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(12) United States Patent (10) Patent No.: US 8,853,493 B2 Ye Et Al

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(12) United States Patent (10) Patent No.: US 8,853,493 B2 Ye Et Al USOO8853493B2 (12) United States Patent (10) Patent No.: US 8,853,493 B2 Ye et al. (45) Date of Patent: Oct. 7, 2014 (54) VIRUS INDUCED GENESILENCING (VIGS) (56) References Cited FOR FUNCTIONAL ANALYSIS OF GENES IN COTTON U.S. PATENT DOCUMENTS 6,369,296 B1* 4/2002 Ratcliff et al. ................ 800,278 (75) Inventors: Jian Ye, Singapore (SG); Nam Hai 7,476.780 B2 * 1/2009 Ryu et al. ...................... 800,285 Chua, Singapore (SG); Jing Qu, 2003/0182684 A1 9/2003 Dinesh Kumar et al. Singapore (SG); Yun Feng Geng, Singapore (SG); Yun Ping Bu, FOREIGN PATENT DOCUMENTS Singapore (SG) WO 2005,103267 A2 11/2005 (73) Assignee: Temasek Life Sciences Laboratory WO WO 2005103267 A2 * 11, 2005 Limited. Singapore (SG) OTHER PUBLICATIONS Notice: Subject to any disclaimer, the term of this Lee et al., 2007, Annals of Botany, 100: 1391-1401.* (*) Ratcliff, F. et al., “Tobacco rattle virus as a vector for analysis of gene patent is extended or adjusted under 35 function by silencing.” The Plant Journal, 2001, vol. 25, No. 2, pp. U.S.C. 154(b) by 247 days. 237-245. Hileman, L.C. et al., “Virus-Induced Gene Silencing is an Effective (21) Appl. No.: 13/376,902 Tool for Assaying Gene Function in the Basal Eudicot Species Papaver somniferum (Opium Poppy).” The Plant Journal. (2005), (22) PCT Fled: Jun. 10, 2010 vol. 44, No. 2, pp. 334-341, (C) 2005 Blackwell Publishing Ltd., XP-002689517. PCT NO.: Caplan, J. et al., “Using Viral Vectors to Silence Endogenous Genes.” (86) Unit 161.6. Current Protocols in Microbiology, vol. Suppl. 1. (Jun. 1, S371 (c)(1), 2006), pp. 1616, 1-161, 6.13, (C) 2006 by John Wiley & Sons, Inc., Dec. 8, 2011 XP-002689519. (2), (4) Date: EP Communication, Extended European Search Report, dated: Jan. PCT Pub. No.: WO2O10/144,058 22, 2013, Reference: RSC/P43020EP; Application No. 10786471.2- (87) 2403 ? 2440666 PCT/SG2010000220, Applicant: Temasek Life Sci PCT Pub. Date: Dec. 16, 2010 ences Laboratory Limited, 9 pages. (65) Prior Publication Data * cited by examiner US 2012/OO96595 A1 Apr. 19, 2012 Primary Examiner — David H Kruse Assistant Examiner — Jason DeVeau Rosen (74) Attorney, Agent, or Firm — Rothwell, Figg, Ernst & Related U.S. Application Data Manbeck P.C. (60) Provisional application No. 61/185,631, filed on Jun. 10, 2009. (57) ABSTRACT The invention relates to the functional analysis of genes in (51) Int. C. cotton by employing the Virus Induced Gene Silencing AOIH I/O (2006.01) (VIGS) method. More specifically this method induces gene CI2N 15/82 (2006.01) silencing in cotton with the help of the Tobacco Rattle Virus (52) U.S. C. (TRV) vectors RNA1 and RNA2 and phenotypic effects on CPC ................................. CI2N 15/8218 (2013.01); the cotton plant can be analysed Moreover this invention also CI2N 15/8261 (2013.01) provides transient expression vector TRV RNA2 in order to USPC ............ 