US 2011 0099668A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0099668A1 Singh et al. (43) Pub. Date: Apr. 28, 2011 (54) EXPRESSING GLKN PLANTS Publication Classification (51) Int. Cl. Inventors: Jasbir Singh, Ottawa (CA); AOIH 5/00 (2006.01) (76) CI2N 5/82 (2006.01) Ghislaine C. Allard, Val des Monts CI2N 15/63 (2006.01) (CA); Leonid V. Savitch, Kanata AOIH I/00 (2006.01) (CA); Rajagopal Subramaniam, CI2N 5/04 (2006.01) Ottawa (CA) C7H 2L/04 (2006.01) (52) U.S. C. ... 800/279; 536/23.1; 800/301; 435/320.1; (21) Appl. No.: 12/151,046 435/419:435/412: 435/.414; 435/417; 435/416; 800/278; 800/298 (22) Filed: May 1, 2008 (57) ABSTRACT The present invention provides, in part, GLK1 nucleic acid Related U.S. Application Data molecules and polypeptides that can be used to confer resis tance to a pathogen in a plant. The present invention also (60) Provisional application No. 60/915,294, filed on May provides methods of detecting disease resistance genes and 1, 2007. plants. Pyu I (8945) EcoRI(876), SacI(8764) Snalt 8752) : xnal $750. Bam H. (8745) 1. Ahal (8739), W Scal (4) SalI (8733). 1 Scal (14) Psi (8731), \ | Scal (24) HindIII (8715), . Scal(34) Psil (8707) 2x P35s NotI(882) EcoRV (8045) XhoI (7948) s \,w \ EW onhancern bar oRF --- Sali (7534). Pyuli (7307) - Koni (7289) pTF101.1 Tvsp - Notl (2415) PyuII (6596) 25 (64.27). LB ClaI(3037) Bell (6190) Bel(5703) ApaL (5626) / Noti (3705) aad A ApaL (4532) ApaL (4034) pBR322 Patent Application Publication Apr. 28, 2011 Sheet 3 of 20 D E E E De Vis Avt t e oss A Figure 1D Patent Application Publication Apr. 28, 2011 Sheet 4 of 20 US 2011/0099668A1 NL Athaliana GKAAK2020 Wheat GK1 AO089 A Rice GLKAAK50393 A liaize. GKAAKO 39) th N G R K. N. G Wheat GK1 ABL 0089 Rice GLKAAK5393 Maize GKAAKS392 Athaiiana GK2AAK2012 Maize GK2 Rice GK2 AAKS-394 40 420 43 Figure 1D Patent Application Publication Apr. 28, 2011 Sheet 9 of 20 US 2011/0099668A1 2-6 5-7 Figure 2A WT WT 2-6 5-7 Figure 2B Patent Application Publication Apr. 28, 2011 Sheet 10 of 20 US 2011/0099668A1 Figure 2C Patent Application Publication Apr. 28, 2011 Sheet 11 of 20 US 2011/0099668A1 Patent Application Publication Apr. 28, 2011 Sheet 12 of 20 US 2011/0099668A1 At2g20570 GLKl At 1g09350 Galactino synthase At 1g09500 Cinnamyl alcohol dehydrogenase Atlg3530 PR10 Atlg73260 Trypsin protease inhibitor At lg77960 Phox/Bem p domain protein Atg8060 Lactoylglutathione lyase At2g4 1050 PQ-loop repeat protein At3g28740 Cytochrome P450 At4g37990 ELI3-2 Atlg18870 Isochorismate synthase At4g21910 MATE efflux protein At2g 14610 PR-1 At lg.02450 NPR1 Actin Figure 4 Patent Application Publication Apr. 28, 2011 Sheet 14 of 20 US 2011/0099668A1 Hindl EcoRV -Daz Bluescript Actin promoter ex intron aGLK1 noster Figure 6 TaGLK1 Oe WT AtGLK1 Oe Figure 7 Patent Application Publication Apr. 28, 2011 Sheet 15 of 20 US 2011/0099668A1 i Patent Application Publication Apr. 28, 2011 Sheet 16 of 20 US 2011/0099668A1 LK1 is Null set days postfinoculation Figure 9 Patent Application Publication Apr. 28, 2011 Sheet 17 of 20 US 2011/0099668A1 Figure 10 Patent Application Publication Apr. 28, 2011 Sheet 18 of 20 US 2011/0099668A1 WT 1 2 3 TaGLK1 Oe AtGLK1 Oe Figure 11 Figure 12 Patent Application Publication Apr. 28, 2011 Sheet 19 of 20 US 2011/0099668A1 PvulI(8945) EcoRI (8766) SacI (8764) Koni (8758) SmaI(8752). \ Anal E. RB1R25 (90.51) Ban H Xbal (8739) l Scal (4) SalI(8733) \ Scal (14) Psil (8731), A Scal (24) Hindu (8715) Scal(34) Psil (8707) \ 2x P35s - NotI (882) EcoRV (8045) XhoI (7948) \ w N \ \ Y a re EV enhancern 1 bar o RF Sali (7534). Y. p VS 1 Pvull (7307) ...-Se \ \- Koni (7289) pTF101.1 Twsp y itsy Up --mirNotI (2415) PyuII (6596) T L25 (6427) - B Clal (3087) Bcl(6.190) Bel(5703) - / ApaLI (5626) / | | \, Noti (3705) A. \ aad A | V ApaL (4532) s ApaL (4034) p BR322 FIG. 13A Patent Application Publication Apr. 28, 2011 Sheet 20 of 20 US 2011/0099668A1 LB HindIII Xbal Kpni Bam Hl RB Bar ORF 2X p35S pTF101.1 2X p35S AtGLK Oster pTF101.1 -70S-AtGLK1-noster 1549 kb Figure 13B US 2011/0099668A1 Apr. 28, 2011 EXPRESSING GLKIN PLANTS that it may play a role in temperature stress response and photosynthetic adaptation (Savitch, L. V., et al., 2005, Plant FIELD OF INVENTION Cell Physiol. 