HK1140910B - Method to improve plant resistance to infections - Google Patents

Description

Method for enhancing resistance of plants to infection

RELATED APPLICATIONS

This application claims priority to U.S. serial No. 60/947,365 filed on day 6, 29 of 2007 and U.S. serial No. 60/947,590 filed on day 7, 2 of 2007. The contents of these applications are incorporated herein by reference in their entirety.

Reference to sequence Listing submitted via EFS-WEB

The entire contents of the sequence listing below, submitted electronically by USPTO EFS-WEB server (approved and listed by MPEP § 1730ii.b.2(a) (C)), are herein incorporated by reference in their entirety for all purposes. The sequence listing is identified from the following electronically submitted text file:

file name	Date of creation	Size (byte)
			549072000740Seqlist.txt	2008, 6 month and 20 days	51,000 bytes

Technical Field

The present invention relates to proteins that enhance the resistance of plants to infection, including infection by pathogens and wounds. The invention also relates to methods for enhancing the resistance of plants to infection by expressing genes encoding these proteins.

Background

Preformed and induced defense mechanisms provide a broad spectrum of resistance to many pathogens encountered by the plant host. Pathogen-specific defense responses are usually initiated by recognition of the pathogen's avirulence (Avr) protein by the corresponding resistance (R) protein of the host. Finally, plant hosts produce a range of defense molecules (including pathogenesis-related proteins) to limit or kill pathogens. The process between resistance initiation and production of resistance proteins involves a complex signal transduction network that has not yet been fully elucidated.

Identification by molecular genetic methods has identified a number of important nodes (hubs) in Arabidopsis thaliana (Arabidopsis thaliana) that defend against signal transduction networks, including EDS 1(Enhanced Disease Susceptibility protein 1), NPR1 (Non-expressor of PR Genes 1), and NDR1 (Non-species-Specific Disease Resistance protein 1). Similar strategies and biochemical studies have confirmed the involvement of plant hormone signals in defense responses in arabidopsis thaliana, particularly Salicylic Acid (SA), and the role of other plant hormones, such as Jasmonic Acid (JA) and Ethylene (ET).

Many known signaling strategies are employed in plant defense responses. For example, some R proteins are receptor kinases, while other protein kinases also play an important role. Biochemical signals such as calcium flux (calcium flux) and oxidative burst are also important. In addition, there are several reports of other signaling components, such as G-protein and RING (novel genes of genuine interest: (Really Interesting New Gene)) zinc finger proteins are involved.

RING zinc finger proteins are a diverse group of proteins with highly conserved zinc binding regions. Depending on the type of cysteine (C) and histidine (H) residue combination, RING zinc finger protein regions can be divided into standard and modified RING zinc fingers. Standard RING fingers can be subdivided into two subclasses: HC subclass (in common: C-X)₂-C-X_9-39-C-X_1-3-H-X_2-3-C-X₂-C-X_4-48-C-X₂-C) (SEQ ID NO: 1) and H2 subclass (consensus: C-X₂-C-X_9-39-C-X_1-3-H-X_2-3-H-X₂-C-X_4-48-C-X₂-C) (SEQ ID NO: 2) (Stone, S.L. et al, Plant Physiology (2005) 137: 13-30). Modified RING zinc fingers include RING-C2, RING-v, RING-D, RING-S/T, and RING-G.

Many members of the RING zinc finger protein family (including HC and H2 subclasses) are E3 ubiquitin ligases. Different subclasses of RING zinc finger regions determine specificity for different E2 ubiquitin-conjugating enzymes. Other RING zinc finger proteins can bind nucleic acids or interact with other protein targets. In addition to ubiquitin-mediated degradation pathways, RING zinc finger proteins also play important roles in organelle transport and transcriptional/translational regulation.

In rice, over 30 resistance loci (Xa loci) against Xanthomonas oryzae pv. oryzae (Xoo) were identified, and 6 Xa genes were cloned mainly by a map-based cloning approach (map-based cloning apreache). Several pathogenesis-related (PR) genes are reported to play a direct role in resistance mechanisms. However, only a few key components have been studied in the signal transduction pathway from the R protein-Avr protein interaction to the actual resistance generation. To obtain a novel Xoo resistance-associated signal transduction component of rice, cDNA clones with the Xa locus differentially expressed in rice lines were investigated.