800/294; 800/285; 800/290: 436/469 transiently express genes in cotton plants and plant tissue (58) Field of Classification Search under the influence of a strong Subgenomic promoter. None See application file for complete search history. 13 Claims, 27 Drawing Sheets U.S. Patent Oct. 7, 2014 Sheet 1 of 27 US 8,853,493 B2 Cotton Virus-induced Gene Silencing Method Different kind of methods to 4-14 days clone gene fragments into seedling TRW vector (2-7 days) Agrobacterium Agrobacterium infiltration (1-3 mins) 1 month Keep trees in 25 C 7-14 days Phenotypes RNA and Chemical Other analysis observation protein level Compound (microscopy) analysis analysis (GC, HPLC, MASS) Figure 1 U.S. Patent Oct. 7, 2014 Sheet 2 of 27 US 8,853,493 B2 Z9.In314 U.S. Patent Oct. 7, 2014 Sheet 3 of 27 US 8,853,493 B2 N1 s O C s a s r 25 R 9 E 3 5 S g g g is is w (%) seAee ouesAsu ea lossadxe angee U.S. Patent Oct. 7, 2014 Sheet 4 of 27 US 8,853,493 B2 3 (%) saaeelalues/su ? eAe-uossada elee CD i s U.S. Patent Oct. 7, 2014 Sheet 5 of 27 US 8,853,493 B2 F 5 1gure U.S. Patent Oct. 7, 2014 Sheet 6 of 27 US 8,853,493 B2 AtAS1 Gh AS1 NtAS1 SkARP Consensus AtAS1 GEKSGSL GhASl GEKSGS) NtAS SkARP Consensus At AS Gh AS1 NtAS1 FERE NNE SkARP Rako Consensus AtAS1 Gh AS1 NtAS1 ASN SkARP SA Consensus AtAS1 Gh AS1 NtAS1 SkARP SDTDTGTHFNNNK Consensus 251 300 At AS1 - - - ---ayies.MSSS--G-SSESSSLSGSELEEGHRiiWiDEKKEAA Gh AS1 GNRBG--FERSEN LESEI NtAS1 ----e LSSFESHGVPECGNPE, ELSEE SkARP KVSTra PKDDEFENEINS DISPGEI Consensus AtAS1 GhAS1 NtAS1 Easy SkARP EE Consensus AtAS1 Gh AS1 35EORGGRPR NtAS1 gEQMGGQSREA SkARP Consensus 401 AtASl GhAS1 NtAS SkARP RGMNSPA Consensus Figure 6 U.S. Patent Oct. 7, 2014 Sheet 7 Of 27 US 8,853,493 B2 89InãIJ U.S. Patent US 8,853,493 B2 69InãIAI pn8 U.S. Patent Oct. 7, 2014 Sheet 9 of 27 US 8,853,493 B2 Original After stained With DMACA Enlarged U.S. Patent Oct. 7, 2014 Sheet 10 of 27 US 8,853,493 B2 stained leaf stained bud Figure 11 U.S. Patent US 8,853,493 B2 U.S. Patent Oct. 7, 2014 Sheet 12 of 27 US 8,853,493 B2 U.S. Patent Oct. 7, 2014 Sheet 13 Of 27 US 8,853,493 B2 Figure 14 1dpa X OO 1dpa X 200 Figure 15 U.S. Patent Oct. 7, 2014 Sheet 14 of 27 US 8,853,493 B2 A Systemic leaves B 0.2 Figure 17 U.S. Patent Oct. 7, 2014 Sheet 15 Of 27 US 8,853,493 B2 Figure 18 U.S. Patent Oct. 7, 2014 Sheet 16 of 27 US 8,853,493 B2 U.S. Patent Oct. 7, 2014 Sheet 17 Of 27 US 8,853,493 B2 Figure 20 U.S. Patent Oct. 7, 2014 Sheet 18 of 27 US 8,853,493 B2 U.S. Patent Oct. 7, 2014 Sheet 19 Of 27 US 8,853,493 B2 U.S. Patent Oct. 7, 2014 Sheet 20 Of 27 US 8,853,493 B2 Z S31I s31-I U.S. Patent Oct. 7, 2014 Sheet 21 of 27 US 8,853,493 B2 U.S. Patent Oct. 7, 2014 Sheet 22 of 27 US 8,853,493 B2 VIGS Of ACtin 1 M WT 1+2 1 2 3 4. 