. The resulting PCR products were analyzed For each of the target genes, the sense T7 template was on agarose gel and purified (QIAquick R. PCR Purification generated using specific T7 forward and specific reverse 10 Kit: Cat. N. 28106, Qiagen). The generated T7 FW and RV primers. The sequences of the respective primers for ampli templates were mixed to be transcribed and the resulting fying the sense template for each of the target genes are RNA strands were annealed, DNase and RNase treated, and given in Table 8-NL. The conditions in the PCR reactions purified by precipitation with sodium acetate and isopropa were as follows: for NL001 & NL002: 4 minutes at 94° C., nol, following the manufacturer's protocol, but with the followed by 35 cycles of 30 seconds at 94° C., 30 seconds 15 following modification: RNA peppet is washed twice in 70% at 60° C. and 1 minute at 72°C., followed by 10 minutes at ethanol. The sense strands of the resulting dsRNA is herein 72° C.; for NL003: 4 minutes at 94° C., followed by 35 represented by SEQ ID NO 235. cycles of 30 seconds at 94° C., 30 seconds at 66° C. and 1 D. Laboratory Trials to Screen dsRNA Targets Using minute at 72° C., followed by 10 minutes at 72° C.; for Liquid Artificial Diet for Activity Against Nilaparvata NL004, NL006, NL008, NL009, NL010 & NL019: 4 min lugens utes at 95°C., followed by 35 cycles of 30 seconds at 95° Liquid artificial diet (MMD-1) for the rice brown plan C., 30 seconds at 54°C. and 1 minute at 72°C., followed by thopper, Nilaparvata lugens, was prepared as described by 10 minutes at 72°C.; for NL005 & NL016: 4 minutes at 95° Koyama (1988) Artificial rearing and nutritional physiol C., followed by 35 cycles of 30 seconds at 95° C., 30 ogy of the planthoppers and leafhoppers (Homoptera: Del seconds at 57° C. and 1 minute at 72° C., followed by 10 25 phacidae and Deltocephalidae) on a holidic diet. JARO 22: minutes at 72°C.; for NL007& NL014: 4 minutes at 95°C., 20-27, but with a modification in final concentration of diet followed by 35 cycles of 30 seconds at 95°C., 30 seconds component Sucrose: 14.4% (weight over Volume) was used. at 51° C. and 1 minute at 72°C., followed by 10 minutes at Diet components were prepared as separate concentrates: 72° C.; for NL011, NL012 & NL022: 4 minutes at 95°C., 10x mineral stock (stored at 4° C.), 2x amino acid stock followed by 35 cycles of 30 seconds at 95°C., 30 seconds 30 (stored at -20°C.) and 10x vitamin stock (stored at -20°C.). at 53° C. and 1 minute at 72°C., followed by 10 minutes at The stock components were mixed immediately prior to the 72° C.; for NL013, NL015, NL018 & NL021: 4 minutes at start of a bioassay to 4/3x concentration to allow dilution 95°C., followed by 35 cycles of 30 seconds at 95°C., 30 with the test dsRNA solution (4x concentration), pH seconds at 55° C. and 1 minute at 72° C., followed by 10 adjusted to 6.5, and filter-sterilised into approximately 500 minutes at 72°C.; for NL023 & NL027: 4 minutes at 95°C., 35 ul aliquots. followed by 35 cycles of 30 seconds at 95°C., 30 seconds Rice brown planthopper (Nilaparvata lugens) was reared at 52° C. and 1 minute at 72°C., followed by 10 minutes at on two-to-three month old rice (Oryza sativa cV Taichung 72° C. The anti-sense T7 template was generated using Native 1) plants in a controlled environment chamber: 27+2 specific forward and specific T7 reverse primers in a PCR C., 80% relative humidity, with a 16:8 hours light:dark reaction with the same conditions as described above. The 40 photoperiod. A feeding chamber comprised 10 first or sec sequences of the respective primers for amplifying the ond instar nymphs placed in a small petri dish (with diameter anti-sense template for each of the target genes are given in 3 cm) covered with a single layer of thinly stretched parafilm Table 8-NL. The resulting PCR products were analyzed on Monto which 50 ul of diet was added. The chamber was agarose gel and purified by PCR purification kit (Qiaquick sealed with a second layer of parafilm and incubated under PCR Purification Kit, Cat. Nr. 28106, Qiagen). The gener 45 the same conditions as the adult cultures but with no direct ated T7 forward and reverse templates were mixed to be light exposure. Diet with dsRNA was refreshed every other transcribed and the resulting RNA strands were annealed, day and the insects survival assessed daily. Per treatment, 5 DNase and RNase treated, and purified by sodium acetate, bioassay feeding chambers (replicates) were set up simul following the manufacturers instructions, but with the fol taneously. Test and control (gfp) dsRNA solutions were lowing modification: RNA peppet is washed twice in 70% 50 incorporated into the diet to a final concentration of 2 mg/ml. ethanol. The sense strand of the resulting dsRNA for each of The feeding chambers were kept at 27+2°C., 80% relative the target genes is given in Table 8-NL. humidity, with a 16:8 hours light:dark photoperiod. Insect The template DNA used for the PCR reactions with T7 survival data were analysed using the Kaplan-Meier survival primers on the green fluorescent protein (gfp) control was curve model and the Survival between groups were com the plasmid pPD96.12 (courtesy of the Andrew Fire Lab, 55 pared using the logrank test (Prism version 4.0). Standford Unversity), which contains the wild-type gfp Feeding liquid artificial diet supplemented with intact coding sequence interspersed by 3 synthetic introns. Double naked dsRNAS to Nilaparvata lugens in vitro using a Stranded RNA was synthesized using the commercially feeding chamber resulted in significant increases in nymphal available kit T7 RiboMAXTM Express RNAi System (Cat. mortalities as shown in four separate bioassays (FIGS. No. P1 700, Promega). First two separate single 5'T7 RNA 60 1(a)-(d)-NL; Tables 10-NL(a)-(d)) (Durham University). polymerase promoter templates were generated in two sepa These results demonstrate that dsRNAs corresponding to rate PCR reactions, each reaction containing the target different essential BPH genes showed significant toxicity sequence in a different orientation relative to the T7 pro towards the rice brown planthopper. moter. For gfp, the sense T7 template was generated using Effect of gfp dsRNA on BPH survival in these bioassays the specific T7 FW primer oGAU183 and the specific RV 65 is not significantly different to survival on diet only primer oGAU182 (represented herein as SEQ ID NO 236 Tables 10-NL(a)-(d) show a summary of the survival of and SEQ ID NO 237, respectively) in a PCR reaction with Nilaparvata lugens on artificial diet Supplemented with 2 US 9,528,123 B2 67 68 mg/ml (final concentration) of the following targets; in Table minute at 72° C., followed by 10 minutes at 72° C. The 10-NL(a): NL002, NL003, NL005, NL010; in Table 10-NL resulting PCR fragments were analyzed on agarose gel. (b): NL009, NL016; in Table 10-NL(c): NL014, NL018; and purified (QIAquick Gel Extraction kit, Cat. Nr. 28706, in Table 10-NL(d): NL013, NL015, NL021. In the survival Qiagen), cloned into the pCR4/TOPO vector (Cat. Nr. analysis column, the effect of RNAi is indicated as follows: 5 K2500-20, Invitrogen), and sequenced. The sequences of the +-significantly decreased Survival compared to gfp dsRNA resulting PCR products are represented by the respective control (alpha<0.05); --no significant difference in survival SEQ ID NOs as given in Table 2-CS and are referred to as compared to gfp dsRNA control. Survival curves were the partial sequences. The corresponding partial amino acid compared (between diet only and diet supplemented with sequences are represented by the respective SEQID NOs as 10 given in Table 3-CS. test dsRNA, gfp dsRNA and test dsRNA, and diet only and B. dsRNA Production of the Chilo suppressalis Genes gfp dsRNA) using the logrank test. dsRNA was synthesized in milligram amounts using the E. Laboratory Trials to Screen dsRNAs at Different Con commercially available kit T7 RibomaxTM Express RNAi centrations. Using Artificial Diet for Activity Against Nilapa System (Cat. Nr. P1700, Promega). First two separate single rvata lugens 15 5' T7 RNA polymerase promoter templates were generated Fifty ul of liquid artificial diet supplemented with differ in two separate PCR reactions, each reaction containing the ent concentrations of target NL002 dsRNA, namely 1, 0.2, target sequence in a different orientation relative to the T7 0.08, and 0.04 mg/ml (final concentration), was applied to promoter. the brown planthopper feeding chambers. Diet with dsRNA For each of the target genes, the sense T7 template was was refreshed every other day and the insects survival generated using specific T7 forward and specific reverse assessed daily. Per treatment, 5 bioassay feeding chambers primers. The sequences of the respective primers for ampli (replicates) were set up simultaneously. The feeding cham fying the sense template for each of the target genes are bers were kept at 27+2°C., 80% relative humidity, with a given in Table 8-CS. The conditions in the PCR reactions 16:8 hours light:dark photoperiod. Insect survival data were were as follows: 4 minutes at 95°C., followed by 35 cycles analysed using the Kaplan-Meier Survival curve model and of 30 seconds at 95°C., 30 seconds at 55° C. and 1 minute the Survival between groups were compared using the 25 at 72°C., followed by 10 minutes at 72°C. The anti-sense logrank test (Prism version 4.0). T7 template was generated using specific forward and Feeding liquid artificial diet supplemented with intact specific T7 reverse primers in a PCR reaction with the same naked dsRNAs of target NL002 at different concentrations conditions as described above. The sequences of the respec resulted in significantly higher BPH mortalities at final tive primers for amplifying the anti-sense template for each concentrations of as low as 0.04 mg dsRNA per ml diet when 30 of the target genes are given in Table 8-CS. The resulting compared with survival on diet only, as shown in FIG. 2-NL PCR products were analyzed on agarose gel and purified by and Table 11-NL. Table 11-NL Summarizes the Survival of PCR purification kit (Qiaquick PCR Purification Kit, Cat. Nilaparvata lugens artificial diet feeding trial Supplemented Nr. 28106, Qiagen) and NaClO precipitation. The generated with 1, 0.2, 0.08, & 0.04 mg/ml (final concentration) of T7 forward and reverse templates were mixed to be tran target NL002. In the survival analysis column the effect of 35 scribed and the resulting RNA strands were annealed, RNAi is indicated as follows: +=significantly decreases DNase and RNase treated, and purified by sodium acetate, survival compared to diet only control (alpha<0.05); -- no following the manufacturers instructions. The sense Strand significant differences in Survival compared to diet only of the resulting dsRNA for each of the target genes is given control. Survival curves were compared using the logrank in Table 8-CS. teSt. C. Recombination of the Chilo suppressalis Genes into 40 the Plant Vector pK7GWIWG2D(II) Example 10 Since the mechanism of RNA interference operates through dsRNA fragments, the target nucleotide sequences Chilo suppressalis (Rice Striped Stem Borer) of the target genes, as selected above, are cloned in anti sense and sense orientation, separated by the intron-CmR A. Cloning of Partial Sequence of the Chilo suppressalis 45 intron, whereby CmR is the chloramphenicol resistance Genes Via Family PCR marker, to form a dsRNA hairpin construct. These hairpin High quality, intact RNA was isolated from the 4 different constructs are generated using the LR recombination reac larval stages of Chilo suppressalis (rice striped stem borer) tion between an attL-containing entry clone (see Example using TRIZol Reagent (Cat. Nr. 15596-026/15596-018, 10A) and an attR-containing destination vector Invitrogen, Rockville, Md., USA) following the manufac 50 (pK7GWIWG2D(II)). The plant vector pK7GWIWG2D turer's instructions. Genomic DNA present in the RNA (II) is obtained from the VIB/Plant Systems Biology with a preparation was removed by DNase treatment following the Material Transfer Agreement. LR recombination reaction is manafacturers instructions (Cat. Nr. 1700, Promega). performed by using LR ClonaseTM II enzyme mix (Cat. Nr. cDNA was generated using a commercially available kit 11791-020, Invitrogen) following the manufacturers (SuperScriptTM III Reverse Transcriptase, Cat. Nr. instructions. These cloning experiments result in a hairpin 18080044. Invitrogen, Rockville, Md., USA) following the 55 construct for each of the target genes, having the promoter— manufacturers instructions. sense-intron-CmR-intron-antisense orientation, and wherein To isolate cDNA sequences comprising a portion of the the promoter is the plant operable 35S promoter. The binary CS001, CS002, CS003, CS006, CS007, CS009, CS011, vector pK7GWIWG2D(II) with the 35S promoter is suitable CS013, CS014, CS015, CS016 and CS018 genes, a series of for transformation into A. tumefaciens. PCR reactions with degenerate primers were performed 60 Restriction enzyme digests were carried out on pCR8/ using Amplitaq Gold (Cat. Nr. N8080240, Applied Biosys GW/TOPO plasmids containing the different targets (see tems) following the manafacturers instructions. Example 10B). The band containing the gene of interest The sequences of the degenerate primers used for ampli flanked by the attl sites using Qiaquick Gel Extraction Kit fication of each of the genes are given in Table 2-CS. These (Cat. Nr. 28706, Qiagen) is purified. An amount of 150 ng primers were used in respective PCR reactions with the 65 of purified fragment and 150 ng pK7GWIWG2D(II) is following conditions: 10 minutes at 95°C., followed by 40 added together with the LR clonase II enzyme and incubated cycles of 30 seconds at 95° C., 1 minute at 55° C. and 1 for at least 1 h at 25° C. After proteinase K solution US 9,528,123 B2 69 70 treatment (10 min at 37° C.), the whole recombination mix cDNA was generated using a commercially available kit is transformed into Top 10 chemically competent cells. (SuperScriptTM III Reverse Transcriptase, Cat. Nr. Positive clones are selected by restriction digest analyses. 18080044. Invitrogen, Rockville, Md., USA) following the D. Laboratory Trials to Test dsRNA Targets. Using Arti manufacturers instructions. ficial Diet for Activity Against Chilo suppressalis Larvae To isolate cDNA sequences comprising a portion of the Rice Striped stem borers, Chilo suppressalis, (origin: PX001, PX009, PX010, PXO15, PX016 genes, a series of Syngenta, Stein, Switzerland) were maintained on a modi PCR reactions with degenerate primers were performed fied artificial diet based on that described by Kamano and using Amplitaq Gold (Cat. Nr. N8080240, Applied Biosys Sato, 1985 (in: Handbook of Insect Rearing. Volumes I & II. tems) following the manufacturers instructions. The P Singh and RF Moore, eds. Elsevier Science Publishers, 10 sequences of the degenerate primers used for amplification Amsterdam and New York, 1985, pp. 448). Briefly, a litre of each of the genes are given in Table 2-PX. These primers diet was made up as follows: 20 g of agar added to 980 ml were used in respective PCR reactions with the following of Milli-Q water and autoclaved; the agar solution was conditions: 10 minutes at 95°C., followed by 40 cycles of cooled down to approximately 55° C. and the remaining 30 seconds at 95°C., 1 minute at 50° C. and 1 minute and ingredients were added and mixed thoroughly: 40 g corn 15 30 seconds at 72° C., followed by 7 minutes at 72° C. (for flour (Polenta), 20 g cellulose, 30 g sucrose, 30 g casein, 20 PX001, PX009, PX015, PX016); 10 minutes at 95° C., g wheat germ (toasted), 8 g Wesson salt mixture, 12 g followed by 40 cycles of 30 seconds at 95°C., 1 minute at VanderZant vitamin mix, 1.8 g. Sorbic acid, 1.6 g nipagin 54°C. and 2 minute and 30 seconds at 72°C., followed by (methylparaben), 0.3 g aureomycin, 0.4 g cholesterol and 7 minutes at 72° C. (for PX010). The resulting PCR frag 0.6 g L-cysteine. The diet was cooled down to approx. 45° ments were analyzed on agarose gel, purified (QIAquick Gel C. and poured into rearing trays or cups. The diet was left to Extraction kit, Cat. Nr. 28706, Qiagen), cloned into the set in a horizontal laminair flow cabin. Rice leaf sections pCR8/GW/TOPO vector (Cat. Nr. K2500-20, Invitrogen) with oviposited eggs were removed from a cage housing and sequenced. The sequences of the resulting PCR products adult moths and pinned to the Solid diet in the rearing cup or are represented by the respective SEQ ID NOs as given in tray. Eggs were left to hatch and neonate larvae were 25 Table 2-PX and are referred to as the partial sequences. The available for bioassays and the maintenance of the insect corresponding partial amino acid sequence are represented cultures. During the trials and rearings, the conditions were by the respective SEQ ID NOs as given in Table 3-PX. 28+2° C. and 80+5% relative humidity, with a 16:8 hour B. dsRNA Production of the Plutella xylostella Genes light:dark photoperiod. dsRNA was synthesized in milligram amounts using the The same artificial diet is used for the bioassays but in this 30 commercially available kit T7 RibomaxTM Express RNAi case the diet is poured equally in 24 multiwell plates, with System (Cat. Nr. P1700, Promega). First two separate single each well containing 1 ml diet. Once the diet is set, the test 5' T7 RNA polymerase promoter templates were generated formulations are applied to the diet's surface (2 cm), at the in two separate PCR reactions, each reaction containing the rate of 50 ul of 1 g/ul dsRNA of target. The dsRNA target sequence in a different orientation relative to the T7 solutions are left to dry and two first instar moth larvae are 35 promoter. placed in each well. After 7 days, the larvae are transferred For each of the target genes, the sense T7 template was to fresh treated diet in multiwell plates. At day 14 (i.e. 14 generated using specific T7 forward and specific reverse days post bioassay start) the number of live and dead insects primers. The sequences of the respective primers for ampli is recorded and examined for abnormalities. Twenty-four fying the sense template for each of the target genes are larvae in total are tested per treatment. 40 given in Table 8-PX. The conditions in the PCR reactions An alternative bioassay is performed in which treated rice were as follows: 1 minute at 95°C., followed by 20 cycles leaves are fed to neonate larvae of the rice striped stem borer. of 30 seconds at 95° C., 30 seconds at 60° C. (-0.5° Small leaf sections of Indica rice variety Taichung native 1 C./cycle) and 1 minute at 72° C., followed by 15 cycles of are dipped in 0.05% Triton X-100 solution containing 1 30 seconds at 95°C., 30 seconds at 50° C. and 1 minute at ug/ul of target dsRNA, left to dry and each section placed in 45 72°C., followed by 10 minutes at 72°C. The anti-sense T7 a well of a 24 multiwell plate containing gellified 2% agar. template was generated using specific forward and specific Two neonates are transferred from the rearing tray to each T7 reverse primers in a PCR reaction with the same condi dsRNA treated leaf section (24 larvae per treatment). After tions as described above. The sequences of the respective 4 and 8 days, the larvae are transferred to fresh treated rice primers for amplifying the anti-sense template for each of leaf sections. The number of live and dead larvae are 50 the target genes are given in Table 8-PX. The resulting PCR assessed on days 4, 8 and 12; any abnormalities are also products were analyzed on agarose gel and purified by PCR recorded. purification kit (Qiaquick PCR Purification Kit, Cat. Nr. 28106, Qiagen) and NaClO precipitation. The generated T7 Example 11 forward and reverse templates were mixed to be transcribed 55 and the resulting RNA strands were annealed, DNase and Plutella xylostella (Diamondback Moth) RNase treated, and purified by sodium acetate, following the manufacturers instructions. The sense strand of the result A. Cloning of a Partial Sequence of the Plutella xylostella ing dsRNA for each of the target genes is given in Table High quality, intact RNA was isolated from all the dif 8-PX. ferent larval stages of Plutella xylostella (Diamondback 60 C. Recombination of the Plutella xylostella Genes into the moth; source: Dr. Lara Senior, Insect Investigations Ltd., Plant Vector pK7GWIWG2D(II) Capital Business Park, Wentloog, Cardiff, CF3 2PX, Wales, Since the mechanism of RNA interference operates UK) using TRIZol Reagent (Cat. Nr. 15596-026/15596-018, through dsRNA fragments, the target nucleotide sequences Invitrogen, Rockville, Md., USA) following the manufac of the target genes, as selected above, are cloned in anti turer's instructions. Genomic DNA present in the RNA 65 sense and sense orientation, separated by the intron-CmR preparation was removed by DNase treatment following the intron, whereby CmR is the chloramphenicol resistance manufacturer's instructions (Cat. Nr. 1700, Promega). marker, to form a dsRNA hairpin construct. These hairpin US 9,528,123 B2 71 72 constructs are generated using the LR recombination reac Invitrogen, Rockville, Md., USA) following the manufac tion between an attL-containing entry clone (see Example turer's instructions. Genomic DNA present in the RNA 11A) and an attR-containing destination vector preparation was removed by DNase treatment following the (=pK7GWIWG2D(II)). The plant vector pK7GWIWG2D manafacturers instructions (Cat. Nr. 1700, Promega). (II) is obtained from the VIB/Plant Systems Biology with a cDNA was generated using a commercially available kit Material Transfer Agreement. LR recombination reaction is (SuperScriptTM III Reverse Transcriptase, Cat. Nr. performed by using LR ClonaseTM II enzyme mix (Cat. Nr. 18080044. Invitrogen, Rockville, Md., USA) following the 11791-020, Invitrogen) following the manufacturers manufacturers instructions. instructions. These cloning experiments result in a hairpin To isolate cDNA sequences comprising a portion of the construct for each of the target genes, having the promoter— 10 AD001, AD002, AD009, AD015 and AD016 genes, a series sense-intron-CmR-intron-antisense orientation, and wherein of PCR reactions with degenerate primers were performed the promoter is the plant operable 35S promoter. The binary using Amplitaq Gold (Cat. Nr. N8080240, Applied Biosys vector pK7GWIWG2D(II) with the 35S promoter is suitable tems) following the manafacturers instructions. for transformation into A. tumefaciens. The sequences of the degenerate primers used for ampli Restriction enzyme digests were carried out on pCR8/ 15 fication of each of the genes are given in Table 2-AD. These GW/TOPO plasmids containing the different targets (see primers were used in respective PCR reactions with the Example 11B). The band containing the gene of interest following conditions: 10 minutes at 95°C., followed by 40 flanked by the attl sites using Qiaquick Gel Extraction Kit cycles of 30 seconds at 95° C., 1 minute at 50° C. and 1 (Cat. Nr. 28706, Qiagen) is purified. An amount of 150 ng minute and 30 seconds at 72°C., followed by 7 minutes at of purified fragment and 150 ng pK7GWIWG2D(II) is 72° C. The resulting PCR fragments were analyzed on added together with the LR clonase II enzyme and incubated agarose gel, purified (QIAquick Gel Extraction kit, Cat. Nr. for at least 1 h at 25° C. After proteinase K solution 28706, Qiagen), cloned into the pCR8/GW/topo vector (Cat. treatment (10 min at 37° C.), the whole recombination mix Nr. K2500 20, Invitrogen) and sequenced. The sequences of is transformed into Top 10 chemically competent cells. the resulting PCR products are represented by the respective Positive clones are selected by restriction digest analyses. 25 SEQ ID NOs as given in Table 2-AD and are referred to as D. Laboratory Trials to Test dsRNA Targets. Using Arti the partial sequences. The corresponding partial amino acid ficial Diet for Activity Against Plutella xylostella Larvae sequence are represented by the respective SEQID NOs as Diamond-back moths, Plutella xylostella, were main given in Table 3-AD. tained at Insect Investigations Ltd. (origin: Newcastle Uni B. dsRNA Production of the Acheta domesticus Genes versity, Newcastle-upon-Tyne, UK). The insects were reared 30 dsRNA was synthesized in milligram amounts using the on cabbage leaves. First instar, mixed sex larvae (approxi commercially available kit T7 RibomaxTM Express RNAi mately 1 day old) were selected for use in the trial. Insects System (Cat. Nr. P1700, Promega). First two separate single were maintained in Eppendorf tubes (1.5 ml capacity). 5' T7 RNA polymerase promoter templates were generated Commercially available Diamond-back moth diet (Bio-Serv, in two separate PCR reactions, each reaction containing the NJ, USA), prepared following the manafacturers instruc 35 target sequence in a different orientation relative to the T7 tions, was placed in the lid of each tube (0.25 ml capacity, promoter. 8 mm diameter). While still liquid, the diet was smoother For each of the target genes, the sense T7 template was over to remove excess and produce an even Surface. generated using specific T7 forward and specific reverse Once the diet has set the test formulations are applied to primers. The sequences of the respective primers for ampli the diet's surface, at the rate of 25 ul undiluted formulation 40 fying the sense template for each of the target genes are (1 lug/ul dsRNA of targets) per replicate. The test formula given in Table 8-AD. The conditions in the PCR reactions tions are allowed to dry and one first instar moth larva is were as follows: 1 minute at 95°C., followed by 20 cycles placed in each tube. The larva is placed on the surface of the of 30 seconds at 95° C., 30 seconds at 60° C. (-0.5° diet in the lid and the tube carefully closed. The tubes are C./cycle) and 1 minute at 72° C., followed by 15 cycles of stored upside down, on their lids such that each larva 45 30 seconds at 95°C., 30 seconds at 50° C. and 1 minute at remains on the surface of the diet. Twice weekly the larvae 72°C., followed by 10 minutes at 72°C. The anti-sense T7 are transferred to new Eppendorf tubes with fresh diet. The template was generated using specific forward and specific insects are provided with treated diet for the first two weeks T7 reverse primers in a PCR reaction with the same condi of the trial and thereafter with untreated diet. tions as described above. The sequences of the respective Assessments are made twice weekly for a total of 38 days 50 primers for amplifying the anti-sense template for each of at which point all larvae are dead. At each assessment the the target genes are given in Table 8-AD. The resulting PCR insects are assessed as live or dead and examined for products were analyzed on agarose gel and purified by PCR abnormalities. Forty single larva replicates are performed purification kit (Qiaquick PCR Purification Kit, Cat. Nr. for each of the treatments. During the trial the test conditions 28106, Qiagen) and NaClO precipitation. The generated T7 are 23 to 26°C. and 50 to 65% relative humidity, with a 16:8 55 forward and reverse templates were mixed to be transcribed hour light:dark photoperiod. and the resulting RNA strands were annealed, DNase and RNase treated, and purified by sodium acetate, following the Example 12 manufacturers instructions. The sense strand of the result ing dsRNA for each of the target genes is given in Table Acheta domesticus (House Cricket) 60 8-AD. C. Recombination of the Acheta domesticus Genes into A. Cloning Acheta domesticus Partial Sequences the Plant Vector pK7GWIWG2D(II) High quality, intact RNA was isolated from all the dif Since the mechanism of RNA interference operates ferent insect stages of Acheta domesticus (house cricket; through dsRNA fragments, the target nucleotide sequences Source: Dr. Lara Senior, Insect Investigations Ltd., Capital 65 of the target genes, as selected above, are cloned in anti Business Park, Wentloog, Cardiff, CF3 2PX, Wales, UK) sense and sense orientation, separated by the intron-CmR using TRIZol Reagent (Cat. Nr. 15596-026/15596-018, intron, whereby CmR is the chloramphenicol resistance US 9,528,123 B2 73 74 marker, to form a dsRNA hairpin construct. These hairpin rodent diet (rat and mouse standard diet, B & K Universal constructs are generated using the LR recombination reac Ltd., Grimston, Aldbrough, Hull, UK). The diet, BK001 P. tion between an attL-containing entry clone (see Example contains the following ingredients in descending order by weight: wheat, soya, wheatfeed, barley, pellet binder, rodent (pK7GWIWG2D(II)).12A) and an attR-containing The plant vector destination pK7GWIWG2D vector 5 S5 vitYinn, min, fatit bledblend, dicalcidealcium phosphatehosph mouldld carb. ThThe (II) is obtained from the VIB/Plant Systems Biology with a pelleted rodent diet is finely ground andd heat-treated in a Material T fer A t. LR binati tion i microwave oven prior to mixing, In order to inactivate any aerial transier Agreement. Ton nation reaction is enzyme components. All rodent diet is taken from the same performed by using LR Clonase II enzyme mix (Cat. Nr. batch in order to ensure consistency. The ground diet and 11791-020, Invitrogen) following the manufacturers 10 dsRNA are mixed thoroughly and formed into small pellets instructions. These cloning experiments result in a hairpin of equal weight, which are allowed to dry overnight at room construct for each of the target genes, having the promoter— temperature. sense-intron-CmR-intron-antisense orientation, and wherein Double-stranded RNA samples from targets and gfp con the promoter is the plant operable 35S promoter. The binary trol at concentrations 10 ugful were applied in the ratio 1 g vector pK7GWIWG2D(II) with the 35S promoter is suitable 15 ground diet plus 1 ml dsRNA solution, thereby resulting in for transformation into A. tumefaciens. an application rate of 10 mg dsRNA per g pellet. Pellets are Restriction enzyme digests were carried out on pCR8/ replaced weekly. The insects are provided with treated GW/TOPO plasmids containing the different targets (see pellets for the first three weeks of the trial. Thereafter Example 12B). The band containing the gene of interest untreated pellets are provided. Insects are maintained within flanked by the attl sites using Qiaquick Gel Extraction Kit 2O lidded plastic containers (9 cm diameter, 4.5 cm deep), ten (Cat. Nr. 28706, Qiagen) is purified. An amount of 150 ng per container. Each arena contains one treated bait pellet and of purified fragment and 150 ng pK7GWIWG2D(II) is one water source (damp cotton wool ball), each placed in a added together with the LR clonase II enzyme and incubated separate small weigh boat. The water is replenished ad lib for at least 1 h at 25° C. After proteinase K solution throughout the experiment. treatment (10 min at 37° C.), the whole recombination mix Assessments are made at twice weekly intervals, with no is transformed into Top 10 chemically competent cells. 25 more than four days between assessments, until all the Positive clones are selected by restriction digest analyses. control insects had either died or moulted to the adult stage D. Laboratory Trials to Test dsRNA Targets. Using Arti- (84 days). At each assessment the insects are assessed as live ficial Diet for Activity Against Acheta domesticus Larvae or dead, and examined for abnormalities. From day 46 House crickets, Acheta domesticus, were maintained at 30 onwards, once moulting to adult has commenced, all insects Insect Investigations Ltd. (origin: Blades Biological Ltd., (live and dead) are assessed as nymph or adult. Surviving Kent, UK). The insects were reared on bran pellets and insects are weighed on day 55 of the trial. Four replicates are cabbage leaves. Mixed sex nymphs of equal size and no performed for each of the treatments. During the trial the test more than 5 days old were selected for use in the trial. conditions are 25 to 33°C. and 20 to 25% relative humidity, Double-stranded RNA is mixed with a wheat-based pelleted with a 12:12 hour light:dark photoperiod. TABLE 1A C. elegans id D. melanogaster id description devgen RNAi screen BO2SO.1 CG1263 large ribosomal Subunit L8 protein. Acute lethal or letha BO336.10 CG3661 large ribosomal Subunit L23 protein. Acute lethal or letha BO336.2 CG838S ADP-ribosylation factor Acute lethal or letha B0464.1 CG3821 Putative aspartyl (D) tRNA synthetase. Acute lethal or letha CO1 G8.5 CG10701 Ortholog of the ERM family of cytoskeletal linkers Acute lethal or letha CO1H6.5 CG331.83 Nuclear hormone receptor that is required in all larval molts Acute lethal or letha CO2C6.1 CG18102 Member of the DYNamin related gene class Acute lethal or letha CO3D6.8 CG6764 Large ribosomal subunit L24 protein (Rp24-p) Acute lethal or letha CO4F12.4 CG6253 rpl-14 encodes a large ribosomal Subunit L14 protein. Acute lethal or letha CO4HS.6 CG10689 Product with RNA helicase activity (EC: 2.7.7.—) involved in nuclear Embryonic lethal or sterile mRNA splicing, via spliceosome which is a component of the spliceosome complex C13B9.3 CG14813 Delta subunit of the coatomer (COPI) complex Acute lethal or letha C17H12.14 CG1088 Member of the Vacuolar HATPase gene class Acute lethal or letha C26E6.4 CG318O DNA-directed RNA polymerase II Acute lethal or letha F23F12.6 CG16916 Triple A ATPase subunit of the 26S proteasome's 19S regulatory particle Acute lethal or letha (RP) base subcomplex F57B9.10 CG10149 Member of the proteasome Regulatory Particle, Non-ATPase-like gene Acute lethal or letha class K11D9.2 CG3725 sarco-endoplasmic reticulum Ca2+ ATPase homolog Embryonic lethal or sterile T20GS.1 CG9012 Clathrin heavy chain Acute lethal or letha T2OH4.3 CGS394 Predicted cytoplasmic prolyl-tRNA synthetase (ProRS) Acute lethal or letha T21E12.4 CGT507 Cytoplasmic dynein heavy chain homolog Acute lethal or letha COSC10.3 CG1140 Orthologue to the human gene 3-OXOACID COATRANSFERASE Acute lethal or letha CO9D4.5 CG2746 Ribosomal protein L19, structural constituent of ribosome involved in Acute lethal or letha protein biosynthesis which is localised to the ribosome CO9E10.2 CG31140 Orthologue of diacylglyerol kinase involved in movement, egg laying, and Acute lethal or letha synaptic transmission, and is expressed in neurons. C13B9.3 CG14813 Delta subunit of the coatomer (COPI) Acute lethal or letha C14B9.7 CG12775 Large ribosomal subunit L21 protein (RPL-21) involved in protein Acute lethal or letha biosynthesis C15H11.7 CG30382 Type 6 alpha subunit of the 26S proteasome's 20S protease core particle Acute lethal or letha (CP) C17E4.9 CG9261 Protein involved with Na+/K+-exchanging ATPase complex Embryonic lethal or sterile US 9,528,123 B2 75 76 TABLE 1 A-continued C. elegans id D. melanogaster id description devgen RNAi screen