The present inventors cloned and characterized a novel RING zinc finger protein gene (OsRHC1) of rice. In the event of trauma, OsRHC1 was differentially expressed in near-equivalent gene lines containing the Xa14 or Xa23 tolerance loci, but not in the corresponding susceptible recurrent parent. Ectopic expression of OsRHC1 in transgenic arabidopsis enhances its resistance to bacterial pathogens, and this protective function depends on the action of 26S proteasome.

Disclosure of Invention

Various genes are known in plants to encode infection resistance proteins, and various transgenic plants modified to produce these proteins have been used to confer resistance to infection. However, these resistance proteins appear to have a limited spectrum of activity with respect to successfully recognized pathogen types. Many also cause adverse side effects (e.g., programmed cell death). The present invention provides materials that can be used to confer resistance to infection to a variety of plants without significant adverse side effects. The present invention provides recombinant material producing a protein designated OsRHC1, a RING zinc finger protein that confers resistance to a broad spectrum of pathogen infections. Since the protein of the present invention derived from a monocotyledonous plant (rice) is also effective in a dicotyledonous plant (Arabidopsis thaliana), it is also applicable to various plants.

In one aspect, the invention relates to expression systems for the production of OsRHC1 protein and its closely related proteins, which are RING zinc finger proteins and which enhance resistance of plants to infection. Transgenic plants modified with the expression system of the invention have enhanced resistance to infection by pathogenic organisms or by trauma.

Thus, in another aspect, the invention relates to a plant cell or plant modified to contain an expression system for producing the RING zinc finger protein. These plants may be heterologous plants derived from OsRHC1, or may be rice plants modified to overexpress the protein.

In yet another aspect, the proteins produced by the expression system can be used to perform screening assays to identify compounds or combinations of compounds that modulate the resistance of a plant to infection.

The invention also relates to an immunospecific antibody of the OsRHC1 protein. These antibodies are useful for detecting and purifying the protein.

Brief Description of Drawings

FIG. 1 shows the nucleotide sequence of the coding region of OsRHC1 gene and the amino acid sequence of OsRHC1 protein (SEQ ID NOS: 42-43).

FIG. 2A shows the full-length amino acid sequence (SEQ ID NO: 43) of OsRHC1 aligned with 7 annotated proteins (SEQ ID NO: 44-50) that showed high similarity. FIG. 2B shows membrane bound and soluble protein fractions extracted from CBB23 and JG30 using anti-OsRHC 1 antibody for Western blot analysis.

FIGS. 3A and 3B show expression of OsRHC1 in bacterial wilt resistant lines CBB14 and CBB23 (carrying the Xa14 locus and Xa23 locus, respectively) and their susceptible recurrent parents (SN 1033 and JG30, respectively).

Fig. 4A shows wound-induced expression of OsRHC1 by real-time PCR. Fig. 4B shows a western blot of the corresponding protein.

FIGS. 5A-C show pathogen inoculation testing of transgenic Arabidopsis expressing OsRHC 1. In fig. 5A, the expression of transgenic OsRHC1 in transgenic arabidopsis thaliana was confirmed by Northern blot analysis. Disease symptoms were visible, as shown in fig. 5B; rosette (rosette) leaves (not the site of infection) were collected to estimate pathogen titer, as shown in fig. 5C.

FIGS. 6A and 6B show expression of defense marker genes in transgenic Arabidopsis thaliana not inoculated with (A) and inoculated with (B) Pseudomonas syringae tomato pathogenic variety (Pseudomonas syringae pv. tomato) DC3000(Pst DC 3000).

FIGS. 7A-D show expression of defense marker genes (PR1(A), PR2(B), PDF1.2(C), and Thi2.1(D)) upon treatment with MG132(26S proteasome inhibitor).

FIGS. 8A and 8B show the results of pathogen inoculation testing of OsRHC1 transgenic Arabidopsis in an npr1-3 background. FIG. 8A shows the expression of OsRHC1 gene, and FIG. 8B shows the expression of 4 defense marker genes.

FIG. 9 shows the results of PCR screening for the OsRHC1 transgene in transgenic rice lines.

FIG. 10A shows the expression of OsRHC1 by real-time PCR, and FIG. 10B shows the production of the corresponding protein in the transgenic rice line.

FIGS. 11A-C show expression of the defense marker genes PR1(A), PBZ1(B) and GRCWP (C) in the OsRHC1 transgenic rice line.

