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(12) United States Patent (10) Patent No.: US 6,852,907 B1 Padidam Et Al

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(12) United States Patent (10) Patent No.: US 6,852,907 B1 Padidam Et Al USOO6852907B1 (12) United States Patent (10) Patent No.: US 6,852,907 B1 Padidam et al. (45) Date of Patent: Feb. 8, 2005 (54) RESISTANCE IN PLANTS TO INFECTION (56) References Cited BY SSDNAVIRUS USING INOVRIDAE PUBLICATIONS VIRUS SSDNA-BINDING PROTEIN, COMPOSITIONS AND METHODS OF USE (Plant Virology, Matthews, R.E.F. 3rd Ed., 1991, Academic Press, San Diego, Calif, p. 424).* (75) Inventors: Malla Padidam, Lansdale, PA (US); Padidam, et al., A phage single-stranded DNA (ssDNA) Roger N. Beachy, St. Louis, MO (US); binding protein complements SSDNA accumulation of a Claude M. Fauquet, Del Mar, CA (US) geminivirus and interferes with viral movement, 1999, J. Virol.., 73(2):1609–1616. 73) AssigSCC Thee Scripps RResearch h Institute,Insti LLa Padidam, et al., Tomato leaf curl geminivirus from India has Jolla, CA (US) a bipartite genome and coat protein is not essential for c: NotiOtice: Subjubject to anyy disclaimer,disclai theh term off thisthi infectivity, 1995, J. Gen. Virol, 76:25-35. patent is extended or adjusted under 35 Horsch, et al., A Simple and general method for transferring genes into plants, 1985, Science, 227: 1229-1231. U.S.C. 154(b) by 0 days. Sanford, et al., Optimizing the biolistic process for different (21) Appl. No.: 09/622,500 biological applications, 1993, Meth. Enzymol., 217:483-509. (22) PCT Filed: Mar. 3, 1999 Bates, Electroporation of plant protoplasts and tissues, 1995, (86) PCT No.: PCT/US99/04716 Meth. Cell Biol., 50:363-373. Timmermans, et al., Geminiviruses and their uses as extra S371 (c)(1), chromosomal replicons, 1994, Annu. Rev. Physiol. Plant (2), (4) Date: Aug. 17, 2000 Mol. Biol. 45:79-112. (87) PCT Pub. No.: WO99/45101 * cited by examiner PCT Pub. Date: Sep. 10, 1999 Primary Examiner Elizabeth McElwain Related U.S. Application Data Assistant Examiner-Georgia L Helmer (60) Provisional application No. 60/076,627, filed on Mar. 3, (74) Attorney, Agent, or Firm Thomas Fitting; Michael J. 1998. McCarthy (57) ABSTRACT (51) Int. Cl." ........................ C12N 15/82; C12N 15/00; The invention describes methods for producing plant resis AO1H 1/00; CO7H 21/04; C12P 21/06 tance to a SSDNA virus, particularly a geminivirus Such as mastrevirus, curtovirus or begomovirus. The method com (52) U.S. Cl. ....................... 800/280; 800/278; 800/279; prises introducing a SSDNA-binding protein of the Inoviri 800/288; 800/295; 800/298; 800/301; 536/23.72; dae Virus into the plant, and includes a phage coat protein, 435/69.1; 435/320.1; 435/410; 435/418; particularly, a coliphage gene 5 protein. The invention also 435/419; 435/468 describes a transgenic plant comprising a gene that expresses the SSDNA-binding protein and vectors for (58) Field of Search ............................. 435/69.1, 320.1, expressing the protein in plants. 435/410, 418, 419,468; 536/23, 72; 800/278, 279, 280, 288, 295, 298,301 28 Claims, 5 Drawing Sheets CR Rep MOVernet Protein Coat Protein Movement Trans eplication MoVement activator Enhancer U.S. Patent Feb. 8, 2005 Sheet 1 of 5 US 6,852,907 B1 Rep MoVernent Protein Coat Protein Movement Trans Replication MOVement activator Enhancer FIG. 1A CP66:Stag:6G:g5 -am-m-O- g5 AV2 -D- CP-O- 200 400 600 800 1000 bp Aft III Styl Sph I Hind I FIG. 1B U.S. Patent Feb. 8, 2005 Sheet 2 of 5 US 6,852,907 B1 N is g 8.8%&gs.So & Cu g N. P S&SSS: 88.9 3.SS c3 : O C SS SC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 FIG. 2 U.S. Patent Feb 8, 2005 Sheet 3 of 5 US 6 9 852.907 B1 U.S. Patent Feb. 8, 2005 Sheet 4 of 5 US 6,852,907 B1 - n C) CO o g Y O CO C) g g l c3 6 g c5 1 U.S. Patent Feb. 8, 2005 Sheet 5 of 5 US 6,852,907 B1 NP40 buffer a e s is a 2 s. 5 2 S 5 O O ym O fro kDa 3 4 5 FIG. 4A SSDNA 3 4 5 6 7 FIG. 4B US 6,852,907 B1 1 2 RESISTANCE IN PLANTS TO INFECTION or by modulating ssDNA synthesis, or both. No evidence is BY SSDNA VIRUS USING INOVIRIDAE available for how CP influences ssDNA accumulation in VIRUS SSDNA-BINDING PROTEIN, geminiviruses. In tomato leaf curl virus from New Delhi COMPOSITIONS AND METHODS OF USE (ToICV-NbE, hereafter referred as ToLCV), a geminivirus with bipartite genome, disrupting the Synthesis of wild type This application is a 371 of PCT/US99/04716, filed Mar. CP resulted in drastic reduction in SSDNA and a three to five 3, 1999, which claims the benefit of U.S. Provisional Appli fold increase in dsDNA accumulation in infected proto cation No. 60/076,627, filed Mar. 3, 1998, now abandoned. plasts. Inoculated plants, however, develop Severe Symp This invention was made with government Support under toms and accumulate wild type levels of