C17H12.14 CG1088 V-ATPase E subunit Acute le 8. O. It 8. C23G10.4 CG11888 Non-ATPase subunit of the 26S proteasome's 19S regulatory paritcle Acute le 8. O. It 8. base subcomplex (RPN-2) C26D10.2 CGT269 Product with helicase activity involved in nuclear mRNA splicing, via Acute le 8. O. It 8. spliceosome which is localized to the nucleus RNA polymerase II 140 kD subunit (RpII140), DNA-directed RNA Acute le 8. O. It 8. polymerase activity (EC: 2.7.7.6) involved in transcription from Pol II promoter which is a component of the DNA-directed RNA polymerase II, core complex CG15697 Product with function in protein biosynthesis and ubiquitin in protein Acute le 8. O. It 8. degradation. C30C11.1 CG12220 Unknown function Acute le 8. O. It 8. C30C11.2 CG10484 Member of the proteasome Regulatory Particle, Non-ATPase-like gene Acute le 8. O. It 8. class CG13977 cytochrome P450 8. CG33103 Orthologous to thrombospondin, papilin and lacunin 8. CG8542 Member of the Heat Shock Protein gene class 8. CG332O Rab-protein 1 involved in cell adhesion 8. CG2331 Transitional endoplasmic reticulum ATPase TER94, Golgi organization arrested in and biogenesis CG8827 ACE-like protein 8. CG1782 Ubiquitin-activating enzyme, function in an ATP-dependent reaction that 8. activates ubiquitin prior to its conjugation to proteins that will Subsequently be degraded by the 26S proteasome. CG1242 Member of the abnormal DAuer Formation gene class Acute le 8. CGS920 Small ribosomal subunit S2 protein. Acute le 8. CG1341 Member of the proteasome Regulatory Particle, ATPase-like gene class Acute le 8. CG8055 Carrier protein with putatively involved in intracellular protein transport Growth arrested in growth CD4.6 CG4904 Type 1 alpha subunit of the 26S proteasome's 20S protease core particle Acu e e 8. (CP). D100.7.1.2 CG9282 Large ribosomal subunit L24 protein. Acu 8. O 8. D1054.2 CGS266 Member of the Proteasome Alpha Subunit gene class Acu 8. O 8. D1081.8 CG6905 MYB transforming protein Acu 8. O 8. FO7D10.1 CG7726 Large ribosomal subunit L11 protein (RPL-11.2) involved in protein Acu 8. O 8. biosynthesis. CG17927 Muscle myosin heavy chain (MHC B) A.C l e e O r e 8. CG4863 Large ribosomal subunit L3 protein (rpl-3) (8. CG998.7 Methanococcus hypothetical protein 0682 like 8. CG17369 V-ATPase B subunit arrested in