FIG. 12 shows the results of a self-ubiquitination assay (autoubiquitination assay) performed on the RING-HC-C-terminal portion of OsRHC 1.

FIG. 13 shows the DNA sequence (SEQ ID NO: 51) and deduced amino acid sequence (SEQ ID NO: 52) of the binding partner of OsRHC 1.

Modes for carrying out the invention

The protein designated Rice RING-HC subclass protein-1 (OsRHC1) is a 409 amino acid protein that is overexpressed in rice in response to infection by pathogens or wounds. These proteins render the plants resistant to the adverse effects of infection when various plants are modified to produce the protein and variants thereof (collectively, the OsRHC1 protein), which variants have at least 90%, preferably 95%, more preferably 98% or 99% sequence identity over the entire length of the 409 amino acid sequence. The present invention provides expression systems that can be used to modify a variety of plants (both monocotyledonous and dicotyledonous) to enhance their ability to resist infection. The general ability of this expression system to confer resistance is demonstrated in the following examples, which show that the protein derived from monocots confers these properties on the dicot Arabidopsis.

Techniques for constructing expression vectors operable in plants, modifying plant cells, regenerating plant cells into intact plants, and recombinant manipulation of plants are generally well known. A review of these techniques is found, for example, in U.S. patent 7,109,033, the contents of which are incorporated herein by reference.

As shown in this patent, promoters useful for plant expression may be constitutive, inducible, and/or tissue specific. Transformation techniques include the use of Agrobacterium, lipofection, electroporation, and the like. Techniques for regenerating plants from transformed plant cells are also well known.

Thus, once the nucleotide sequence encoding the OsRHC1 protein is available, methods for making transgenic plants that produce these proteins are well known to those of ordinary skill in the art. The nucleotide sequence naturally producing this protein is deposited in GenBank under accession number EF584506, and synthetic alternatives with differences in codon usage are possible.

Thus, in accordance with the present invention, a suitable expression system operable in plants is constructed in which a nucleotide sequence encoding a protein of the present invention is operably linked to suitable control sequences operable in plants. The expression system is used to modify plant cells or plants so that the protein can be produced in plant tissues either universally or exclusively at the desired location in the plant, depending on the choice of control system and transformation method. The resulting plants (whether monocotyledonous or dicotyledonous) then produce the protein in response to infection by pathogens or wounds, thereby enhancing their ability to protect against damage caused by these infectious events.

As shown below, OsRHC1 is an E3 ubiquitin ligase that enhances the disruption of undesirable proteins by directing them to the proteasome. This property is identical to other RING proteins and represents one aspect of their protective function. This protein is the first E3 with a transmembrane domain in the N-terminal region and a RING-HC in the C-terminal cytoplasmic tail, which was found to be involved in plant disease resistance.

Furthermore, the protein itself, produced in sufficient quantity and isolated and purified to a suitable degree (purity of at least 50%, preferably 75%, more preferably 90% or 95% by weight), can be used as a screening tool. Compounds or combinations of compounds that bind to the protein are candidates for modulating the ability of a plant to fight infection. A compound or combination of compounds that stimulate the activity of the protein by binding to it will enhance the infection-fighting capabilities of the plant that produces the protein.

Thus, binding assays can be used for preliminary screening. Since OsRHC1 has been demonstrated to be an E3 ubiquitin ligase, the ubiquitin ligase activity assay described below in example 9, or an analogous assay for that activity, can be used to demonstrate agonist activity of candidate compounds. Thus, suitable candidate compounds will enhance the ability of OsRHC1 to achieve ubiquitination.

Antibodies having specific immunological activity to the proteins may also be used to purify the proteins of the invention and detect them. The term "antibody" is understood to mean an intact antibody (polyclonal or monoclonal), an immunospecific fragment thereof, such as Fab fragments, and recombinantly produced forms thereof, e.g. single-chain F_vAn antibody. Thus, the term "antibody" refers to any form of antibody and any portion that retains its immunospecific characteristics. These antibodies can be used, for example, on affinity columns for purification.

In the following examples, the nucleotide sequence encoding the OsRHC1 protein shown in FIG. 1 was recovered from Oryza sativa and deposited. Furthermore, it was demonstrated that OsRHC1 expression can be induced at the mRNA level and at the protein level in rice lines that show resistance in response to pathogens and in response to trauma.