46:1525-1539). SUMMARY OF THE INVENTION 0001. The present invention relates to expressing GLK in plants. More specifically, the invention provides GLK1 mol 0006. The present invention relates to expressing GLK in ecules for conferring disease resistance to plants, increasing plants. More specifically, the invention provides GLK1 mol nitrogen status in plants, or both. ecules for conferring disease resistance to plants, increasing nitrogen status in plants, or both. 0007. The present invention provides a method of confer BACKGROUND OF THE INVENTION ring resistance to a pathogen in a plant, the method compris ing: 0002 The economic impact of plant pathogens in relation 0008 i) introducing a nucleic acid molecule encoding a to agriculture and horticulture is considerable. For example, GLK1 polypeptide or fragment or variant thereof into a plant Fusarium Head Blight (FHB) is a major disease of concern cell to produce a transgenic plant cell. Such that the nucleic for Canadian cereals. acid molecule is positioned for expression in the transgenic 0003 Global gene expression analyses suggest that plant cell; and induced disease resistance can result from pathogen or patho 0009 ii) growing a transgenic plant from the transgenic gen derived molecule-induced reprogramming of gene net plant cell, works (Liet al., 2006; Bae et al., 2006). The role of transcrip wherein the GLK1 polypeptide or fragment or variant thereof tion factors in the modulation of these networks has been is over-expressed relative to wild type GLK1 at least 2 fold, whereby the 2 fold over-expression is sufficient to confer Suggested for WRKY domain containing trans-factors (Liet resistance to the pathogen in the plant. al, 2006) and in the slh1 mutation (Naoutoshi et al., 2005). 0010. The GLK1 may comprises the nucleic acid 0004 Non-host resistance is the term used to describe the sequence set forth in SEQ ID NO: 1, a nucleic acid that phenomenon where all members of a plant species exhibit exhibits an identity of from about 70 to about 100% with SEQ resistance to all members of a given pathogen species. Non ID NO:1, or a nucleic acid molecule that hybridizes under host resistance is thought to be multigenic; therefore the conditions of high Stringency to the nucleic acid sequence set inactivation of a single gene may not be sufficient to render a forth in SEQID NO: 1. Furthermore, the GLK1 polypeptide plant Susceptible to a pathogen. Non-host resistance has been may comprise an amino acid sequence Substantially identical studied in Salicylate hydroxylase (NahG) over-expressing to the amino acid sequence set forth in SEQ ID NO: 2. plants, which convert Salicylic acid to catechol and are defec Furthermore, the plant may be selected from the group con tive in non-host response to pathogen. NahG plants expresses sisting of canola, Brassica spp., maize, tobacco, alfalfa, the salicylic acid degrading enzyme salicylate hydroxylase, potato, ginseng, pea, oat, rice, soybean, wheat, barley, Sun resulting in indirect and side effects in plant defense (Hecket flower, and cotton. The pathogen may be selected from the al, 2003; van Wees and Glazebrook 2003). Furthermore, it is group consisting of Fusarium graminearum, Sclerotinia scle known that part of non-host resistance is derived from “prim rotium, Puccinia graminis, Puccinia triticinia, Puccinia ing of the plant to produce a readiness to infection. recondite, and Phytopthora infestans. 0011. The present invention also provides a pathogen 0005. The maize Golden2-like or GLK genes encode pro resistant plant comprising a GLK1 nucleic acid molecule that teins belonging to a class of GARP domain of transcriptional encodes a GLK1 polypeptide, wherein expression of the activators (Hall, L. N. et al., 1998, Plant Cell 10:925-936; GLK1 produces at least 2 fold greater GLK1 polypeptide Fitter, W., et al., 2002, The Plant Journal 31: 713-727: relative to a wild-type plant. Rossini, D. L., et al., 2001, Plant Cell 13: 1231-1244: US 0012. The present invention also provides a genetic con 2007/0022495)There are 56 predicted members of the GARP struct comprising a regulatory region operably linked to a domain containing transcriptional activators in Arabidopsis nucleic acid molecule encoding a GLK1 polypeptide or frag (Riechmann, J. L., et al., 2000, Science 290: 2105-2110: ment or variant thereof. The nucleic acid molecule may com which is incorporated herein by reference) and are involved in prise a nucleic acid sequence as set forth in SEQID NO: 1, a plant specific processes. These include the G2 or GLK pro nucleotide sequence that exhibits from about 70% to about teins that are involved in chloroplast development (Cribb, L. 100% sequence identity with the sequence of SEQID NO:1, et al., 2001, Genetics 159:787-797:Yasumura, Y., et al., 2005, or a nucleotide sequence that hybridizes to the nucleic acid Plant Cell 17: 1894-1907), the Arabidopsis type-B phospho sequence set forth in SEQID NO: 1 under conditions of high relay response regulators (ARR family) that mediate cytoki stringency. Furthermore, the GLK1 polypeptide may com nin signaling (Sakai, H., et al.