5 A. Tubulin Primer 1kb -> 500bp 200bp l 1+2 WGS- IGS-2 WIGS-3 WIGS-4 WIGS-5 Figure 28 U.S. Patent Oct. 7, 2014 Sheet 23 Of 27 US 8,853,493 B2 14 12 10 g 8 t 8 4 2 O 1+2 WIGS-1 WIGS-2 WIGS-3 WIGS-4 WIGS-5 Figure 29 7 8 5 s 3 : 2 l O 1+2 WIGS-1 WIGS-2 WIGS-3 WIGS-4 WIGS-5 Figure 30 U.S. Patent Oct. 7, 2014 Sheet 24 of 27 US 8,853,493 B2 3 1+2 WIGS-1 WIGS-2 WIGS-3 WIGS-4 WIGS-5 Figure 31 & 88: : 8& : 383 s - MSS- WGS-2 VGS-3 WGS- WGS-5 Figure 32 U.S. Patent Oct. 7, 2014 Sheet 25 Of 27 US 8,853,493 B2 -2 WGS-1 WGS- GS-3 WGS-4 WGS-5 Figure 33 1948 7522 1 S enp +2 WGS- W1GS-2 WGS-3 WGS-4 WGS-5 Figure 34 U.S. Patent Oct. 7, 2014 Sheet 26 of 27 US 8,853,493 B2 1-2 WGS-1 WGS-2 WGS-3 VGS-4 WGS-5 Figure 35 O 1 6 O O4. 1 WGS- WGS-2 WGS-3 WGS-4 VGS-5 Figure 36 U.S. Patent Oct. 7, 2014 Sheet 27 Of 27 US 8,853,493 B2 Misc Feature 4 Sna BI (9628) Misc Feature 3 TRV2-MF3 Sna I (9247) Ala I (9245) Xma I (9245) ti R Nco I (9208) \\ Xba I (9196) \ Eco RI (919O) Misc Feature 2 Misc Feature 2 TRV2-MF1, Sph I (490) Sna BI (8962) Ala I (508) M N Bgl II (8744) Ala I (1138) CP STRV2001 Misc Feature 3 Misc Feature 1/ p . Nde I(2038) ; 9696 bp . Nde I (8298) f i N. Aua I (2269) Bgl II (8193) -2 Ala I (2749) HindIII (6786) A Misc Feature 5 SNhe I(3493) Bisp HI (5964) Y.. - ... : Rep. Origin 1 CDS 1 g y- Cia I (3573) / Apa LI (5O18) \ Apa LI(452O) Rep. Origin 2 Nde I (4527) Misc Feature 4 Figure 37 US 8,853,493 B2 1. 2 VIRUS INDUCED GENESILENCING (VIGS) Cotton is an important crop that is widely grown and is FOR FUNCTIONAL ANALYSIS OF GENESN used to produce both natural textile fiber and cotton seed oil. COTTON Cotton fiber is a model system for the study of cell elongation and cell wall and cellulose biosynthesis. And it is unicellular, CROSS-REFERENCE TO RELATED therefore cell elongation can be evaluated independently APPLICATION from cell division. One of the most significant benefits for using cotton fiberas a model system for plant development is The present application is a national stage filing under 35 that a culture method for cotton ovules was perfected by U.S.C. S371 of PCT/SG2010/000220, filed on 10 Jun. 2010 Beasley and Ting (1973). which in turn claims priority to U.S. provisional patent appli 10 Gossypium includes approximately 45 diploid (2n=2x=26) and five tetraploid (2n=4x=52) species, all exhibiting disomic cation Ser. No. 61/185,631 filed on 10 Jun.
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