US 2015025.9700A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0259700 A1 Elling et al. (43) Pub. Date: Sep. 17, 2015 (54) TRANSGENC PLANTS WITH RNA Publication Classification INTERFERENCE-MEDIATED RESISTANCE AGAINST ROOT-KNOT NEMATODES (51) Int. Cl. CI2N 5/82 (2006.01) (71) Applicant: WASHINGTON STATE CI2N IS/II3 (2006.01) UNIVERSITY, PULLMAN, WA (US) (52) U.S. Cl. CPC .......... CI2N 15/8285 (2013.01); C12N 15/I 13 (72) Inventors: Axel A. Elling, Pullman, WA (US); (2013.01); C12N 23 10/141 (2013.01); C12N Charles R. Brown, Pullman, WA (US) 2310/531 (2013.01) (21) Appl. No.: 14/626,070 (57) ABSTRACT Transgenic plants that are stably resistant to the nematode (22) Filed: Feb. 19, 2015 Meloidogyne Chitwoodi are provided, as are methods of mak ing such transgenic plants. The transgenic plants (such as Related U.S. Application Data potatoes) are genetically engineered to express interfering (60) Provisional application No. 61/948,761, filed on Mar. RNA that targets the Meloidogyn effector protein 6, 2014. Mc16D1OL. Patent Application Publication US 2015/025.9700 A1 OIGI9I?JÄI TOIGI9IDJÄI OIGI9I?IAI TOICI9IDJÄI OIC19I?IN TOICI9IDWI Patent Application Publication Sep. 17, 2015 Sheet 2 of 11 US 2015/025.9700 A1 e h; Figure 2A Figure 2B Figure 2C Figure 2D Figure 3 Patent Application Publication Sep. 17, 2015 Sheet 3 of 11 US 2015/025.9700 A1 COL E2 D1 D2 D4 COL E2 D1 D2 D4 Figure 4A Figure 4B 25000 ;20000 15000 s 10000 2 5000 DES E29 D54 D56 D57 DES E29 D54 D56 D57 Figure 5A Figure 5B 60 1800 50 3. 1600 1400 1200 1000 30 b 8OO 2O 6OO 400 1O 2OO O DES E29 D54 D56 D57 DES E29 D54 D56 D57 Figure 5C Figure 5D Patent Application Publication Sep. 17, 2015 Sheet 4 of 11 US 2015/025.9700 A1 Arabidopsis Potato COL E2 D1 D2 D4 D54 D56 D57 DES Figure 6A Arabidopsis Potato COL E2 D1 D2 D4 D54 D56 D57 DES 2OOnt ) 10Ont - ) Figure 6B Arabidopsis POtatO COL E2 D1 D2 D4 D54 D56 D57 DES Figure 6C Patent Application Publication Sep. 17, 2015 Sheet 5 of 11 US 2015/025.9700 A1 1.8 a c 1.2 1.6 C 1 it do 14 3 a 21.2 a 92 0.8 Q81 bC b : 0.6 to - 0.2. dcus' O.2 Y O Y O DES E29 D54 D56 D57 DES E29 D54 D56 D57 Figure 7A Figure 7B F?h & 3G?) ?h SS() onY 5 g?h 3() ()& 2() Ndid S ?)S 3?) 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N 3' Y--N-m-- - - -1 -w- 11 12 13 14 20N transcription N-Y-N--m-1-Y- 21 22 23 24 ! folded shRNA 32 30 31 35 33 5 334 3' Figure 14 US 2015/025.9700 A1 Sep. 17, 2015 TRANSGENC PLANTS WITH RNA has been estimated that as little as one juvenile per 250 g soil INTERFERENCE-MEDIATED RESISTANCE at the beginning of the growing season can lead to a total loss AGAINST ROOT-KNOT NEMATODES of marketability at harvest. To limit spread, M. Chitwoodi has been designated as a quarantine pest, which has a significant CROSS-REFERENCE TO RELATED impact on the international trade of potato tubers. APPLICATIONS 0009 Meloidogyne spp. are obligate parasites that depend 0001. This application claims benefit of U.S. provisional on their host plant for survival. The pathogenic part of the life patent application 61/948,761, filed Mar. 6, 2014, the com cycle of root-knot nematodes begins with the second-stage plete contents of which is hereby incorporated by reference. juveniles (J2), which are the infective life stage and invade plant roots, rhizomes and tubers. The J2 migrate intercellu STATEMENT OF FEDERALLY SPONSORED larly through host tissue until they become sedentary and RESEARCH AND DEVELOPMENT induce the formation of giant cells, which constitute the nematodes feeding site and sole source of nutrition. The 0002 This invention was made with government support exact mechanisms that lead to the formation and maintenance undergrant number 58-5354-1-467 awarded by United States of giant cells are unknown, but generally it is thought that Department of Agriculture/Agricultural Research Service. secretory proteins from the nematode esophageal gland cells, The government has certain rights