CG16916 Triple A ATPase subunit of the 26S proteasome's 19S regulatory particle O. It 8. (RP) base Subcomplex (RPT-3) CG-2238 Translation elongation factor 2 (EF-2), a GTP-binding protein involved in elay or arrested in protein synthesis CG4264 Member of the Heat Shock Protein gene class O. It 8. CG6846 Large ribosomal subunit L26 protein (RPL-26) involved in protein ethal or sterile biosynthesis CG841S Small ribosomal subunit S23 protein (RPS-23) involved in protein Acute le O. It 8. biosynthesis CGS289 Member of the proteasome Regulatory Particle, ATPase-like gene class Acute le 8. O. It 8. CG3523 Mitochondrial protein Acute le 8. O. It 8. CG-2986 Small ribosomal subunit S21 protein (RPS-21) involved in protein Acute le 8. O. It 8. biosynthesis CGT622 Large ribosomal subunit L36 protein (RPL-36) involved in protein Acute le 8. O. It 8. biosynthesis CG1527 Small ribosomal subunit S14 protein (RPS-14) involved in protein Acute le 8. O. It 8. biosynthesis CG6699 beta' (beta-prime) subunit of the coatomer (COPI) complex Acute le 8. O. It 8. CG10305 Small ribosomal subunit S26 protein (RPS-26) involved in protein Acute le 8. O. It 8. biosynthesis Member of the Proteasome Beta Subunit gene class Acute le 8. O. It 8. Ribosomal protein S9 (RpS9), structural constituent of ribosome involved Acute le 8. O. It 8. in protein biosynthesis which is a component of the cytosolic Small ribosomal subunit Small ribosomal subunit S8 protein (RPS-8) involved in protein Acute le 8. O. It 8. biosynthesis Member of the proteasome Regulatory Particle, Non-ATPase-like gene Acute le 8. O. It 8. class Small ribosomal subunit S15a protein. Acute le 8. O. It 8. arge ribosomal subunit L7 protein (rpl-7) Acute le 8. O. It 8. CG8977 Unknown function Acute le 8. O. It 8. CG1915 Product with Sallimus (Sls), myosin-light-chain kinase activity (EC: 2.7.1.117) involved in mitotic chromosome condensation which is ocalized to the nucleus CG11271 Small ribosomal subunit S12 protein (RPS-12) involved in protein Acute le 8. O. It 8. biosynthesis FSSA11.2 CG4214 Member of the SYNtaxin gene class Acute le 8. O. It 8. US 9,528,123 B2 77 78 TABLE 1 A-continued C. elegans id D. melanogaster id description devgen RNAi screen FSSA3.3 CG1828 transcritpion factor Acute lethal or letha F55C10.1 CG11217 Ortholog of calcineurin B, the regulatory subunit of the protein Acute lethal or letha phosphatase 2B F56F3.5 CG21.68 rps-1 encodes a small ribosomal subunit S3A protein. Acute lethal or letha F57B9.10 CG10149 Member of the proteasome Regulatory Particle, Non-ATPase-like gene Acute lethal or letha class F58F12.1 CG2968 ATP synthase Acute lethal or letha F59E10.3 CG3948 Zeta subunit of the coatomer (COPI) complex Acute lethal or letha C8.3 CG3195 Large ribosomal subunit L12 protein (rpl-12) Acute lethal or letha KO1GS4 CG1404 Putative RAN Small monomeric GTPase (cell adhesion) Acute lethal or letha KO4F10.4 CG18734 Subtilase Acute lethal or letha KOSC41 CG12323 Member of the Proteasome Beta Subunit gene class Acute lethal or letha KO7D4.3 CG18174 Putative proteasome regulatory particle, lid Subcomplex, rpn11 Acute lethal or letha K11D9.2 CG3725 Sarco-endoplasmic reticulum Ca2+ ATPase Embryonic lethal or sterile; Acute lethal or letha MO3F4.2 CG4O27 An actin that is expressed in body wall and vulval muscles and the Acute lethal or letha spermatheca. RO6A4.9 CG1109 six WD40 repeats Acute lethal or letha R1OE1.1.1 CG15319 Putative transcriptional cofactor Acute lethal or letha R12E2.3 CG3416 Protein with endopeptidase activity involved in proteolysis and Acute lethal or letha peptidolysis F10C1.2 CG101.19 Member of the Intermediate Filament, B gene class Embryonic lethal or sterile F35G12.8 CG11397 Homolog of the SMC4 subunit of mitotic condensin Embryonic lethal or sterile FS3G12.1 CG5771 GTPase homologue Embryonic lethal or sterile F54E7.3 CG5055 PDZ domain-containing protein Embryonic lethal or sterile H28O16.1 CG3612 ATP synthase Growth delay or arrested in growth K12C11.2 CG4494 Member of the SUMO (ubiquitin-related) homolog gene class Embryonic lethal or sterile R12E2.3 CG3416 Member of the proteasome Regulatory Particle, Non-ATPase-like gene Acute lethal or letha class R13A5.8 CG6141 Ribosomal protein L9, structural constituent of ribosome involved in Acute lethal or letha protein biosynthesis which is localised to the ribosome TO1C3.6 CG4046 rps-16 encodes a small ribosomal subunit S16 protein. Acute lethal or letha TO1H3.1 CG7007 proteolipid protein PPA1 like protein Acute lethal or letha TO5C12.7 CG5374 Cytosolic chaperonin Acute lethal or letha TOSH4.6 CG5605 eukaryotic peptide chain release factor Subunit 1 Acute lethal or letha T1OH9.4 CG17248 N-synaptobrevin; V-SNARE, vesicle-mediated transport, synaptic vesicle T14F9.1 CG17332 ATPase subunit Growth delay or arrested in growth T20GS.1 CG9012 Clathrin heavy chain Acute lethal or letha T21B10.7 CG7033 -complex protein 1 Embryonic lethal or sterile WO9B12.1 CG17907 Acetylcholineesterase T27F2.1 CG8264 Member of the mammalian SKIP (Ski interacting protein) homolog gene Acute lethal or letha class ZC434.5 CGS394 predicted mitochondrial glutamyl-tRNA synthetase (GluRS) Acute lethal or letha BOS11.6 CG6375 helicase Embryonic lethal or sterile DY3.2 CG101.19 Nuclear lamin; LMN-1 protein Growth delay or arrested in growth R13 G10.1 CG11397 homolog of the SMC4 subunit of mitotic condensin Wild Type T26E3.7 CG3612 Predicted mitochondrial protein. Growth delay or arrested in growth Y113G-7A3 CG1250 GTPase activator, ER to Golgi prot transport, component of the Golgi Acute lethal or lethal stack Y43B11 AR4 CG11276 Ribosomal protein S4 (RpS4), structural constituent of ribosome involved Acute lethal or lethal in protein biosynthesis which is a component of the cytosolic Small ribosomal subunit Y46GSA4 CGS931 Y46G5A.4 gene Acute lethal or lethal Y71F9AL.17 CGT961 Alpha subunit of the coatomer (COPI) complex Acute lethal or lethal Y76B12C.7 CG10110 Gene cleavage and polyadenylation specificity factor Embryonic lethal or sterile Y37D8A.10 CG1751 Unknown function Embryonic lethal or sterile CGT765 CO6G3.2 Member of the Kinesin-Like Protein gene class CG10922 C44E4.4 RNA-binding protein Embryonic lethal or sterile CG4145 FO1 G12.5 alpha-2 type IV collagen Embryonic lethal or sterile CG13391 F28H1.3 apredicted cytoplasmic alanyl-tRNA synthetase (AlaRS) Growth delay or arrested in growth CGT765 ROSD3.7 Member of the UNCoordinated gene class Embryonic lethal or sterile CGA398 RO6A4.4 Member of the IMportin Beta family gene class Embryonic lethal or sterile CGA436 T17E9.2 Unknown function Embryonic lethal or sterile CG2666 T2SG3.2 putative chitin synthase Embryonic lethal or sterile CG17603 WO4A8.7 TATA-binding protein associated factor TAF1L (TAFII250) Embryonic lethal or sterile US 9,528,123 B2 79 80 TABLE 1-LD Dm SEQ ID SEQ ID Target ID identifier NONA NOAA Function (based on Flybase) LDOO1 CG11276 1 2 Ribosomal protein S4 (RpS4), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal subunit LDOO2 CG8055 3 4 Carrier protein with putatively involved in intracellular protein transport LDOO3 CG3395 5 6 Ribosomal protein S9 (RpS9), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal subunit LDOO6 CG318O 7 8 RNA polymerase II 140 kD subunit (RpII140), DNA-directed RNA polymerase activity (EC: 2.7.7.6) involved in transcription from Pol II promoter which is a component of the DNA-directed RNA polymerase II, core complex LDOO7 CGT269 9 10 Helicase at 25E (Hel25E), also known in FlyBase as Dbp25F, Hel, I(2)25Eb and I(2)k11511, pre mRNA splicing factor activity involved in nuclear mRNA splicing, via spliceosome which is localized to the nucleus LDO10 CG1250 11 12 GTPase activator, ER to Golgi prot transport, component of the Golgi stack LDO11 CG1404 13 14 Tutative RAN Small monomeric GTPase (cell adhesion) LDO14 CG1088 15 16 V-ATPase E subunit LDO15 CG2331 17 18 Transitional endoplasmic reticulum ATPase TER94, Golgi organization and biogenesis LDO16 CG17369 19 2O V-ATPase B subunit LDO18 CG1915 21 22 Sallimus (Sls), myosin-light-chain kinase activity (EC: 2.7.1.117) involved in mitotic chromosome condensation which is localized to the nucleus LDO27 CG6699 23 24 Beta-coatamer protein, Subunit of a multimeric complex that forms a membrane vesicle coat

TABLE 1-PC Target Dm SEQ ID SEQ ID ID identifier NONA NOAA Function (based on Flybase) PCOO1 CG11276 247 248 Ribosomal protein S4 (RpS4), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal Subunit PCOO3 CG3395 249 250 Ribosomal protein S9 (RpS9), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal Subunit PCOOS CG2746 251 252 Ribosomal protein L19, structural constituent of ribosome involved in protein biosynthesis which is localised to the ribosome PCO10 CG1250 253 254 GTPase activator, ER to Golgi prot transport, component of the Golgi stack PCO14 CG1088 255 256 V-ATPase E Subunit PCO16 CG17369 257 258 V-ATPase B Subunit PCO27 CG6699 259 260 Beta-coatamer protein, Subunit of a multimeric complex that forms a membrane vesicle coat

TABLE 1-EV Target Dm SEQ ID SEQ ID ID identifier NONA NOAA Function (based on Flybase) EVOOS CG2746 513 514 Ribosomal protein L19, structural constituent of ribosome involved in protein biosynthesis which is localised to the ribosome EVOO9 CG9261 515 516 Protein involved with Na+/K+-exchanging ATPase complex EVO10 CG1250 517 518 GTPase activator, ER to Golgi prot transport, component of the Golgi stack EVO1S CG2331 519 520 Transitional endoplasmic reticulum ATPase TER94, Golgi organization and biogenesis EVO16 CG17369 521 522 V-ATPase B Subunit

TABLE 1-AG Target Dm SEQ ID SEQ ID ID identifier NONA NOAA Function (based on Flybase) AGOO1 CG11276 6O1 602 Ribosomal protein S4 (RpS4), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal Subunit AGOOS CO2746 603 604 Ribosomal protein L19, structural constituent of ribosome involved in protein biosynthesis which is localised to the ribosome AGO 10 CG1250 60S 606 GTPase activator, ER to Golgi prot transport, component of the Golgi stack AGO14 CG1088 607 608 V-ATPase E Subunit AGO16 CG17369 609 610 V-ATPase B Subunit

TABLE 1-TC Target Dm SEQ ID SEQ ID ID identifier NONA NOAA Function (based on Flybase) TCOO1 CG11276 793 794 Ribosomal protein S4 (RpS4), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal Subunit US 9,528,123 B2 81 82 TABLE 1-TC-continued