Transgenic arabidopsis plants were obtained using expression constructs for the OsRHC1 protein, and these transgenic plants showed enhanced expression of 4 defense marker genes both under normal growth conditions and upon addition of salicylic acid or jasmonic acid. OsRHC1 transgenic Arabidopsis thaliana also showed constitutive expression of OsRHC 1-encoding DNA, which is protected by this expression when challenged with Pseudomonas (Pseudomonas). Similarly, overexpression of this DNA in rice results in the expression of several defense marker genes.

The following examples are provided to illustrate, but not to limit, the present invention.

Example 1

Identification and cloning of OsRHC 1-encoding cDNA

Partial cDNA clones were obtained by PCR selective cDNA subtraction kit of cloning technology company (Clontech 637401) using total RNA extracted from 6-8 weeks old CBB14 and SN1033, a susceptible parent rice line of CBB14 harvested 4 days after inoculation with a pathogen (Xoo LN44), by inhibition subtractive hybridization technique, where CBB14 is a bacterial wilt resistant strain (tester) and SN1033 (driver). The compounds were prepared by Zhang, q. et al, Acta agr.sin. (1996) 22: 135-141 for inoculation. Using the DNA sequence information of this partial clone, 5 '-Rapid-amplification cDNA Ends (5' -RACE) experiments and subsequent PCR amplifications were performed using specific primers.Gene-specific primers 5'-TTCTCCATGTTCGGTAAACCTTTC-3' (SEQ ID NO: 3), 5'-TAAAGTTGTGAT TGAGACTACATGG-3' (SEQ ID NO: 4) and 5'-ACATTGCACAACCAACATGTAC-3' (SEQ ID NO: 5) were used in the 5 ' RACE reaction. To amplify the full-length coding region, PCR was performed using primer pair 5'-CCTCACTTTTGTCTCCCAC-3' (SEQ ID NO: 6) and 5'-CGACATTGCACAACCAAC-3' (SEQ ID NO: 7). All clones were stored in plasmid vectors of Stelata Gene Co (Stratagene)KSII (+) was propagated in E.coli strain DH5 alpha.

The resulting cDNA clone (GenBank accession No. EF584506) encodes a complete open reading frame of 409 amino acid residues (fig. 1). EF584506 is 99% identical to the rice cDNA clone deposited directly (accession No.: NM 001057564). The corresponding gene in the rice genome appears to be a single copy gene located on chromosome 3. The BlastP search showed that the protein encoded by our clone was 99% identical to the rice clone annotated as a zinc finger family protein (accession number: ABF98464), but 64 amino acid residues less at the N-terminus. Other analyses using the Conserved Domain Database (CDD) showed that the predicted protein has RING zinc finger regions. The pattern of conserved cysteine and histidine residues in the RING zinc finger region is shown to be a marker for the RING-HC subclass. Thus, this clone was designated as OsRHC 1.

The predicted amino acid sequence of OsRHC1 protein was compared with two rice RING zinc finger proteins, EL5(RING-H2 subclass) and XB3(RING-HC subclass), involved in disease resistance. No significant homology was found except in the RING finger region (data not shown). The RING finger zinc finger region of OsRHC1 is located at the C-terminus (FIG. 2A), whereas in EL5 and XB3, the regions are located in the middle or near the C-terminus of the protein, respectively. Predictions made by the TopPred and ipsert programs suggest that OsRHC1 may have multiple transmembrane regions (fig. 2A), whereas EL5 has only one, and XB3 may be almost certainly devoid of any transmembrane region (data not shown).

BlastP analysis showed OsRHC1 and others of various plant species deposited in GenBankThe 7 annotated proteins have a high degree of amino acid sequence homology (fig. 2A). These proteins showed greater than 50% identity to OsRHC1 (over their full length), multiple transmembrane domains in the N-terminal half and a RING-HC domain at the C-terminal. The consensus sequence of the RING-HC region of this group of proteins is Cys-X₂-Cys-X₁₁-Cys-X-His-X₃-Cys-X₂-Cys-X₆-Cys-X₂Cys (SEQ ID NO: 8). The function of these homologues is clearly undisclosed.