in the invention. i.e., effectors, play a key role in the underlying processes. After a feeding site has been established, the J2 increase in SEQUENCE LISTING size and undergo Subsequent molts into third- and fourth 0003. This application includes as the Sequence Listing stage juveniles (J3, J4). After a final molt they develop into the complete contents of the accompanying text file “Sequen adult females or males. The females remain sedentary, but the ce.txt, created Feb. 16, 2015, containing 6.14 kilobytes, males regain their motility, exit the plant tissue and fertilize hereby incorporated by reference. the females. Many Meloidogyne spp. are parthenogenic and not all species form males. M. Chitwoodi reproduces by fac DESCRIPTION ultative meiotic parthenogenesis. Adult females deposit eggs into egg masses, a gelatinous matrix that protects eggs from BACKGROUND OF THE INVENTION desiccation. Inside the eggs, first-stage juveniles (J1) develop and molt into J2, which hatch under favorable conditions that 0004. 1. Field of the Invention are mostly dictated by soil moisture and temperature. 0005. The invention generally relates to stable transgenic 0010 Nematode control in most potato growing areas is plants that are resistant to the nematode Meloidogyne Chit based on routine applications of synthetic nematicides. This woodi. In particular, the invention provides stable transgenic practice not only is costly but also potentially harmful to the plants (such as potatoes) in which the Mc16D10L gene, environment because Some products have been linked to which encodes a putative M. Chitwoodi effector protein, is negative effects on the atmosphere's OZone layer. Host resis targeted using RNA interference. tance against root-knot nematodes would be an ideal control 0006 2. BACKGROUND OF THE INVENTION strategy, but is difficult to achieve. In spite of the extremely 0007. The potato (Solanum tuberosum L.) is the most rich genetic resources found in Solanum sect. Petota, which important non-cereal food crop worldwide and makes up the consists of wild and cultivated potatoes and includes up to 232 staple diet of over 1 billion people. Global potato production tuber-bearing and non-tuber-bearing species, exploiting this per year has been estimated at over 374 million metric tons in 2012, but varies widely on a country-by-country basis and genetic diversity and introducing M. Chitwoodi resistance into depends on local growing conditions and agricultural prac cultivated potato (S. tuberosum ssp. tuberosum) has proven to tices. The highest yields are reached in Western Europe and be challenging. In spite of over two decades of breeding the USA at about 50 t/ha, whereas average yields in Africa efforts no commercial potato cultivar with resistance to M. and most of Asia are no higher than 13 t?ha. Potato production Chitwoodi is available to date. The R gene from S. bulb has increased dramatically over the past two decades and Ocastanurn has been used to develop advanced S. tuberosum developing countries now account for most of the market breeding clones, but M. Chitwoodi populations are very vari volume, which underscores the importance of this crop for able and some isolates are able to overcome the R gene, global food security. presenting a significant problem for traditional resistance 0008 Potatoes are attacked by a wide variety of diseases breeding. Furthermore, hairy nightshade (S. Sarrachoides and pests that can reduce tuber yield and quality significantly. Sendtn.), a common weed in potato producing areas in the Plant-parasitic nematodes area major threat to potato produc western USA and a host for M, Chitwoodi, was found to tion worldwide, with root-knot (Meloidogyne spp.) and cyst undermine resistance undergreenhouse and field conditions nematodes (Globodera spp.) being the most widespread and when it co-occurs with potato breeding lines carrying the causing most of the damage.