Target Dm SEQ ID SEQ ID ID identifier NONA NOAA Function (based on Flybase) TCOO2 CG8055 795 796 Protein with putatively involved in intracellular protein transport TC010 CG1250 797 798 GTPase activator, ER to Golgi prot transport, component of the Golgi stack TC014 CG1088 799 800 V-ATPase E Subunit TCO15 CG2331 8O1 802 Transitional endoplasmic reticulum ATPase TER94, Golgi organization and biogenesis

TABLE 1.-MP Target Dm SEQ ID SEQ ID ID identifier NONA NOAA Function (based on Flybase) MPOO1 CG11276 888 889 Ribosomal protein S4 (RpS4), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal Subunit MPOO2 CG8OSS 890 891 Carrier protein with putatively involved in intracellular protein transport MPO10 CG1250 892 893 GTPase activator, ER to Golgi prot transport, component of the Golgi stack MPO16 CG17369 894 895 V-ATPase B Subunit MPO27 CG6699 896 897 Beta-coatamer protein, subunit of a multimeric complex that forms a membrane vesicle coat

TABLE 1-NL Dm SEQ ID SEQ ID Target ID identifier NONA NOAA Function (based on Flybase) NLOO1 CG11276 O71 072 Ribosomal protein S4 (RpS4), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal Subunit NLOO2 CG8055 O73 074 Protein with putatively involved in intracellular protein transport NLOO3 CG3395 O75 076 Ribosomal protein S9 (RpS9), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal Subunit NLOO4 CG6141 O77 078 Ribosomal protein L9, structural constituent of ribosome involved in protein biosynthesis which is ocalised to the ribosome NLOOS CG2746 O79 080 Ribosomal protein L19, structural constituent of ribosome involved in protein biosynthesis which is ocalised to the ribosome NLOO6 CG318O O81 082 RNA polymerase II 140 kD subunit (RpII140), DNA-directed RNA polymerase activity (EC: 2.7.7.6) involved in transcription from Pol II promoter which is a component of the DNA-directed RNA polymerase II, core complex NLOO7 CGT269 O83 084 Helicase at 25E (Hel25E), also known in FlyBase as Dbp25F, Hel, I(2)25Eb and I(2)k11511, pre mRNA splicing factor activity involved in nuclear mRNA splicing, via spliceosome which is localized to he nucleus NLOO8 CG3416 O85 086 Protein with endopeptidase activity involved in proteolysis and peptidolysis which is a component of he proteasome regulatory particle, lid Subcomplex (sensu Eukarya) NLOO9 CG9261 O87 088 Protein involved with Na+/K+-exchanging ATPase complex NLO10 CG1250 O89 090 GTPase activator, ER to Golgi prot transport, component of the Golgi stack NLO11 CG1404 O91 092 Putative RAN small monomeric GTPase (cell adhesion) NLO12 CG17248 093 094 N-synaptobrevin; V-SNARE, vesicle-mediated transport, synaptic vesicle NLO13 CG18174 O95 096 Putative proteasome regulatory particle, lid Subcomplex, rpn11 NLO14 CG1088 O97 098 V-ATPase E Subunit NLO15 CG2331 O99 100 Transitional endoplasmic reticulum ATPase TER94, Golgi organization and biogenesis NLO16 CG17369 101 102 V-ATPase B Subunit NLO18 CG1915 103 104 Sallimus (sils), myosin-light-chain kinase activity (EC: 2.7.1.117) involved in mitotic chromosome condensation which is localized to the nucleus NLO19 CG332O 105 106 Rab-protein 1 involved in cell adhesion NLO21 CG10110 107 108 Gene cleavage and polyadenylation specificity factor NLO22 CG10689 109 110 Product with RNA helicase activity (EC: 2.7.7.—) involved in nuclear mRNA splicing, via spliceosome which is a component of the spliceosome complex NLO23 CG17907 111 112 Acetylcholineesterase NLO27 CG6699 113 114 Beta-coatomer protein

TABLE 1-CS Dm SEQ ID SEQ ID Target ID identifier NONA NOAA Function (based on Flybase) CSOO1 CG11276 1682 1683 Ribosomal protein S4 (RpS4), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal subunit CSOO2 CG8055 1684 1685 Carrier protein with putatively involved in intracellular protein transport CSOO3 CG3395 1686 1687 Ribosomal protein S9 (RpS9), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal subunit CSOO6 CG318O 1688 1689 RNA polymerase II 140 kD subunit (RpII140), DNA-directed RNA polymerase activity (EC: 2.7.7.6) involved in transcription from Pol II promoter which is a component of the DNA-directed RNA polymerase II, core complex US 9,528,123 B2

TABLE 1-CS-continued

Dm SEQ ID SEQ ID Target ID identifier NONA NOAA Function (based on Flybase)

CSOO7 CGT269 1690 1691 Helicase at 25E (Hel25E), also known in FlyBase as Dbp25F, Hel, I(2)25Eb and I(2)k11511, pre mRNA splicing factor activity involved in nuclear mRNA splicing, via spliceosome which is localized to the nucleus CSOO9 CG9261 1692 1693 Protein involved with Na+/K+-exchanging ATPase complex CSO11 CG1404 1694 1695 Tutative RAN Small monomeric GTPase (cell adhesion) CSO13 CG18174 1696 1697 Putative proteasome regulatory particle, lid subcomplex, rpn11 CSO14 CG1088 1698 1699 V-ATPase E Subunit CSO15 CG2331 1700 1701 Transitional endoplasmic reticulum ATPase TER94, Golgi organization and biogenesis CSO16 CG17369 1702 1703 V-ATPase B Subunit CSO18 CG1915 1704 1705 Sallimus (sils), myosin-light-chain kinase activity (EC: 2.7.1.117) involved in mitotic chromosome condensation which is localized to the nucleus

TABLE 1-PX Dm SEQ ID SEQ ID Target ID identifier NONA NOAA Function (based on Flybase) PXOO1 CG11276 2100 2101 Ribosomal protein S4 (RpS4), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal subunit PX009 CG9261 2102 2103 Protein involved with Na+/K+-exchanging ATPase complex PXO10 CG1250 2104 2105 GTPase activator, ER to Golgi prot transport, component of the Golgi stack PXO15 CG2331 2106 2107 Transitional endoplasmic reticulum ATPase TER94, Golgi organization and biogenesis PXO16 CG17369 2108 2109 V-ATPase B Subunit

TABLE 1-AD Dm SEQ ID SEQ ID Target ID identifier NONA NOAA Function (based on Flybase) ADOO1 CG11276 2364 2365 Ribosomal protein S4 (RpS4), structural constituent of ribosome involved in protein biosynthesis which is a component of the cytosolic Small ribosomal subunit ADOO2 CG8055 2366 2367 Carrier protein with putatively involved in intracellular protein transport ADOO9 CG9261 2368 2369 Protein involved with Na+/K+-exchanging ATPase complex ADO15 CG2331 2370 2371 Transitional endoplasmic reticulum ATPase TER94, Golgi organization and biogenesis ADO16 CG17369 2372 2373 V-ATPase B Subunit

TABLE 2 -LD Target Primer Forward Primer Reverse cDNA. Sequence (sense strand) ID s" -> 3 s" -> 3 s" -> 3

LDOO1 SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 1 GGCCCCAAGAA TAGCGGATGGT GGCCCCAAGAAGCATTTGAAGCGTTTGAATGCCCCAAAAGCATGGATGTTGGATAAATTGG GCATTTGAAGC GCGDCCRTCRT GAGGTGTTTTCGCACCTCGCCCATCTACAGGACCTCACAAATTGCGAGAGTCTTTGCCCTT G G GGTGATCTTCCTACGTAACCGATTGAAGTATGCTTTGACTAACAGCGAAGTTACTAAGATTG TTATGCAAAGGTTAATCAAAGTAGATGGAAAAGTGAGGACCGACTCCAATTACCCTGCTGG GTTTATGGATGTTATTACCATTGAAAAAACTGGTGAATTTTTCCGACT CATCTATGATGTTAA AGGACGATTTGCAGTGCATCGTATTACTGCTGAGGAAGCAAAGTACAAACTATGCAAAGTC AGGAGGATGCAAACTGGCCCCAAAGGAATTCCCTTCATAGTGACACACGACGGCCGCACC ATCCGCTA

LDO O2 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 3 GAGCGGCCAT GCAATGTCATC GCAATGTCATCCATCATGTCGTGTACATTGTCCACGTCCAAGTTTTTATGGGCTTTCTTAAG GCAAGCWCTBA CATCAKRTCRT AGCTTCAGCTGCATTTTTCATAGATTCCAATACTGTGGTGTTCGTACTAGCTCCCTCCAGAG ARMRRAAG GCAC CTTCTCGTTGAAGTTCAATAGTAGTTAAAGTGCCATCTATTTGCAACTGATTTTTTTCTAATC GCTTCTTCCGCTTCAGCGCTTGCATGGCCGCTC

LDOO3 SEQ ID NO : 29 SEQ ID NO : 30 SEO ID NO : 5 TCGGTCT TOTC CAGGTTCTTCC CAGGTTCTTCCTCTTGACGCGTCCAGGGCGACCACCACCGAATGGAGATTTGAGCGAGAA GAAGACNTAYG TCTTKACRCGD GTCAATATGCTTCTGGGAATCAAGTCTCACAATGAAGCTTGGAATATTCACGACCTGCTTAC TKAC CC GAACCCTGATATGTCTTTGACGGACCAGCACACGAGCATGATGGATTGATTTTGCAAGCCC CAACTTGAAAACTTGTGTTTGGAGACGTCGTTCCAAGAAATCTTCAATCTTCAAACCCAAGA CGTAATCAAGCTTCATACGGGTTTCATCCAACACTCCAATACGCACCAACCGACGAAGAAG AGCATTGCCTTCAAACAACCTGCGCTGATCTTTCTCTTCCAAAGTCAGAAGTTCTCTGGCAG CTTTACGGATTTTTGCCAAGGTATACTTGACTCGCCACACTTCACGTTTGTTCCTAAGACCA TATTCTCCTATGATTTTCAACTCCTGATCAAGACGTGCCTTTTCATAAGGTCGCCTGGGA

US 9,528,123 B2 133 134 TABLE 4-LD - continued

SEQ Target ID ID NO Sequences* Example Gi-number and species 76.552910 (Spodoptera frugiperda), 92.959651 (Drosophila ananassae), 9246.7993 (Drosophila erecta) LDOO3 58 TTGAGCGAGAAGTCAATATGCTTCT 4955893 O (Boophilus microplus) LDOO3 59 TTCCAAGAAATCTTCAATCTTCAAACCCAA 62238.687 (Diabrotica virgifera), 761699 O7 (Diploptera punctata), 678.72253 (Drosophila pseudoobscura), 55877642 (Locusta migratoria), 66548956 (Apis mellifera) LDOO3 6 O TTCATCCAACACTCCAATACG 2204 O140 (Ctenocephalides felis)

LDOO3 61 AAGAGCATTGCCTTCAAACAACCT 2459311 (Antheraea yamamai) LDOO3 62 AGTTCTCTGGCAGCTTTACGGATTTT 76.1699 O7 (Diploptera punctata)

LDOO3 63 CCACACTTCACGTTTGTTCCT 57963694 (Heliconius melpomene) LDOO3 64 CCGTATGAAGCTTGATTACGT 108742527 (Gryllius rubens), 108742525 (Gryllius pennsylvanicus), 108742523 (Gryllius veletis), 108742521 (Gryllius bimaculatus), 108742519 (Gryllius firmus), 109194897 (Myzus persicae) DOO3 65 AGGAACAAACGTGAAGTGTGGCG 109194897 (Myzus persicae)

DOO6 66 AGCGCTATGGGTAAGCAAGCTATGGG 2781997O () DOO6 67 TGTTATACTGGTTATAATCAAGAAGAT 558O1622 (Acyrthosiphon pisum), 6653.513 O (Apis mellifera)

LDO Of 68 GAAGTTCAGCACGAATGTATTCC 50563 603 (Homalodisca coagulata)

LDO Of 69 CAAGCAAGTGATGATGTTCAGTGCCAC 50563 603 (Homalodisca coagulata)

LDO Of 70 TGCAAGAAATTCATGCAAGATCC 21068658 (Chironomus tentans)

LDO Of 71 AAATGAAAAGAATAAAAAATT 492O1437 (Drosophila melanogaster)

LDO Of 72 CAGAATTTCCCAGCCATAGGAAT 67895.225 (Drosophila pseudoobscura)

LDO Of 73 AGCAGTTCAAAGATTTCCAGAAG 77848709 (Aedes aegypti)

LDO Of 74 TTCCAAATCAGCAAAGAGTACGAG 91083250 (Tribolium castaneum)

DO1 O 75 TACCCGCAGTTCATGTACCAT 29558345 (Bombyx mori)

DO1 O 76. CAGTCGCTGATCATGATCCAGCC 49559866 (Boophilus microplus)

DO1 O 77 CTCATGGACACGTTCTTCCAGAT 60293559 (Homalodisca coagulata)

DO1 O 78 GGGGCTGCATACAGTTCATCAC 92971.011 (Drosophila mojavensis)

DO1 O 79 CCCGCAGTTCATGTACCATTTG 92952825 (Drosophila ananassae)

DO1 O 8 O GACAATGCCAAATACATGAAGAA 92.92.1253 (Drosophila virilis)

DO1 O 81 TTCGATCAGGAGGCAGCCGCAGTG 92.92.1253 (Drosophila virilis) DO 82 AGCAGGGCTGGCATGGCGACAAA. 28317118 (Drosophila melanogaster)

DO 83 TTCTCAAAGTTGTAGTTAGATTTGGC 37951963 (Ips pini)

DO 84 TACTGCAAATTCTTCTTCCTATG 55883.846 (Locusta migratoria)

DO 8. GGTACATTCTTGTATGTAACTC 67885713 (Drosophila pseudoobscura)

DO 86 TCAAACATGATAATAGCACACTG 68771114 (Acanthos curria domesiana) DO 87 TCTCCTGACCGGCAGTGTCCCATA 1794 4197 (Drosophila melanogaster), 77843 537 (Aedes aegypti), 9446.9127 (Aedes aegypti), 24664595 (Drosophila melanogaster)

DO 88 GCTACTTTGGGAGTTGAAGTCCATCC 101.410 627 (Plodia interpuntella)

DO 89 TAACTACAACTTTGAGAAGCCTTTCCT 90813103 (Nasonia vitripennis)

DO 9 O AAGTTTGGTGGTCTCCGTGATGG 84267747 (Aedes aegypti) S 9,528,123 B2 135 136 TABLE 4-LD - continued

SEQ Target ID ID NO Sequences* Example Gi-number and species DO14 91 GCAGATCAAGCATATGATGGC 9732 (Manduca sexta), 90814338 (Nasonia vitripennis), 87266590 (Choristoneura fumiferana) DO14 92 ATCAAGCATATGATGGCTTTCATTGA 7547 O953 (Tribolium castaneum), 7616939 O (Diploptera punctata)

DO14 93 AATATTGAAAAGGGGCGCCTTGT 78055682 (Heliconius erato)

DO14 94 CAACGTCTCAAGATTATGGAATA 37659584 (Bombyx mori)

DO14 95 ATTATGGAATATTATGAGAAGAAAGA 66556286 (Apis mellifera)

DO14 96 AACAAAATCAAGATCAGCAATACT 25958976 (Curculio glandium)

DO16 97 ATGTCGTCGTTGGGCATAGTCA 27372076 (Spodoptera littoralis) DO16 98 GTAGCTAAATCGGTGTACATGTAACCTGGG 27372076 (Spodoptera littoralis), 55797 015 (Acyrthosiphon AAACCACGACG pisum), 73 615307 (Aphis gossypii), 4 680479 (Aedes aegypti), 9713 (Manduca sexta), 76.555122 (Spodoptera frugiperda), 237458 (Heliothis virescens), 53883819 (Plutella xylostella), 22038926 (Ctenocephalides felis), 1014 03557 (Plodia interpuntella), 92.96.9578 (Drosophila grimshawi), 91.82.9127 (Bornbyx mori)

DO16 99 GCAGATAC CTCACGCAAAGCTTC 62239897 (Diabrotica virgifera) DO16 OOGGATCGTTGGCCAAATTCAAGAACAGGCA 67882712 (Drosophila pseudoobscura), 92.985.459 (Drosophila grimshawi) DO16 O1 TTCTCCATAGAACCGTTCTCTTCGAAATCCT 4680479 (Aedes aegypti), 27372O76 (Spodoptera littoralis) G