Example 2

OsRHC1 was shown to be membrane bound

To confirm that OsRHC1 was membrane bound as shown in the bioinformatics tool, a fractionation protocol was used to separate membrane bound and soluble proteins (modified from the method described by Jiang and Rogers, J.cell Biol. (1998) 143: 1183-1199). For Western blot analysis, proteins were separated by electrophoresis on a polyacrylamide gel (4% concentration; 10% resolution) followed by the use of the Trans-SD semi-dry electrophoresis transfer tank (Trans-SD Semi-Dry Electrophoretic Transfer Cell, Bio-Rad 170-3949) proteins were transferred to activated PVDF membrane (pretreated with anhydrous methanol for 20 minutes followed by protein Transfer buffer for 15 minutes). According to the manufacturer's Invitrogen company's manual (Western Breeze)^TMImmunoassay kit, InvitroGen WB 7106). A first antibody (polyclonal) targeting the OsRHC1 protein was prepared by injecting synthetic peptide ('N' -CGYPPEVVRKMPKRD- 'C') (SEQ ID NO: 9) into rabbits from a commercial service (Invitrogen, custom antibody) and used after purification on an affinity column. Using anti-rabbit secondary antibodies conjugated to alkaline phosphatase (in Western Breeze)^TMThe immune detection kit is provided in the technical field of biological detection,invitrogen WB7106) recognizes the primary antibody. Western blot analysis confirmed that OsRHC1 protein was tightly associated with the membrane (fig. 2B).

Example 3

OsRHC1 is wound-induced in rice lines CBB14 and CBB23

To study the expression pattern of OsRHC1, real-time PCR analysis was performed on reverse transcribed RNA samples using two near-identical gene pairs (CBB 14 containing Xa14 and its susceptible recurrent parent SN 1033; CBB23, resistant strain containing Xa23 and its susceptible recurrent parent JG 30). The rice line grows in the soil of a conventional field in a greenhouse (temperature is 24-28 ℃, RH is 70-80 percent, and natural light is emitted). Xoo strains LN44 and P6 were inoculated by the splicing method described by Zhang, Q.et al, supra (1996). Simulated inoculation and wound treatment follow the same protocol except that water is replaced with the pathogen. For the time course experiments, samples were collected at approximately the same time (8-10 am) on days 0, 2, 4, and 6. Day 0 samples were collected prior to treatment.

To assess the expression of OsRHC1 by real-time PCR, by Ausubel et al, latest methods in molecular biology (R) ((R))Current Protocols in Molecular Biology) (1995) JW father and son (J.Wiley)&Sons) in new york, the phenol extraction method described in new york, extracts total RNA. Reverse transcription of RNA samples (18-mer oligo-dT; SUPERSCRIPT) by DNase I (Invitrogen 18068-^TMIIRNase H (Invitrogen 18064-071)) produced cDNA samples.

Real-time PCR amplification of cDNA was performed in 96-well PCR plates with domes using ABI PRISM 7700 sequence detection System from Applied Biosystems. The reaction was carried out in a 20. mu.l reaction volume containing 10. mu.l SYBR Green PCR master mix (applied biosystems, Inc. 4309155) and 0.3. mu.M of each of the forward and reverse primers. The primers OsRHC1 of the real-time PCR are as follows: 5'-AAAGAAGAGCAAGCCCGG TTAT-3' (SEQ ID NO: 10) and 5'-GCCTCCATACCTCTTCTGCAA-3' (SEQ ID NO: 11). All reactionsAt least 4 times, at least 3 sets of consistent data were performed independently for analysis. The expression levels of actin (rice (O.sativa) OsAc 1D; accession number X15865) using primer sets 5'-CTTCATAGGAATGGAAGCTGCGGGTA-3' (SEQ ID NO: 12) and 5'-GACCACCTTGATCTTCATGCTGCTA-3' (SEQ ID NO: 13) were used to normalize the results. Through 2^-ΔΔCTThe method (Livak and Schmittgen, Methods (2001) 25: 402-.

To confirm the reliability data, the amplification efficiency between the target gene and the housekeeping gene of all real-time PCR reactions was compared, and the dissociation curves of all PCR products were examined to ensure the quality of PCR. Consistent gene expression patterns were observed with at least two independent batches of plant samples. All PCR products were sequenced at least once to confirm that the correct target was quantitated.