dsDNA and low Contract No. 2630152-1-3036-00 by the Agency for Inter levels of SSDNA. national Development. The government has certain rights in There remains a need to better understand the role of CP the invention. in geminivirus replication. BRIEF SUMMARY OF THE INVENTION TECHNICAL FIELD We have now discovered that a heterologous ssDNA The invention relates methods and compositions for pro 15 binding protein can complement CP function in geminivirus ducing plants which are resistant to infection by plant SSDNA accumulation. It is also discovered that ToLCV Viruses. modified to express the SSDNA binding gene 5 protein (g5p) from E. coli phage M13 in place of CP accumulates ssDNA BACKGROUND to wild type levels in protoplasts, but fails to move effi Geminiviruses are plant pathogens that cause significant ciently in plants, providing key insight into the present yield losses in crop plants in many countries of the World invention. Exemplary heterologous SSDNA-binding proteins (Briddon et al., “Geminiviridae”, p. 158-165. In F. A. Mur are found in the Inoviridae virus family. phy (ed.), Virus Taxonomy, Sixth Report of International Thus, in one embodiment, the invention describes a Committee On Taxonomy of Viruses, Springer-Verlag, Vienna 25 method for producing in a plant resistance to a single & New York, 1995; Frischmuth et al, Semin. Virol., stranded DNA (ssDNA) virus comprising introducing a 4:329–337, 1993; Harrison et al, Ann. Rev. Phytopathol., SSDNA-binding protein of the Inoviridae virus family into 23:55-82, 1985; Polston et al, Plant Dis., 81:1358–1369, the plant. The Inoviridae family virus ssDNA-binding pro 1997). Different members are transmitted by whiteflies or tein is Selected from the group consisting of the Inovirus and leafhoppers (Davies et al, Genet., 5:77-81, 1989; Lazarow Plectrovirus genuses, and the Inovirus genus virus is itzetal, Crit. Rev. Plant Sci., 11:327–349, 1992). Most of the Selected from the group consisting of Coliphage, enterobac whitefly-transmitted geminiviruses (WTGs) have bipartite teria phage, Pseudomonas phage, Vibrio phage and Xanth genomes while all the leafhopper-transmitted geminiviruses Omonas phage species. A preferred Coliphage Species of and some of the WTGs have monopartite genomes. The Virus is Selected from the group consisting of AE2, dA, Ec9, monopartite genomes (2566-3028 nt) encode proteins 35 f1, fa, HR, M13, ZG/2 and ZJ/2 coliphages, with a coat required for replication, encapsidation and movement, while protein or a gene 5 protein being more preferred. Particularly in the case of the bipartite viruses the movement functions preferred is the Coliphage M13 gene 5 protein. are encoded by a Second genome component of Similar size The method of introduction of the ssDNA-binding protein (Davies et al, Genet., 5:77-81, 1989; Ingham et al, Virology, into the plant can include producing a transgenic plant 207:191-204, 1995; Timmermans et al, Annu. Rev. Plant containing an expression vector for expressing the protein, Physiol. Plant Mol. Biol, 45:79–112, 1994). contacting a plant with an expression vector for expressing Geminiviruses have circular single-stranded (ss) DNA the protein, infecting the plant with a carrier vector, Such as genomes encapsidated in double icosahedral particles. an Agrobacterium vector, and the like methods. Geminiviruses replicate via a rolling circle mechanism The invention also describes a DNA expression vector analogous to replication of bacteriophages with SSDNA 45 comprising a nucleotide Sequence that encodes a SSDNA genomes. The incoming geminivirus single-Stranded (SS) binding protein of the Inoviridae virus family, wherein the DNA is converted by host enzymes to double-stranded (ds) vector is capable of expressing the protein in plants. The DNA which in turn serves as a template for transcription of vector is used in the methods described herein. early, replication associated genes on the complementary Also described is a composition for producing resistance Sense Strand. Replication initiator protein (Rep or AC1) is 50 to a SSDNA virus that infects plants comprising an effective the only viral protein required for replication. In bipartite amount of a DNA expression vector comprising a nucleotide geminiviruses, a Second protein (AC3) enhances replication. Sequence that encodes a SSDNA-binding protein of the AC2, another early gene product, transactivates expression Inoviridae virus family, wherein the vector is capable of of the coat protein (CP) gene on the virion-sense Strand. expressing the protein in the plant. In preferred While the CP is not required for replication of the virus in 55 embodiments, the vector is a carrier vector which can infect protoplasts or plants, mutations in CP lead to dramatic the plant.
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