DO16 O2 GCTGTTTCCATGTTAACACCCAT 49558344 (Boophilus microplus) DO16 O3TCCATGTTAACACCCATAGCAGCGA 622.38871 (Diabrotica virgifera) DO16 O4 CTACAGATCTGGGCAGCAATTTCATTGTG 22O38926 (Ctenocephalides felis), 16898595 (Ctenocephalides felis) DO16 O5 GGCAGACCAGCTGCAGAGAAAAT 22O38926 (Ctenocephalides felis), 16898595 (Ctenocephalides felis) DO16 O6 GAGAAAATGGGGATCTTCTGACCACGAGCA 4680479 (Aedes aegypti), 9713 (Mandu.ca sexta), ATGGAGTTCATCACGTC 22O38926 (Ctenocephalides felis), 16898595 (Ctenocephalides felis), 67877903 (Drosophila pseudoobscura), 10763875 (Manduca sexta), 76.554661 (Spodoptera frugiperda), 77905105 (Aedes aegypti), 50562965 (Homalodisca coagulate), 27372076 (Spodoptera littoralis) LDO16 107 ATGGAGTTCATCACGTCAATAGC 9713 (Manduca sexta), 237458 (Heliothis vires cens), 76.5546 61 (Spodoptera frugiperda), 22474331 (Helicoverpa armigera)

LDO16 108 GTCTGGATCATTTCCTCAGGATAGATACGG 16898595 (Ctenocephalides felis), GACCACGGATTGATTGGTTGACCCTGGATG 22O38926 (Ctenocephalides felis), TCCAAGAAGTCTTCAGCCAAAATTGGGGGA 50562965 (Homalodisca coagulate), CCTTTGTC 493951.65 (Drosophila melanogaster), 6901845 (Bombyx mori), 92.931OOO (Drosophila virilis)

LDO16 109 ATTGGGGGACCTTTGTCGATGGG 10763875 (Manduca sexta)

LDO16 11 OATGGGTTTTCCTGATCCATTGAAAACACGTC 493951.65 (Drosophila melanogaster), CCAACATATCTTCAGAAACAGGAGTCCTCA 55905051 (Locusta migratoria) AAATATCTCCTGTGAATTCACAAGCGGTGTT TTTGGCGTCGATTCCTGATGTGCCCTCGAA CACTTGAACCACAGCTTT LDO16 111ACAGCTTTTGACCCACTGACTTCCAG 216422 66 (Amblyomma variegatum)

LDO16 112 GACCCACTGACTTCCAGAACTTGTCCCGAA 493951.65 (Drosophila melanogaster) CGTATAGTGCCATCAGCCAGTTTGAGT

LDO16 113. GGACCGTTCACACCAGACACAGT 24 64 6342 (Drosophila melanogaster) US 9,528,123 B2 137 138 TABLE 4-LD - continued

SEQ Target ID ID NO Sequences* Example Gi-number and species DO16 14 GACTGTGTCTGGTGTGAACGGTCCTCT 1037691.63 (Drosophila melanogaster), 92 O48971 (Drosophila Willistoni) DO16 15 TTCTCTTCGAAATCCTGTTTGAA 84.116133 (Dermatophagoides fairinae) DO16 16 GACTGTGTWTGGTGTGAACGGTCC 24 64 6342 (Drosophila melanogaster) DO16 17 GGTCGTCGTGGTTTCCCAGGTTACATGTAC 922.31646 (Drosophila Willistoni), 91755555 (Bombyx mori), ACCGATTT 8422 8226 (Aedes aegypti)

DO16 18 TGACAGCTGCCGAATTCTTGGC 92231646 (Drosophila Willistoni)

DO18 19 CAAGTCACCGACGACCACAACCACAA 91080016 (Tribolium castaneum)

DO18 2OATCGCGATTGACGGTGGAGCC 91080016 (Tribolium castaneum)

DO27 21AGACGATCGGTTGGTTAAAATC 665O1387 (Apis mellifera)

DO27 22 GATATGGGAGCATGTGAAATATA 77326476 (Chironomus tentans)

DO27 2.3TTAGAGAATTGTTTGAATTAT 9012 9719 (Bicyclius any nana)

TABLE 4 - PC Target SEQ ID ID NOSequence * Example Gi-number and species PCOO 275 AAAATTGTCATGCAAAGGTTGAT 37952206 (Ips pini)

PCOO 276 AAAGCATGGATGTTGGACAAA. 98.994282 (Antheraea my litta) 10997 8109 (Gryllius pennsylvanicus) 55904580 (Locusta migratoria) PCOO 277 AAAGCATGGATGTTGGACAAATT 313 66663 (Toxoptera citricida)

PCOO 278 AAAGCATGGATGTTGGACAAATTGGG 60311985 (Papilio dairdanus) PCOO 279 AAAGCATGGATGTTGGACAAATTGGGGGGTGT 37951951 (Ips pini)

PCOO 28 O AAATACAAGTTGTGTAAAGTAA 84647793 (Myzus persicae) PCOO 281 AAGCATGGATGTTGGACAAATTGGGGGGTGT 709 O9486 (Mycetophagus quadripustulatus)

PCOO 282 ATGGATGTCATTACTATTGAGAA 25957367 () PCOO 283 CATCAAATTTGAATCTGGCAACCT 37952206 (Ips pini)

PCOO 284 CATGATGGCAGAACCATTCGTTA 603 03405 (Julodis onopordi) PCOO 285 CCAAAGCATGGATGTTGGACAA 9 O1381.64 (Spodoptera frugiperda)

PCOO 286 CCATTTTTGGTAACACATGATGG 11101.1915 (Apis mellifera)

PCOO 287 CCCAAAGCATGGATGTTGGACAA 50565112 (Homalodisca coagulata)

PCOO 288 CCCAAAGCATGGATGTTGGACAAA. 103790417 (Heliconius erato) 101.419954 (Plodia interpunctella) PCOO 289 CCCAAAGCATGGATGTTGGACAAATT 73 612809 (Aphis gossypii)

PCOO 29 O CCCAAAGCATGGATGTTGGACAAATTGGG 77329254 (Chironomus tentans) PCOO 291 CCCAAAGCATGGATGTTGGACAAATTGGGGGGTGT 603 O542O (Mycetophagus quadripustulatus)

PCOO 292 CCCAAAGCATGGATGTTGGACAAATTGGGGGGTGTCTTCGC 846.47995 (Myzus persicae) PCOO 293 CGTTACCCTGACCCCAACATCAA 73 613 O65 (Aphis gossypii) PCOO 294 GCAAAATACAAGTTGTGTAAAGTAA 836 62334 (Myzus persicae) PCOO 295 GCATGGATGTTGGACAAATTGGG 929 69396 (Drosophila grimshawi)

PCOO 296 GCATGGATGTTGGACAAATTGGGGG 67885868 (Drosophila pseudoobscura) US 9,528,123 B2 139 140 TABLE 4 - PC-continued Target SEQ ID ID NOSequence * Example Gi-number and species

PCOO 297 GCATGGATGTTGGACAAATTGGGGGGTGT 25.956.479 (Biphyllius lunatus

PCOO 298 GCATGGATGTTGGACAAATTGGGGGGTGTCT 90814901 (Nasonia vitripennis)

PCOO 299 GCTCCCAAAGCATGGATGTTGGA 11026O785 (Spodoptera frugiperda)

PCOO GCTCCCAAAGCATGGATGTTGGACAA 76.551269 (Spodoptera frugiperda)

PCOO 3O1 GCTCCCAAAGCATGGATGTTGGACAAA 56O85210 (Bombyx mori)

PCOO GCTCCCAAAGCATGGATGTTGGACAAATTGGG 22474.232 (Helicoverpa armigera)

PCOO GGTCCCAAAGGAATCCCATTTTTGGT SOS 65.112 (Homalodisca coagulata)

PCOO GGTGTCTTCGCCCCTCGTCCA 82575022 (Acyrthosiphon pisum)

PCOO 305 GTGAAGTCACTAAAATTGTCATGCAAAG 25.95682O (Biphyllius lunatus

PCOO 3 O 6 TCCACCGGGCCTCACAAGTTGCG 58371410 (Lonomia obliqua)

PCOO 3. Of TCCCAAAGCATGGATGTTGGA 11026.3957 (Spodoptera frugiperda)

PCOO TGCTCCCAAAGCATGGATGTTGGACAA 489.27129 (Hydropsyche sp.)

PCOO 309 TGGATGTTGGACAAATTGGGGGGTGTCT 90814 560 (Nasonia vitripennis)

PCOO3 31 O AAAATTGAAGATTTCTTGGAA 108742519 (Gryllius firmus) 1099.78291 (Gryllius pennsylvanicus) (Lysiphlebus testacelipes) (Rhynchosciara americana)

PCOO3 AACAAACGTGAAGTGTGGAGAGT 579 63 755 (Heliconius melpomene)

PCOO3 AAGTCGCCCTTCGGGGGTGGCCG 77884 O26 (Aedes aegypti)

PCOO3 ACTTCTCCCTGAAGTCGCCCTTCGG 929 92.453 (Drosophila mojavensis)

PCOO3 AGATTGTTTGAAGGTAATGCACTTCT 6O2.98816 (Diaphorina citri)

PCOO3 ATCCGTAAAGCTGCTCGTGAA 33373 689 (Glossina morsitans

PCOO3 ATCGACTTCTCCCTGAAGTCGCC 92987113 (Drosophila grimshawi)

PCOO3 ATCGACTTCTCCCTGAAGTCGCCCT 18995.48 (Drosophila melanogaster)

PCOO3 ATGAAGCTTGATTATGTTTTGGGTCTGAAAATTGAAGATTTCT 71539,459 (Diaphorina citri) TGGAAAGA

PCOO3 ATTGAAGATTTCTTGGAAAGA 6224 OO69 (Diabrotica virgifera)

PCOO3 32O CACATCGACTTCTCCCTGAAGTC 715.50961 (Oncome topia nigricans)

PCOO3 321 CAGAAGCACATCGACTTCTCCCTGAAGTCGCCCTTCGG 682 6715.1 (Drosophila simulians) 33355 OOO (Drosophila yakuba)

PCOO3 322 CAGAAGCACATCGACTTCTCCCTGAAGTCGCCCTTCGGGGG 215.2719 (Drosophila melanogaster)

PCOO3 323 CGACTTCTCCCTGAAGTCGCC 107324 644 (Drosophila melanogaster)

PCOO3 324 CTCCCTGAAGTCGCCCTTCGG 154 61311 (Drosophila melanogaster)

PCOO3 3.25 CTGGACTCGCAGAAGCACATCGACTTCTCCCTGAA 38624772 (Drosophila melanogaster)

PCOO3 326 GACTTCTCCCTGAAGTCGCCCTTCGG 92.959 651 (Drosophila ananassae 92.981958 (Drosophila mojavensis) 76.552 4 67 (Spodoptera frugiperda)

PCOO3 327 GCTAAAATCCGTAAAGCTGCTCGTGA 6O296953 (Diaprepes abbreviatus)

PCOO3 328 GCTAAAATCCGTAAAGCTGCTCGTGAACT 773.293 41 (Chironomus tentans)

PCOO3 329 GTGCGCAAGCAGGTGGTGAACATCCC 6 O312414 (Papilio dairdanus)

PCOO3 33 O TACACTTTGGCTAAAATCCGTAAAGCTGC 22O4. O14 O (Ctenocephalides felis)

PCOO3 331 TCGCAGAAGCACATCGACTTCTC 18883211 (Anopheles gambiae US 9,528,123 B2 141 142 TABLE 4 - PC-continued Target SEQ ID ID NOSequence * Example Gi-number and species

PCOO3 332 TCGCAGAAGCACATCGACTTCTCCCTGAAGTCGCCCTTCGG 92.963738 (Drosophila grimshawi)

PCOO3 333 TCTCCCTGAAGTCGCCCTTCGG 380.47836 (Drosophila yakuba) 272 60897 (Spodoptera frugiperda)

PCOO3 334 TGAAAATTGAAGATTTCTTGGAA 61646980 (Acyrthosiphon pisum) 73 61.5225 (Aphis gossypii) 836 61890 (Myzus persicae) 37804775 (Rhopalosiphum padi 3.0049209 (Toxoptera citricida)

PCOO3 335 TGAAAATTGAAGATTTCTTGGAAAGA 90813959 (Nasonia vitripennis)

PCOO3 336 TGGACTCGCAGAAGCACATCGACTTCTCCCT 25959.408 (Meliadema coriacea)

PCOO3 337 TGGCTAAAATCCGTAAAGCTGC 76.169907 (Diploptera punctata)

PCOO3 338 TGGGTCTGAAAATTGAAGATTTCTTGGA 3478804. 6 (Calilosobruchus maculatus)

PCOO3 339 TTCTCCCTGAAGTCGCCCTTCGG 10733 13 62 (Drosophila melanogaster) 11024 O861 (Spodoptera frugiperda)

PCOO3 34 O TTGGGTCTGAAAATTGAAGATTTCTTGGAAAG 37952.462 (Ips pini)

PCOO3 341 GGGTGCGCAAGCAGGTGGTGAAC 110887729 (Argas monoliakensis) PCOOS 342 CTCCTCAAAAAGTACAGGGAGGCCAAGAA 635.12537 (Ixodes scapularis)

PCOOS 343 AAAAAGAAGGTGTGGTTGGATCC 33491424 (Trichoplusia ni)

PCOOS 344 AAAAAGAAGGTGTGGTTGGATCCAAATGAAATCAA 91759273 (Bombyx mori) 55908261 (Locusta migratoria)

PCOOS 345 AAAGAAGGTGTGGTTGGATCCAAATGAAATCA. 101.414 616 (Plodia interpunctella)

PCOOS 346 AACACCAACTCAAGACAAAACAT 25957531 ( campestris)

PCOOS 347 AACACCAACTCAAGACAAAACATCCGTAA 25958948 (Curculio glandium)

PCOOS 348 AACT CAAGACAAAACATCCGTAA 60314333 (Panorpa cf. vulgaris APV-2005)

PCOOS 349 AAGAACACTGAAGCCAGAAGGAAGGGAAGGCATTGTGG 25958948 (Curculio glandium)

PCOOS 350 AATGAAATCAACGAAATCGCCAACAC 9297916O (Drosophila grimshawi) 92232O72 (Drosophila Willistoni)

PCOOS 351 ATGGAGTACATCCACAAGAAGAAGGC 15454802 (Drosophila melanogaster)

PCOOS 352 CAAGATGCTGTCTGACCAGGC 67872905 (Drosophila pseudoobscura)

PCOOS 353 CGCCTCCT CAAAAAGTACAGGGAGGC 754.71260 (Tribolium cast aneum)

PCOOS 3.54 CGTATCGCCACCAAGAAGCAG 6.82 67374 (Drosophila simulians)

PCOOS 355 CTGTACATGAAAGCGAAGGGTAA 2595724 6 (Carabus granulatus)

PCOOS 356 GAACAAGAGGGTCCTTATGGAG 90977107 (Aedes aegypti)

PCOOS 357 GAACAAGAGGGTCCTTATGGAGTACATCCA 40544432 (Tribolium cast aneum)

PCOOS 358 GAGCGTATCGCCACCAAGAAGCA 924.80972 (Drosophila erecta) 3335.4497 (Drosophila yakuba)

PCOOS 359 GAGTACATCCACAAGAAGAAGGC 15516174 (Drosophila melanogaster)

PCOOS 360 GATCCAAATGAAATCAACGAAAT 56149737 (Rhynchosciara americana)

PCOOS 361 GCCAACACCAACT CAAGACAAAACATCCG 103 019061 (Tribolium castaneum)

PCOOS 362 GCCAACACCAACT CAAGACAAAACATCCGTAAGCTCAT 56149737 (Rhynchosciara americana)

PCOOS 363 GGCAAAAAGAAGGTGTGGTTGGATCCAAATGAAATCA 101.417 O42 (Plodia interpunctella)

PCOOS 364 GGGTCCTTATGGAGTACATCCACAAGAA 67885759 (Drosophila pseudoobscura)