Rice lines containing Xa14 or Xa23 showed induction of OsRHC1 gene expression when inoculated with incompatible Xoo strains (LN 44 for Xa14 and P6 for Xa 23), while the susceptible recurrent parent did not respond, as shown in fig. 3A and 3B, respectively. However, this induction was also observed in mock-inoculated samples subjected to trauma, suggesting that OsRHC1 may be trauma-inducible. In the case of CBB23 containing the Xa23 locus conferring broad spectrum resistance, the magnitude of induction was stronger.

The effect of trauma on OsRHC1 expression in CBB23 strain and its susceptible recurrent parent JG30 was further analyzed. RNA and protein samples were collected after wounding of the (plants) by leaf cutting. CBB23 and JG30 rice lines (8-week old plants) were wounded by shearing. Leaf samples were collected on day 0 prior to injury. Leaf tissue approximately 6-8mm from the wound site was collected on days 2, 4 and 6 after shearing. Total RNA and membrane bound protein samples were prepared in parallel. Real-time PCR experiments were performed as described above. Western blot analysis was performed using anti-OsRHC 1 antibody as described in example 2.

In CBB23, the peak in induction of OsRHC1 gene expression appeared at day 4 post-treatment (fig. 4A). Western blot analysis of membrane-bound proteins showed that the production of OsRHC1 protein in CBB23 was greatly increased at day 6 after induction of gene expression at day 4 (fig. 4B). The response in JG30 was not evident compared to CBB23, indicating that the presence of the Xa23 locus may play a role in wound induction of OsRHC 1.

Example 4

Generation of transgenic Arabidopsis lines

To examine whether OsRHC1 could mediate resistance in dicotyledonous plants, arabidopsis was modified to produce OsRHC1 protein and challenged with pseudomonas syringae solanaceous variety DC 3000. The OsRHC1cDNA is inserted into a binary vector, and transgene expression is driven by a cauliflower mosaic virus 35S promoter. Agrobacterium-mediated transformation of wild-type Col-0 Arabidopsis lines was performed using the vacuum infiltration method (Bechtold, N. et al, Methods mol. biol. (1998) 82: 259-266). Transgenic plants with a single insertion locus were screened for the kanamycin-tolerant phenotype of the progeny (encoded by the selectable marker gene in the binary vector). Kappa²The test demonstrated that a 3: 1 ratio (resistance: susceptibility) in the T1 generation suggested a single insertion event.

Only positive transformants containing a single insertion locus were propagated to obtain homozygous lines for further experiments. Transgene expression was detected in 3 independent homozygous transgenic lines by Northern blot analysis. As shown in FIG. 5A, 3 transformed lines, B-1-4, G-1-5 and H-2-9, showed high levels of mRNA production. However, wild-type Col-0 lines showed no mRNA production.

Arabidopsis thaliana was grown in a growth chamber (temperature 22-24 ℃; RH 70-80%; light intensity 80-120. mu.E, 16 hours day-8 hours night cycle). Preparation of PstDC3000 culture medium, inoculation (by the dipping method) and subsequent titer determination were carried out as described previously (modified by Kim, H.S. et al, Plant Cell (2002) 14: 1469-1482; Uknes, S. et al, Plant Cell (1992) 4: 645-656). With 10mM MgSO 10 supplemented with 0.02% (v/v) Silwet L-77₄Prepared at a concentration of 10⁸Colony forming units/ml of Pst DC3000 challenged 6-week old seedlings (P.i. eterse, C.M.J. et alPlant Cell (1998) 10: 1571-1580; ton, j, et al, mol.plant-Microbe Interact (2002) 15: 27-34).

Inoculation of Pst DC3000 in infected Col-0 and transgenic plants transformed with empty vector V7 resulted in severe yellowing and necrosis, while disease symptoms were very mild in all OsRHC1 transgenic lines, as shown in FIG. 5B.

Pathogen titers within rosette leaves were consistent with the observed phenotype (fig. 5C). In addition, pathogen titers were also lowest for H-2-9 lines that showed the highest level of transgene expression (compare FIGS. 5A and 5C).

Example 5

Expression enhancement of OsRHC1 for defense marker gene in transgenic Arabidopsis

Expression of 4 defense marker genes, PR1, PR2, PDF1.2, and thi2.1, in transgenic arabidopsis thaliana was examined. In arabidopsis, these genes are markers of different phytohormone-mediated defense pathways including SA, JA and ET.