PCOOS 365 TGCGATGCGGCAAAAAGAAGGT 56149531 (Rhynchosciara americana) US 9,528,123 B2 143 144 TABLE 4 - PC-continued Target SEQ ID ID NOSequence * Example Gi-number and species PCOOS 366 TGGTTGGATCCAAATGAAATCAACGAAAT 15355452 (Apis mellifera) 83662749 (Myzus persicae)

PCOOS 367 TTGGATCCAAATGAAATCAACGAAAT 110985.444 (Apis mellifera) 111158.439 (Myzus persicae)

PCO1 O 368 CCGCAGTTCATGTACCATTTG 92952 825 (Drosophila ananassae

PCO1 O 369 CTGATGGAGATGAAGCAGTGCTGCAATTC 583 95529 (Anopheles gambiae str. PEST)

PCO1 O 37 O GACGTGCTCAGATGGGTGGACAG 56152422 (Rhynchosciara americana)

PCO1 O 3.71 GCCCGAGCCTGTGTTGTTGGA 9293982O (Drosophila virilis

PCO1 O 372 GGCACATGCTGATGCGTGAGGAT 83937570 (Lutzomyia longipalpis)

PCO1 O 373. GGGCACATGGTCATGGGCGATTC 3337934 (Drosophila melanogaster)

PCO14 374. AAGATCATGGAGTACTACGAGAA 85577611 (Aedes aegypti)

PCO14 375 ACGAGAAAAAGGAGAAGCAAG 67838315 (Drosophila pseudoobscura)

PCO14 376 ATGGAGTACTACGAGAAAAAGGAGAAGCAAGT 92.928915 (Drosophila virilis

PCO14 377 CAAAAACAAATCAAACACATGATGGC 82574 OO1 (Acyrthosiphon pisum) 11116 0670 (Myzus persicae)

PCO14 378 CTCAAGATCATGGAGTACTACGA 55692554 (Drosophila yakuba)

PCO14 379 CTCAAGATCATGGAGTACTACGAGAA 929 423 O1 (Drosophila ananassae 92.4761.96 (Drosophila erecta) 53884266 (Plutella xylostella)

PCO14 38 O GAACAAGAAGCCAATGAGAAAGC 11116 0670 (Myzus persicae)

PCO14 381 GACT CAAGATCATGGAGTACT 112432414 (Myzus persicae)

PCO14 382 GATGTTCAAAAACAAATCAAACACATGATGGC 736.18688 (Aphis gossypii)

PCO14 383 TACTACGAGAAAAAGGAGAAGC 62239529 (Diabrotica virgifera)

PCO14 384 TTCATTGAACAAGAAGCCAATGA 15357365 (Apis mellifera)

PCO16 385 ACACGACCGGCGCGCTCGTAAAT 75710699 (Tribolium cast aneum)

PCO16 386 ACCAGCACGTGCTTCTCGCACTGGTAGGCCAAGAATTCGGC 92.048971 (Drosophila willistoni)

PCO16 387 AGCACGTGCTTCTCGCACTGGTAGGC 92.985.459 (Drosophila grimshawi)

PCO16 388 ATACGCGACCACGGGTTGATCGG 18868609 (Anopheles gambiae 312O6154 (Anopheles gambiae str. PEST)

PCO16 389 ATCGGTGTACATGTAACCGGGGAAACC 292 1501 (Culex pipiens) 62239897 ((Diabrotica virgifera) 92.957249 (Drosophila ananassae) 92477818 ((Drosophila erecta) 92965. 644 (Drosophila grimshawi) 246.46342 (Drosophila melanogaster) 67896654 (Drosophila pseudoobscura) 75710699 (Tribolium cast aneum)

PCO16 390 ATCGTTGGCCAAGTTCAAGAACAG 92950254 (Drosophila ananassae)

PCO16 391 CACGTGCTTCTCGCACTGGTAGGCCAAGAA 4680479 (Aedes aegypti)

PCO16 392 CCAGTCTGGATCATTTCCTCGGG 67884 189 (Drosophila pseudoobscura)

PCO16 393 CCAGTCTGGATCATTTCCTCGGGATA 929 40287 (Drosophila virilis

PCO16 394 CGCTCGATGGTCGGATCGTTGGCCAAGTTCAAGAACA 292 1501 (Culex pipiens)

PCO16 395 CGCTCGATGGTCGGATCGTTGGCCAAGTTCAAGAACAGACA 92477818 (Drosophila erecta) CACGTTCTCCAT 15061308 (Drosophila melanogaster)

PCO16 396 CGTGCTTCTCGCACTGGTAGGCCAAGAA 13752998 (Drosophila melanogaster)

PCO16 397 CTGGCAGTTTCCATGTTGACACCCATAGC 16898595 (Ctenocephalides felis) US 9,528,123 B2 145 146 TABLE 4 - PC-continued Target SEQ ID ID NOSequence * Example Gi-number and species PCO 6 398 CTTAGCATCAATACCTGATGT 61646 107 (Acyrthosiphon pisum)

PCO 399 GACATGTCGGTCAAGATGACCAGCACGTG 9713 (Mandu.ca sexta)

PCO 4 OO GACATGTCGGTCAAGATGACCAGCACGTGCTTCTCGCACTG 92.933 153 (Drosophila virilis)

PCO GACATGTCGGTCAAGATGACCAGCACGTGCTTCTCGCACTG 292 1501 (Culex pipiens) GTA

PCO GAGCCGTTCTCTTCGAAGTCCTG 237458 (Heliothis virescens)

PCO GATGACCAGCACGTGCTTCTC 18883474 (Anopheles gambiae

PCO GATGACCAGCACGTGCTTCTCGCACTG 92477818 (Drosophila erecta)

PCO 405 GATGACCAGCACGTGCTTCTCGCACTGGTAGGCCAAGAA 15061308 (Drosophila melanogaster) 67883 622 (Drosophila pseudoobscura)

PCO GATGACCAGCACGTGCTTCTCGCACTGGTAGGCCAAGAATTC 312O6154 (Anopheles gambiae str. PEST)

PCO 4. Of GATGGGGATCTGCGTGATGGA 101403557 (Plodia interpunctella)

PCO 408 GATGGGGATCTGCGTGATGGAGCCGTTGCGGCCCTCCAC 53883819 (Plutella xylostella)

PCO GGAATAGGATGGGTGATGTCGTCGTTGGGCATAGT 11024 O379 (Spodoptera frugiperda)

PCO GGAATAGGATGGGTGATGTCGTCGTTGGGCATAGTCA 27372076 (Spodoptera littoralis)

PCO GGATCGTTGGCCAAGTTCAAGAA 917 57299 (Bombyx mori)

PCO GGATCGTTGGCCAAGTTCAAGAACA 103 O2O3 68 (Tribolium castaneum)

PCO GGATCGTTGGCCAAGTTCAAGAACAG 237458 (Heliothis virescens)

PCO GGATGGGTGATGTCGTCGTTGGGCAT 101403557 (Plodia interpunctella)

PCO GGCAGTTTCCATGTTGACACCCATAGC 4680479 (Aedes aegypti)

PCO GGCATAGTCAAGATGGGGATCTG 929 24977 (Drosophila virilis)

PCO GTCTGGATCATTTCCTCGGGATA 929 66144 (Drosophila grimshawi)

PCO GTGATGATGCGCTCGATGGTCGGATCGTTGGCCAAGTTCAA 1551475 O (Drosophila melanogaster) GAACAGACACACGTTCTCCAT

PCO GTGTACATGTAACCGGGGAAACC 929 24977 (Drosophila virilis)

PCO GTTTCCATGTTGACACCCATAGC 91826 756 (Bombyx mori)

PCO 421 TCAATGGGTTTTCCTGATCCATTGAA 493 95.165 (Drosophila melanogaster) 990 O9492 (Leptinotarsa decemlineata)

PCO 422 TCATCCAGCACAGACTTGCCAG 10763875 (Manduca sexta)

PCO 423 TCATCCAGCACAGACTTGCCAGG 9713 (Mandu.ca sexta)

PCO 424 TCCATGTTGACACCCATAGCAGC 929 62.756 (Drosophila ananassae)

PCO 425 TCCATGTTGACACCCATAGCAGCAAACAC 6O2956O7 (Homalodisca coagulata)

PCO 426 TCGAAGTCCTGCTTGAAGAACCTGGC 101403557 (Plodia interpunctella)

PCO 427 TCGATGGTCGGATCGTTGGCCAAGTTCAAGAACAGACACAC 4680479 (Aedes aegypti) GTTCTCCAT

PCO 428 TCGGATCGTTGGCCAAGTTCAAGAACAGACACACGTTCTCCA 2.793275 (Drosophila melanogaster)

PCO 429 TCGTTGGCCAAGTTCAAGAACAG 90.1375O2 (Spodoptera frugiperda)

PCO 43 O TGGGTGATGTCGTCGTTGGGCAT 53883819 (Plutella xylostella)

PCO 431 TTCTCGCACTGGTAGGCCAAGAA 11024 O379 (Spodoptera frugiperda) 27372076 (Spodoptera littoralis)

PCO 432 TTCTCTTCGAAGTCCTGCTTGAAGAACCTGGC 9713 (Mandu.ca sexta) US 9,528,123 B2 147 148 TABLE 4 - PC-continued

Target SEQ ID ID NOSequence * Example Gi-number and species

PCO16 433 TTGGCCAAGTTCAAGAACAGACACACGTT 559 O5051 (Locusta migratoria)

PCO16 434 GTTTCCATGTTGACACCCATAGCAGCAAA 841.16133 (Dermatophagoides farinae)

TABLE 4-EW Target ID SEQ ID NO Sequence * Example Gi-number and species EWOOS 533 AAGCGACGTGAAGAGCGTATCGC 765532O6 (Spodoptera frugiperda)

EWOOS 534 ATTAAAGATGGTCTTATTATTAA 15355452 (Apis mellifera)

EWOOS 535 CGTAAGCGACGTGAAGAGCGTATCGC 33.491424 (Trichoplusia ni) EWOOS 536 GGTCGTCATTGTGGATTTGGTAAAAG 60314333 (Panorpa cf. vulgaris APV-2005) EWOOS s37 TGCGATGCGGCAAGAAGAAGGT 1504893 O (Drosophila melanogaster) EWOOS 538 TGCGGCAAGAAGAAGGTTTGG 93 OO2524 (Drosophila mojavensis 92.93 0455 (Drosophila virilis) 92 044532 (Drosophila Willistoni) EWOOS 539 TTGTGGATTTGGTAAAAGGAA 603 O 6723 ( sp.)

EWO1 O 54 O CAAGTGTTCAATAATTCACCA 83.937567 (Lutzomyia longipalpis)

EWO1 O 541 CATTCTATAGGCACATGTTGATG 29558345 (Bombyx mori)

EWO1 O 542 CTGGCGGCCACATGGTCATGGG 92476940 (Drosophila erecta) 9297.7931 (Drosophila grimshawi) 2871327 (Drosophila melanogaster)

EWO15 543 AACAGGCCCAATTCCATCGACCC 92.947821 (Drosophila ananassae)

EWO15 544 AGAGAAAAAATGGACCTCATCGAC 62239128 (Diabrotica virgifera) EWO15 545 CGCCATCCGTCGCTGTTCAAGGCGATCGG 1886 6954 (Anopheles gambiae)

EWO15 546 CTGGCAGTTACCATGGAGAACTTCCGTTACGCCATG 62239128 (Diabrotica virgifera)

EWO15 547 GTGATCGTGATGGCGGCCACGAA 18887285 (Anopheles gambiae)

EWO15 548 GTGATCGTGATGGCGGCCACGAAC 83423460 (Bombyx mori)

EWO15 549 TGATGGACGGCATGAAGAAAAG 91086234 (Tribolium castaneum) EWO16 550 AATATGGAAACAGCCAGATTCTT 109193 659 (Myzus persicae)

EWO16 551 ATGATCCAGACTGGTATTTCTGC 92.938857 (Drosophila virilis)

EWO16 552 ATTGATGTGATGAATTCCATTGCC 55905051 (Locusta migratoria)

EWO16 553 GAAATGATCCAGACTGGTATTTCTGC 50562965 (Homalodisca coagulata) EWO16 554 GAAGAAATGATCCAGACTGGTAT 92.96.9748 (Drosophila mojavensis) EWO16 555 GACTGTGTCTGGTGTGAACGG 2286 639 (Drosophila melanogaster) 92 O42621 (Drosophila Willistoni) EWO16 556 GATATGTTGGGTCGTGTGTTTAA 92.96.9748 (Drosophila mojavensis)

EWO16 sist GATCCTACCATTGAAAGAATTAT 99.01.1193 (Leptinotarsa decemlineata)

EWO16 558 GTGTCTGAAGATATGTTGGGTCGTGT 76.554661 (Spodoptera frugiperda)

EWO16 559 GTGTCTGGTGTGAACGGACCG 22474331 (Helicoverpa armigera)

EWO16 560 TCTGAAGATATGTTGGGTCGTGT 27372076 (Spodoptera littoralis) EWO16 561 TGGCATATCAATGTGAGAAGCA 6033 6595 (Homalodisca coagulata)

EWO16 562 TTGAACTTGGCCAATGATCCTACCAT 91827863 (Bombyx mori) US 9,528,123 B2 149 150 TABLE 4 - AG Target SEQ ID ID NOSequence * Example Gi-number and species AGOO 621 AAAACTGGTGAATTCTTCCGTTTGAT 37953169 (Ips pini)

AGOO 622 AAAGCATGGATGTTGGACAAA. 98.994.282 (Antheraea myiitta) 109 978109 (Gryllius pennsylvanicus) 559 O458 O (Locusta migratoria)

AGOO 623 AAAGCATGGATGTTGGACAAATT 313 66663 (Toxoptera citricida)

AGOO 624 AAAGCATGGATGTTGGACAAATTGGG 60311985 (Papilio dairdanus)

AGOO 625 AAAGCATGGATGTTGGACAAATTGGGGGGTGT 37951951 (Ips pini) 1091951 07 (Myzus persicae)

AGOO 626 AAATACAAATTGTGCAAAGTCCG 25958703 (Curculio glandium)

AGOO 627 AACTTGTGCATGATCACCGGAG 22O39624 (Ctenocephalides felis)

AGOO 628 AAGCATGGATGTTGGACAAATTGGGGG 112433559 (Myzus persicae)

AGOO 629 AAGCATGGATGTTGGACAAATTGGGGGGTGTGTT 709 O9486 (Mycetophagus quadripustulatus)

AGOO 63 O ACTGGTGAATTCTTCCGTTTGAT 773273O3 (Chironomus tentains

AGOO 631 ATTGAAAAAACTGGTGAATTCTTCCGTTTGATCTATGATGTTA 22O39624 (Ctenocephalides felis) A.