Leaf tissues of 6-week-old Arabidopsis transgenic lines (B-1-4, G-1-5 and H-2-9) expressing OsRHC1 and untransformed wild type (Col-0) were collected to prepare total RNA, followed by real-time PCR as described in example 3. The primers used are as follows:

PR 1: 5 'TCAAGATAGCCCACAAGATTATC-3' (SEQ ID NO: 14) and 5 'CTTCTCGTTCACATAATTCCCAC-3' (SEQ ID NO: 15);

PR 2: 5 'ACCACCACTGATACGTCTCCTC-3' (SEQ ID NO: 16) and 5 'AACTTCATACTTAGACTGTCGATC-3' (SEQ ID NO: 17);

PDF 1.2: 5 'CCCTTATCTTCGCTGCTCTTGT-3' (SEQ ID NO: 18) and 5 'CCCTGACCATGTCCCACTTG-3' (SEQ ID NO: 19);

thi2.1: 5 'AGCACTGCAAGTTAGGGTGTGA-3' (SEQ ID NO: 20) and 5 'ACATTGTTCCGACGCTCCAT-3' (SEQ ID NO: 21).

Tubulin (Arabidopsis beta-tubulin 4, accession number: M21415) using primer sets 5'-GAAGGTGCTGAGTTGATTG-3' (SEQ ID NO: 22) and 5'-GGACTTGACGTTGTTTGG-3' (SEQ ID NO: 23) was used to normalize the results.

The expression levels of PR1 (filled), PR2 (open), PDF1.2 (hatched) and thi2.1 (dotted) in each transgenic line shown in fig. 6A were compared with those of Col-0 (expression level set to 1).

In 6-week old seedlings under conventional growth conditions, expression of all 4 defense marker genes was enhanced compared to wild type Col-0 (FIG. 6A). The fold induction of the PR1 and PDF1.2 genes mediated by two different signaling pathways is particularly high. Of the 3 independent transgenic lines tested, the H-2-9 line that showed the highest transgene expression and the best protection in the pathogen inoculation experiment also had the highest fold induction of PR1 and PDF1.2 (compared to fig. 5 and 6A).

When plants were subjected to Pst DC3000 challenge, the levels of PR1 and PR2 transcripts increased in Col-0 (data not shown), but the expression levels of these genes were even higher in the transgenic lines (fig. 6B). Although pathogen inoculation did not significantly alter thi2.1 levels in Col-0 (data not shown), its expression was elevated in the transgenic lines (fig. 6B). On the other hand, expression of PDF1.2 in both Col-0 and transgenic lines was inhibited by inoculation with Pst DC3000 (FIG. 6B).

To show the relationship between OsRHC1 and ubiquitin-mediated protein degradation, the effect of MG132, a 26S proteasome inhibitor, on the expression of defense marker genes was studied in transgenic lines. 4-week-old seedlings were treated with MG 132. Adopts the method which is reported from the past (Abas, L. et al, Nature CellBiol. (2006) 8: 249-doped 256; Dong, C.H. et al, Proc.Nat' l Acad.Sci.USA (2006) 103: 8281-doped 8286; Guo, H. et al, Cell (2003) 115: 667-doped 677;-S n chez, L, etcPlant Physiol. (2002) 128: 1313-1322) 26S proteasome inhibitor (MG132) was applied to transgenic arabidopsis thaliana. Briefly, 50MG/L MG132 dissolved in 1% (v/v) DMSO supplemented with 0.01% (v/v) Silwet L-77 was poured onto MS square plates to cover the roots of the seedlings, but not the aerial tissue. The simulation was performed in the same manner except that MG132 was not added. After 4 hours, the seedlings were harvested for RNA extraction and then subjected to real-time PCR.

This treatment did not affect transgene expression (data not shown). In Col-0, no significant effect of MG132 on expression of the defense marker gene was observed. On the other hand, under the treatment of MG132, the induction of the 4 defense marker genes by the overexpressed OsRHC1 is reduced, and PR1, PR2, PDF1.2 and Thi2.1 are shown in FIGS. 7A-7D, respectively, wherein the open bars represent the simulation treatment and the solid bars represent the MG132 treatment.

In summary, it appears that in all cases, the ability of the OsRHC1 protein to enhance expression of defense marker genes is inhibited by 26S proteasome inhibitors. Thus, it appears that the ability of the proteins of the invention to enhance expression of defense marker genes may depend on 26S proteasome activity.