AGOO 632 CCAAAGCATGGATGTTGGACAA 9 O1381.64 (Spodoptera frugiperda)

AGOO 633 CCCAAAGCATGGATGTTGGACAA 4892 7129 (Hydropsyche sp.) 76551269 (Spodoptera frugiperda)

AGOO 634 CCCAAAGCATGGATGTTGGACAAA. 91835558 (Bombyx mori) 103783745 (Heliconius erato) 101.419954 (Plodia interpunctella)

AGOO 635 CCCAAAGCATGGATGTTGGACAAATT 73619372 (Aphis gossypii) 773.2925.4 (Chironomus tentans)

AGOO 636 CCCAAAGCATGGATGTTGGACAAATTGGG 22474.232 (Helicoverpa armigera)

AGOO 637 CCCAAAGCATGGATGTTGGACAAATTGGGGG 84647382 (Myzus persicae)

AGOO 638 CCCAAAGCATGGATGTTGGACAAATTGGGGGGTGT 846.47995 (Myzus persicae)

AGOO 639 CCCAAAGCATGGATGTTGGACAAATTGGGGGGTGTGTT 603 O542O (Mycetophagus quadripustulatus)

AGOO 64 O CTGGATTCATGGATGTGATCA 27617172 (Anopheles gambiae

AGOO 641 GAATTCTTCCGTTTGATC ATGATGT SOS 65.112 (Homalodisca coagulata) 71049326 (Oncome topia nigricans)

AGOO 642 GCATGGATGTTGGACAAA TGGG 929 69396 (Drosophila grimshawi) 93 OO1617 (Drosophila mojavensis) 92.929 731 (Drosophila virilis

AGOO 643 GCATGGATGTTGGACAAA TGGGGG 67885868 (Drosophila pseudoobscura)

AGOO 644 GCATGGATGTTGGACAAA TGGGGGGTGT 90814901 (Nasonia vitripennis)

AGOO 645 GCATGGATGTTGGACAAA TGGGGGGTGTGTTCGCCCC 25956.479 (Biphyllius lunatus

AGOO 646 GCCCCCAAAGCATGGATG TGGACAA 50565112 (Homalodisca coagulata)

AGOO 647 GCTGGATTCATGGATGTGATC 103775903 (Heliconius erato)

AGOO 648 GGATCATTCGATATTGTCCACAT 113 017118 (Bemisia tabaci)

AGOO 649 GGCAACTTGTGCATGATCACCGGAGG 25958703 (Curculio glandium)

AGOO 650 TACAAATTGTGCAAAGTCCGCAA 56161193 (Rhynchosciara americana)

AGOO 651 TATCCTGCTGGATTCATGGATGT 4093.4103 (Bombyx mori)

AGOO 652 TCACCATTGAAAAAACTGGTGAATTCTTC 62O83410 (Lysiphlebus testacelipes)

AGOO 653 TGCATGATCACCGGAGGCAGGAA 347855O (Antheraea yamamai) US 9,528,123 B2 151 152 TABLE 4-AG-Continued Target SEQ ID ID NOSequence * Example Gi-number and species

AGOO1 654 TGCATGATCACCGGAGGCAGGAATTTGGG 14627585 (Drosophila melanogaster) 33355 OO8 (Drosophila yakuba)

AGOO1 655 TGGATGTTGGACAAATTGGGGGGTGT 90814560 (Nasonia vitripennis)

AGOO1 656 TGTGCATGATCACCGGAGGCAG 929.49859 (Drosophila ananassae 929.993 O6 (Drosophila grimshawi)

AGOO1 657 TGTGCATGATCACCGGAGGCAGGAATTTGGG 6784.2487 (Drosophila pseudoobscura)

AGOOs 658 AAGATCGACAGGCATCTGTACCACG 83935 651 (Lutzomyia longipalpis)

AGOOs 659 AAGATCGACAGGCATCTGTACCACGCCCTGTACATGAAGGC 76.552995 (Spodoptera frugiperda)

AGOOs 660 AAGGGTAACGTGTTCAAGAACAA 18932248 (Anopheles gambiae 603 06 606 (Sphaerius sp.)

AGOOs 661 AAGGGTAACGTGTTCAAGAACAAG 18953 735 (Anopheles gambiae 25957811 () 60311920 (Euclidia glyphica)

AGOOs 662 AAGGGTAACGTGTTCAAGAACAAGAGAGT 25958948 (Curculio glandium) 90812513 (Nasonia giraulti)

AGOOs 663 ACAAGAAGAAGGCTGAGAAGGC 603117OO (Euclidia glyphica)

AGOOs 664 ATCAAGGATGGTTTGATCATTAA 25957811 (Cicindela campestris)

AGOOs 665 ATGGAATACATCCACAAGAAGAAG 56149737 (Rhynchosciara americana)

AGOOs 666 CAAAACATCCGTAAATTGATCAAGGATGGT 60314333 (Panorpa cf. vulgaris APV-2005)

AGOOs 667 CAAAACATCCGTAAATTGATCAAGGATGGTTTGATCAT 25958948 (Curculio glandium)

AGOOs 668 CAAGGGTAACGTGTTCAAGAA 476608 (Drosophila melanogaster) 380483 OO (Drosophila yakuba)

AGOOs 669 CAAGGGTAACGTGTTCAAGAACAAG 92.946023 (Drosophila ananassae) 2871633 (Drosophila melanogaster) 6.82 67374 (Drosophila simulians) 3335.4497 (Drosophila yakuba) 83937.096 (Lutzomyia longipalpis)

AGOOs 670 CATCTGTACCACGCCCTGTACATGAAGGC 101.417 O42 (Plodia interpunctella)

AGOOs 671 GAAGAAGGCTGAGAAGGCCCG 40874303 (Bombyx mori)

AGOOs 672 GACAGGCATCTGTACCACGCCCTGTACATGAAGGC 901.35865 (Bicyclus any nana)

AGOOs 673 GAGAAGGCCCGTGCCAAGATGTTG 82572137 (Acyrthosiphon pisum)

AGOOs 674 GATCCAAATGAAATCAATGAGATTGC 60312128 (Papilio dairdanus)

AGOOs 675 GCTCGTATGCCTCAAAAGGAACTATGG 2595724 6 (Carabus granulatus)

AGOOs 676 GGGTAACGTGTTCAAGAACAAG 4447348 (Drosophila melanogaster)

AGOOs 677 GGTAACGTGTTCAAGAACAAG 18948649 (Anopheles gambiae

AGOOs 678 TACATCCACAAGAAGAAGGCTGAGAAG 2871633 (Drosophila melanogaster)

AGOOs 6.79 TACCACGCCCTGTACATGAAGGC 10764114 (Manduca sexta)

AGOOs TCAATGAGATTGCCAACACCAACTC 83935 651 (Lutzomyia longipalpis)

AGOOs 681 TGATCAAGGATGGTTTGATCAT 77642775 (Aedes aegypti) 276.15052 (Anopheles gambiae 929 82271 (Drosophila grimshawi) 67896.961 (Drosophila pseudoobscura)

AGOOs 682 TGATCAAGGATGGTTTGATCATTAAGAA 92O42883 (Drosophila Willistoni)

AGOOs 683 TGGTTGGATCCAAATGAAATCA 40867709 (Bombyx mori) 101.417 O42 (Plodia interpunctella)

AGOOs 684 TGGTTGGATCCAAATGAAATCAA 15355452 (Apis mellifera) 83662749 (Myzus persicae) US 9,528,123 B2 153 154 TABLE 4-AG-continued Target SEQ ID ID NOSequence * Example Gi-number and species

AGOOs 685 TGGTTGGATCCAAATGAAATCAATGAGAT 63 013469 (Bombyx mori) 55908261 (Locusta migratoria)

AGOOs 686 TGTACCACGCCCTGTACATGAAGGC 23573 622 (Spodoptera frugiperda)

AGOOs 687 TTGATCAAGGATGGTTTGATCA 113019292 (Bemisia tabaci)

AGOOs 688 TTGATCAAGGATGGTTTGATCAT 61674956 (Aedes aegypti) 41576849 (Culicoides sonorensis)

AGOOs 689 TTGATGGAATACATCCACAAGAAGAAGGC 92.225847 (Drosophila willistoni)

AGOOs 69 O. AGGATGCGTGTCTTGAGGCGTCT 110887217 (Argas monoliakensis) AGOOs 691 AAGGCCAAGGGTAACGTGTTCAAGAACAAG 110887217 (Argas monoliakensis)

AGO1 O 692 CGTTTGTGTCAAAAGTTTGGAGAATA 785397O2 (Glossina morsitans)

AGO1 O 693 GATGTTTTAAGATGGGTCGATCG 110759793 (Apis mellifera)

AGO1 O 694 TTTTACAGGCATATGCTTATGAGGGAAGATTT 55902158 (Locusta migratoria)

AGO1 O 695 TTTTTCGAGGTGGTCAATCAGCATTCGGC 92.92.5934 (Drosophila virilis

AGO14 696 AACATGCTGAACCAAGCCCGT 754.668O2 (Tribolium cast aneum)

AGO14 697 AACATGCTGAACCAAGCCCGTCT 87266590 (Choristoneura fumiferana) 103779114 (Heliconius erato)

AGO14 698 AAGATCATGGAATACTATGAGAAGAA 101.403826 (Plodia interpunctella)

AGO14 699 AAGATCATGGAATACTATGAGAAGAAGGAGAA 8152O 950 (Lutzomyia longipalpis)

AGO14 7 OO AATGAAAAGGCCGAGGAAATTGATGC 62239529 (Diabrotica virgifera)

AGO14 ATGGAATACTATGAGAAGAAGGA 16901350 (Ctenocephalides felis)

AGO14 CAATCCTCCAACATGCTGAACCA 53148,472 (Plutella xylostella)

AGO14 CAGATCAAGCATATGATGGCCTTCAT 53148,472 (Plutella xylostella)

AGO14 704 GCAGATCAAGCATATGATGGCCTTCAT 87266590 (Choristoneura fumiferana) 9732 (Mandu.ca sexta) 90814338 (Nasonia vitripennis)

AGO14 GCGGAAGAAGAATTTAACATTGAAAAGGG 50558386 (Homalodisca coagulata) 7155217 O (Oncometopia nigricans)

AGO16 706 AACGACGACATCACCCATCCTATTC 1102481.86 (Spodoptera frugiperda) 27372076 (Spodoptera littoralis)

AGO16 707 AACGGTTCCATGGAGAACGTGTG 292 1501 (Culex pipiens) 92950254 (Drosophila ananassae) 11024 O379 (Spodoptera frugiperda)

AGO16 7 OS AACGGTTCCATGGAGAACGTGTGTCT 246.46342 (Drosophila melanogaster)

AGO16 AACGGTTCCATGGAGAACGTGTGTCTCTTCTTGAA 91829127 (Bombyx mori)

AGO16 ATGATCCAGACCGGTATCTCCGC 22474 04 O (Helicoverpa armigera)

AGO16 ATGCCGAACGACGACATCACCCATCC 312O6154 (Anopheles gambiae str. PEST)

AGO16 CAATGCGAGAAACACGTGCTGGT 9713 (Mandu.ca sexta)

AGO16 CCGCACAACGAAATCGCCGCCCAAAT 754 69507 (Tribolium cast aneum)

AGO16 CGTTTCTTCAAGCAGGACTTCGA 83937868 (Lutzomyia longipalpis)

AGO16 CTTGGACATCCAAGGTCAACCCATCAACCCATGGTC 104530890 (Belgica antarctica)

AGO16 GAAATGATCCAGACCGGTATCTC 292 1501 (Culex pipiens) 929 66144 (Drosophila grimshawi)

AGO16 GAAATGATCCAGACCGGTATCTCCGCCATCGACGTGATGAAC 312O6154 (Anopheles gambiae str. PEST) TC US 9,528,123 B2 155 156 TABLE 4-AG-Continued Target SEQ ID ID NOSequence * Example Gi-number and species

AGO16 718 GAAGAAATGATCCAGACCGGTAT 754.695. Of (Tribolium cast aneum)

AGO16 719 GAAGAAGTACCCGGACGTCGTGG 22O38926 (Ctenocephalides felis)

AGO16 72 O GACATCCAAGGTCAACCCATCAA 16898595 (Ctenocephalides felis)

AGO16 721 GCCCGTTTCTTCAAGCAGGACTTCGA 312O6154 (Anopheles gambiae str. PEST)

AGO16 722 GCCGCCCAAATCTGTAGACAGGC (Homalodisca coagulata)

AGO16 723 GGATCAGGAAAACCCATTGACAAAGGTCC 493951.65 (Drosophila melanogaster) 99 OO94.92 (Leptinotarsa decemlineata)

AGO16 724 GGTTACATGTACACCGATTTGGC 91.829 127 (Bombyx mori)

AGO16 72 GGTTACATGTACACCGATTTGGCCACCAT 777s. Of 65 (Aedes aegypti) 9713 (Manduca sexta) 110248.186 (Spodoptera frugiperda) 273.72 Of 6 (Spodoptera littoralis)

AGO16 726 GGTTACATGTACACCGATTTGGCCACCATTTACGAA 922.31646 (Drosophila Willistoni)

AGO16 727 GTGTCGGAGGATATGTTGGGCCG 924 6 O25 O (Drosophila erecta) 24 646 342 (Drosophila melanogaster) 55694673. (Drosophila yakuba)

AGO16 728 TACATGTACACCGATTTGGCCACCAT 312O6154 (Anopheles gambiae str. PEST)

AGO16 729 TTCAACGGATCAGGAAAACCCATTGACAAAGGTCC 99.01.0653 (Leptinotarsa decemlineata)

AGO16 73 O TTCCCCGGTTACATGTACACCGATTTGGCCAC 292 15 O1. (Culex pipiens) 75710699 (Tribolium cast aneum)

AGO16 731 TTCCCCGGTTACATGTACACCGATTTGGCCACCAT 6223.9897 Diabrotica virgifera) 92.95.7249 Drosophila ananassae 924f7 149 Drosophila erecta) 67896654 Drosophila pseudoobscura)

AGO16 732 TTCCCCGGTTACATGTACACCGATTTGGCCACCATTTA 929 69.578 (Drosophila grimshawi)

AGO16 733 TTCCCCGGTTACATGTACACCGATTTGGCCACCATTTACGA 1037447.58 (Drosophila melanogaster)

AGO16 734 TTCGCCATCGTGTTCGCCGCCATGGGTGT 312O6154 (Anopheles gambiae str. PEST)

AGO16 73 TTCTTCAAGCAGGACTTCGAAGA 9713 (Manduca sexta)

AGO16 736 TTCTTGAATTTGGCCAACGATCC 92.972.277 (Drosophila grimshawi) 990111.93 (Leptinotarsa decemlineata)

AGO16 737 TTCTTGAATTTGGCCAACGATCCCACCATCGAG 67839381 (Drosophila pseudoobscura)

AGO16 738 GCCGAATTTTTGGCTTATCAATG 841-16133 (Dermatophagoides farinae

TABLE 4-TC Target SEQ D ID NOSequence * Example Gi-number and species TCOO1 813 AAAGCATGGATGTTGGATAAA 709 O948 O (Carabus granulatus) 16898 765 (Ctenocephalides felis) 60298 OOO (Diaprepes abbreviatus) TCOO1 814 AATTTGTGTATGATTACTGGAGG 55904576 (Locusta migratoria) TCOO1 815 ACTGGAGGTCGTAACTTGGGGCGTGT 60298 OOO (Diaprepes abbreviatus)

TCOO1 816 ATGATTACTGGAGGTCGTAACTTGGGGCGTGT 73619372 (Aphis gossypii) 37804548 (Rhopalosiphum padi TCOO1 817 ATGCAAAGATTGATTAAAGTTGACGG 709 O9478 (Biphyllius lunatus)

TCOO1 818 ATTAAAGTTGACGGAAAAGTT 110763874 (Apis mellifera) US 9,528,123 B2 157 158 TABLE 4 -TC-continued Target SEQ ID NOSequence * Example Gi-number and species COO 819 ATTGAGAAAACTGGGGAATTCTTCCG 37952206 (Ips pini)

COO ATTGTTATGCAAAGATTGATTAAAGTTGACGGAAAAGT 709 O9486 (Mycetophagus quadripustulatus)

COO 821 CCAAGAAGCATTTGAAGCGTCT 55904580 (Locusta migratoria)

COO 822 CCAAGAAGCATTTGAAGCGTCTC 83935971 (Lutzomyia longipalpis)

COO 823 GCGCCCAAAGCATGGATGTTGGA 103790417 (Heliconius erato) 101.419954 (Plodia interpunctella)

COO 824 GGCCCCAAGAAGCATTTGAAGCGT 147OO 642 (Drosophila melanogaster)

COO 825 TGATTACTGGAGGTCGTAACTTGGGGCGTGT 7361.2212 (Aphis gossypii)

COO 826 TGTATGATTACTGGAGGTCGTAACTTGGGGCGTGT 709 O9478 (Biphyllius lunatus

COO 827 TTGATTTATGATGTTAAGGGA 773254.85 (Chironomus tentains

COO 828 TTGTGTATGATTACTGGAGGTCGTAA 603 0581.6 (Mycetophagus quadripustulatus)

COO2 829 AAAAACAAACGAGCGGCCATCCAGGC 1892O284 (Anopheles gambiae

COO2 83 O ATCGACCAAGAGATCCTCACAGCGAAGAAAAACGCGTCGAAA 75717966 (Tribolium cast aneum) AACAAACGAGCGGCCATCCAGGCC

COO2 831 CTCCAGCAGATCGATGGCACCCT 92475657 (Drosophila erecta) 1376322O (Drosophila melanogaster)

COO2 832 TCAAGAGGAAGAAACGCTACGAAAAGCAGCTCCAGCAGATC 75717966 (Tribolium cast aneum) GATGGCACCCTCAGCACCATCGAGATGCAGCGGGAGGCCCT CGAGGGGGCCAACACCAACACAGCCGTACT CAAAACGATGA AAAACGCAGCGGACGCCCT CAAAAATGCCCACCTCAACATG GATGTTGATGAGGT

CO1 O 833 AACCTCAAGTACCAGGACATGCCCGA 90973566 (Aedes aegypti)

CO1 O 834 AGCCGATTTTGTACAGTTATA 92.944 62O (Drosophila ananassae)

CO1 O 835 ATGGACACATTTTTCCAAATT 33427937 (Glossina morsitans

CO1 O 836 ATGGACACATTTTTCCAAATTTTGATTTTCCACGG 56151768 (Rhynchosciara americana)

CO1 O 837 CAAGTACCAGGACATGCCCGA 1891.1059 (Anopheles gambiae

CO1 O 838 CACATGCTGATGCGGGAGGACCTC 678.93321 (Drosophila pseudoobscura)

CO1 O 839 CCTCAAGTACCAGGACATGCCCGA 678.93324 (Drosophila pseudoobscura)

CO1 O 84 O TCAAGTACCAGGACATGCCCGA 678.93321 (Drosophila pseudoobscura)

CO1 O 841 TTCATGTACCATTTGCGCCGCTC 92952 825 (Drosophila ananassae

CO14 842 AAAATTCAGTCGTCAAACATGCTGAA 7616939 O (Diploptera punctata)

CO14 843 AACATGCTGAACCAAGCCCGT 87266590 (Choristoneura fumiferana) 103779114 (Heliconius erato)

CO14 844 CACAGCAACTTGTGCCAGAAAT 92.923 718 (Drosophila virilis)

CO14 845 GAGAAAGCCGAAGAAATCGATGC 773.2583 O (Chironomus tentains

CO14 846 GCCCGCAAACGTCTGGGCGAA 92232132 (Drosophila Willistoni)

CO14 847 TAAAAGTGCGTGAAGACCACGT 58371699 (Lonomia obligua)

CO15 848 ACACTGATGGACGGCATGAAGAA 78531609 (Glossina morsitans

CO15 849 ATCGGCGGTTGTCGCAAACAACT 6904417 (Bombyx mori)

CO15 850 CCCGATGAGAAGATCCGGATGAA 83922.984 (Lutzomyia longipalpis)

CO15 851 CTGCCCCGATGAGAAGATCCG 929 48836 (Drosophila ananassae

CO15 852 AACGAAACCGGTGCTTTCTTCTT 841.16975 (Dermatophagoides farinae US 9,528,123 B2 159 160 TABLE 4-MP

SEQ Target ID ID NO Sequence * Example Gi-number and species MPOO1 908 AAAGCATGGATGTTGGACAAA. 98.994282 (Antheraea my litta) 10878.9768 (Bombyx mori) 1099 78109 (Gryllius pennsylvanicus) 55904580 (Locusta migratoria)

MPOO1 909 AAAGCATGGATGTTGGACAAAT 773254.85 (Chironomus tentans) 37951951 (Ips pini) 60311985 (Papilio dairdanus) 30.031258 (Toxoptera citricida)

POO 910 AAGAAGCATTTGAAGCGTTTAAACGCACC 3658572 (Manduca sexta)

POO 911 AAGCATTTGAAGCGTTTAAACGC 103790417 (Heliconius erato) 22474.232 (Helicoverpa armigera) POO 912 AAGCATTTGAAGCGTTTAAACGCACC 25957217 (Carabus granulatus)

POO 913 AAGTCCGTACCGACCCTAATTATCCAGC 46994131 (Acyrthosiphon pisum) POO 914 ACGCACCCAAAGCATGGATGTT 46999 037 (Acyrthosiphon pisum)

POO 915 ACTATTAGATACGATATTGCA 46998791 (Acyrthosiphon pisum)

POO 916 ACTGGACCCAAAGGTGTGCCATTTTTAACTACTCATGATGGC 46.997137 (Acyrthosiphon pisum) CGTACTAT

POO 917 AGAAGCATTTGAAGCGTTTAAA 2762O566 (Anopheles gambiae)

POO 918 AGAAGCATTTGAAGCGTTTAAACGCACC 98.994282 (Antheraea my litta) POO 919 AGAAGCATTTGAAGCGTTTAAACGCACCCAAAGCATGGATGT 73 619191 (Aphis gossypii) TGGACAAAT

POO 92O AGTAAGGGAGTTAAATTGACTA. 46998791 (Acyrthosiphon pisum)

POO 921 ATACAAGTTGTGTAAAGTAAAG 29553519 (Bombyx mori) POO 922 ATGGATGTTATATCTATCCAAAAGACCAGTGAGCACTTTAGAT 46998791 (Acyrthosiphon pisum) TGATCTATGATGTGAAAGGTCGTTTCAC

POO 923 ATTGATCTATGATGTGAAAGGTCGTTTCAC 46999 037 (Acyrthosiphon pisum)

POO 924 CAAAAGACCAGTGAGCACTTTAGATTGAT 30.031258 (Toxoptera citricida) POO 925 CACAGAATTACTCCTGAAGAAGC 73 619191 (Aphis gossypii) POO 926 CACAGAATTACTCCTGAAGAAGCAAAATACAAG 46998791 (Acyrthosiphon pisum) 30.031258 (Toxoptera citricida) POO 927 CATCCAGGATCTTTTGATATTGTTCACATTAA 31364848 (Toxoptera citricida) POO 928 CATCCAGGATCTTTTGATATTGTTCACATTAAGGATGCAAATG 37804548 (Rhopalo siphum padi) AACATATTTTTGCTAC

POO 929 CATCTAAAATTTTGGATCATATCCGTTTTGAAACTGGAAACTT 46998791 (Acyrthosiphon pisum) GTGCATGAT

POO 930 CATTTGAAGCGTTTAAACGCACC 30.031258 (Toxoptera citricida)

POO 931 CATTTGAAGCGTTTAAACGCACCCAAAGCATGGATGTT 46998791 (Acyrthosiphon pisum) POO 932 CCAAAGCATGGATGTTGGACAA 9 O1381.64 (Spodoptera frugiperda) POO 933 CCAAGGAGTAAGGGAGTTAAATTGACTA. 73 615238 (Aphis gossypii) 31364848 (Toxoptera citricida) POO 934 CCCAAAGCATGGATGTTGGAC 10878.9768 (Bombyx mori)

POO 935 CCCAAAGCATGGATGTTGGACAA 50565112 (Homalodisca coagulata) 4892 7129 (Hydropsyche sp.) 76551269 (Spodoptera frugiperda) POO 936 CCCAAAGCATGGATGTTGGACAAA. 56085210 (Bombyx mori) 103792.451 (Heliconius erato) 1014 19954 (Plodia interpunctella) US 9,528,123 B2 161 162 TABLE 4 -MP - continued

SEQ Target ID ID NO Sequence * Example Gi-number and species POO 937 CCCAAAGCATGGATGTTGGACAAAT 22474O95 (Helicoverpa armigera)

POO 938 CGTCCAAGCACCGGTCCACACAAACT 4753.7863 (Acyrthosiphon pisum)

POO 939 CTGGAAACTTGTGCATGATAACTGGAGG 78524585 (Glossina morsitans)

POO 94 O GAAAGACATCCAGGATCTTTTGATATTGTTCACATTAAGGATG 46.997137 (Acyrthosiphon pisum) CAAATGAACATATTTTTGCTACCCGGATGAACAATGTTTTTAT TATTGGAAAAGGTCAAAAGAACTACATTTCTCTACCAAG

POO 941 GATCATATCCGTTTTGAAACTGGAAACTTGTGCATGAT 73 614725 (Aphis gossypii)

POO 942 GATGCAAATGAACATATTTTTGCTAC 31364848 (Toxoptera citricida) POO 943 GCACCCAAAGCATGGATGTTGGA 709094.86 (Mycetophagus quadripustulatus)

POO 944 GCACCCAAAGCATGGATGTTGGACAAAT 77329254 (Chironomus tentans) 6 O30542O (Mycetophagus quadripustulatus) POO 945 GGATCTTTTGATATTGTTCACAT 60303405 (Julodis onopordi) POO 946 GGATCTTTTGATATTGTTCACATTAAGGATGCAAATGAACATA 73 619191 (Aphis gossypii) TTTTTGCTAC

POO 947 GGCCCCAAGAAGCATTTGAAGCGTTTAA 14693528 (Drosophila melanogaster)

POO 948 GGGCGTGTTGGTATTGTTACCAACAG 31365398 (Toxoptera citricida) POO 949 GGGCGTGTTGGTATTGTTACCAACAGGGAAAG 73 61.2212 (Aphis gossypii) 37804548 (Rhopalo siphum padi)

POO 950 GGTACAAACTGGACCCAAAGG 6O297.572 (Diaprepes abbreviatus) POO 951 GTTTTTATTATTGGAAAAGGTCAAAAGAACTACATTTCTCT 73 619191 (Aphis gossypii) 31364848 (Toxoptera citricida) POO 952 TGAAGTATGCACTTACTGGTGC 73 619191 (Aphis gossypii) POO 95.3 TGTAAAGTAAAGAGGGTACAAACTGGACCCAAAGGTGT 73 619191 (Aphis gossypii)

POO 954 TGTGTAAAGTAAAGAGGGTACAAACTGGACCCAAAGGTGT 30.031258 (Toxoptera citricida) POO 955 TTCTTGCGTAATCGTTTGAAGTATGCACTTACTGGTGCCGAA 46998791 (Acyrthosiphon pisum) GT CACCAAGATTGTCATGCAAAGATTAATCAAGGTTGATGGC AAAGTCCGTACCGACCCTAATTATCCAGC

POO 956 TTGGAAAAGGTCAAAAGAACTACATTTCTCT 73 615060 (Aphis gossypii) POO 95.7 TTGGATCATATCCGTTTTGAAACTGGAAACTTGTGCATGAT 37804548 (Rhopalo siphum padi)

POO2 958 AAAAAAAATGGTACAACTAATAAACGAGCTGCATTGCAAGC 47537017 (Acyrthosiphon pisum)

POO2 959 AAGAAACGGTACGAACAACAA 15363283 (Apis mellifera) POO2 96.O ACAAGAATTTTTAGAAAAAAAAATTGAACAAGAAGTAGCGATA 47537017 (Acyrthosiphon pisum) GC

POO2 961 CAAATTGATGGTACCATGTTAACTATTGAACAACAGCG 47537017 (Acyrthosiphon pisum)

POO2 962 GAAGATGCGATACAAAAGCTTCGATCCAC 47537017 (Acyrthosiphon pisum) POO2 963 GAGTTTCTTTAGTAAAGTATTCGGTGG 110762 684 (Apis mellifera)

PO1 O 964 AAAAGATGATCCAAATAGTTT 110759 793 (Apis mellifera)

PO1 O 965 AAAATATTATTGATGGACACATTTTTCCATATTTTGATATTCCA 4752O567 (Acyrthosiphon pisum)

PO1 O 966 AATAGTCCTGATGAAACATCATATTATAG 4752O567 (Acyrthosiphon pisum)

PO1 O 967 CAAAAAGATGATCCAAATAGTTTCCGATTGCCAGAAAACTTCA 4752O567 (Acyrthosiphon pisum) GTTTATATCCACAGTTCATGTATCATTTAAGAAGGTCT CAATTT CTACAAGTTTTTAA

PO1 O 968 CAACATTCCAGTGGCTATAAACGAAT 4752O567 (Acyrthosiphon pisum)

PO1 O 969 CACATGTTGATGCGTGAAGATGTTAC 4752O567 (Acyrthosiphon pisum) US 9,528,123 B2 163 164 TABLE 4 -MP - continued

SEQ Target ID ID NO Sequence * Example Gi-number and species

PO1 O 97 O CCAATTCTGTATAGCTATAGTTTTAATGGTAGGCCAGAACCTG 4752O567 (Acyrthosiphon pisum) TACTTTTGGATACCAG

PO1 O 971 CCATCTCAAACACATAATAATATGTATGCTTATGGAGG 5581494.2 (Acyrthosiphon pisum)

PO1 O 972 CTCAAAACTCGATTCCCAATGCCTCGGTATATTGACACAGAA 5581494.2 (Acyrthosiphon pisum) CAAGGTGGTAGTCAGGCAAGATTTTTACTATGCAAAGT

PO1 O 973. GGTGATGGTGGAGCACCAGTTTTGACAGATGATGTAAGCTTG 5581494.2 (Acyrthosiphon pisum) CA

PO1 O 974. GTGGCTGCATACAGTTCATTACGCAGTA 28571527 (Drosophila melanogaster)

PO1 O 975 TAATGGCTCGTATGGTAGTGAACCGTGCTGAAACTGA 4752O567 (Acyrthosiphon pisum)

PO1 O 976 TATAGGCACATGTTGATGCGTGAAGAT 4O924332 (Bombyx mori)

PO1 O 977 TGGGCTGATCGTACGCTTATACGCTTGTGTCA 4752O567 (Acyrthosiphon pisum)

PO1 O 978 TTAGCTAGGAATTGGGCAGACCCTGT 4752O567 (Acyrthosiphon pisum)

PO16 979 AAACAAGATTTTGAGGAAAATGG 3550.8791 (Acyrthosiphon pisum)

PO16 98 O AACCTGGTAAATCAGTTCTTGA 3550.8791 (Acyrthosiphon pisum) PO16 981 AACGACGACATCACCCATCCTATTC 11024 O379 (Spodoptera frugiperda) 27372076 (Spodoptera littoralis) PO16 982 AATTTAGCTAATGATCCTACTATTGA 1536 6446 (Apis mellifera)

PO16 983 ACTATGCCTAACGACGACATCACCCATCC 237458 (Heliothis vires cens) PO16 984 ATAGTATTTGCTGCTATGGGTGTTAATATGGAAAC 3 O 124460 (Toxoptera citricida)

PO16 985 CAAATTTGTAGACAAGCTGGTCT 10302O368 (Tribolium cast aneum) PO16 986 CATGAAGACAATTTTGCTATAGTATTTGCTGCTATGGGTGTTA 3550.8791 (Acyrthosiphon pisum) ATATGGAAAC

PO16 987 CCGATAGATAAAGGACCTCCTATTTTGGCTGAAGATTATTTGG 3550.8791 (Acyrthosiphon pisum) ATATTGAAGGCCAACCTATTAATCCATA

PO16 988 CCTATTTTGGCTGAAGATTAT 55905051 (Locusta migratoria) PO16 989 CGTATCATTACACCACGTCTTGCTTTAACTGCTGCTGAATTTT 3 O 124460 (Toxoptera citricida) TAGCTTA

PO16 990 CGTCTTGCTTTAACTGCTGCTGAATTTTTAGCTTA 3550.8791 (Acyrthosiphon pisum) PO16 991 GAAGAAGTACCTGGGCGTCGTGGTTTCCCTGGTTACATGTAC 3 O 124460 (Toxoptera citricida) AC PO16 992 GAAGGAAGAAATGGTTCTATCACACAAATACCTATTTTAACTA 3 O 124460 (Toxoptera citricida) TGCCTAA PO16 993 GAAGGAAGAAATGGTTCTATCACACAAATACCTATTTTAACTA 73 6153 O7 (Aphis gossypii) TGCCTAACGA

PO16 994 GATTTAGCTACAATTTATGAACG 3 O 124460 (Toxoptera citricida)

PO16 995 GCCAGATTCTTTAAACAAGATTTTGAGGAAAATGG 3 O 124460 (Toxoptera citricida)

PO16 996 GCTATGGGTGTTAATATGGAAAC 754.69507 (Tribolium castaneum)

PO16 997 GCTGCAGGTTTACCACATAATGAGATTGCTGCTCAAATTTG 3550.8791 (Acyrthosiphon pisum)

PO16 998 GCTGGGCGTGTAGAAGGAAGAAATGGTTCTATCACACAAATA 55813096 (Acyrthosiphon pisum) CCTATTTTAACTATGCCTAACGA

PO16 999 GGTTACATGTACACCGATTTAGCTACAATTTATGAACG 55813096 (Acyrthosiphon pisum) 73 6153 O7 (Aphis gossypii)

PO16 1 OOO GTGGACAAAAAATTCCAATATTTTC 55813096 (Acyrthosiphon pisum)