Example 6

The protective function of the OsRHC1 clone in transgenic Arabidopsis depends on the function of NPR1

Model plant systems (model plant systems) were used to locate the function of OsRHC1 associated with a known node in the defense signaling network. NPR1, which mediates SA and JA/ET, signals and plays a central role in the defense signaling of Arabidopsis thaliana. OsRHC1 was transformed into an NPR1-3 Arabidopsis mutant with elimination of NPR1 as described above. Independent transformants containing the single insertion locus were selected. At inoculation, real-time PCR as described in example 3 was used to detect transgene expression (under the control of the cauliflower mosaic virus 35S promoter) in each line. The steady state levels of OsRHC1 in the npr1-3 background were found to be comparable to those in the transgenic line with the Col-0 background (data not shown).

8-week old transgenic lines (D-2, E-1, F-1, G-1 and G-2) expressing OsRHC1, the untransformed npr1-3 mutant and wild type Col-0 were challenged with Pst DC3000 and pathogen titers were subsequently evaluated, as shown in FIG. 8A. Expression of defense marker genes was determined as described in example 5. The expression of PR1 (filled), PR2 (open), PDF1.2 (hatched) and thi2.1 (dotted) in each line was comparable to Col-0 (expression level set to 1), as shown in fig. 8B. No significant increase in expression of the 4 selected defense marker genes was found in any of these transgenic lines.

When the npr1-3 transgenic line was subjected to Pst DC3000 challenge, no protection was observed in the transgenic line. Disease symptoms developed in these transgenic lines (data not shown) and pathogen titers were similar to those of the untransformed npr1-3 mutant. Thus, the protective effect appears to require NPR 1.

Example 7

Construction of OsRHC1 transgenic rice

The nucleotide sequence encoding OsRHC1 shown in FIG. 1 was subcloned into the dual T-DNA binary vector, pSB130 (professor Liu Qiaoquan, university of Chinese, hong Kong, and Samuel Sun). The vector pSB130 carries two portions of T-DNA. One T-DNA has a hygromycin resistance gene (selectable marker) and the other has a multiple cloning site downstream of the maize ubiquitin promoter for cloning the target gene. The recombinant construct was transformed into Agrobacterium EHA105 for rice transformation to construct transgenic rice lines.

FIG. 9 shows PCR screening of the OsRHC1 transgene (parent: Aichi Asahi) in a T2 transgenic rice line. The forward and reverse primers for PCR were from the maize ubiquitin promoter and OsRHC1 coding regions, respectively, as shown below:

a forward primer: 5 'CTGATGCATATACATGATGG-3' (SEQ ID NO: 24);

reverse primer: 5'-ACATTGCACAACCAACATGTAC-3' (SEQ ID NO: 25).

A total of 5 OsRHC1 transgenic rice lines were obtained.

Example 8

Overexpression of OsRHC1 and defense marker gene in rice

The expression of OsRHC1 and 3 rice defense marker genes (PR1, glycine-rich cell wall protein coding Gene (GRCWP) and PBZ1) were investigated by real-time PCR as described in example 3. PR1 is a well-known PR protein. Glycine-rich cell wall proteins (encoded by GRCWP) are common structural proteins in the enhanced cell wall to hinder pathogen attack. PBZ1 was induced by Probenazole (PBZ), N-cyanomethyl-2-chloro-isonicotinamide (a compound known to induce disease resistance) and the fungal blight pathogen magnaporthe oryzae (m. Incompatible strains of Pyricularia oryzae induced PBZ1 faster than compatible strains. Overexpression of NH1, a critical signaling component in rice defense responses, induced PR1 and PBZ 1.

RNA was extracted from 8-week old transgenic rice lines (T3 generation) carrying a single insertion of OsRHC1 and their wild type parent (Aiyaxu). The primers used for real-time PCR are shown below:

rice (o.sativa) OsRHC1 forward primer: 5'-AAAGAAGAGCAAGCCCGGTTAT-3' (SEQ ID NO: 26);

rice OsRHC1 reverse primer: 5'-GCCTCCATACCTCTTCTGCAA-3' (SEQ ID NO: 27);

forward primer of rice PR1(BF 889437): 5'-CGGACAGAGGCCTTACTAAGTTATTT-3' (SEQ ID NO: 28);

rice PR1(BF889437) reverse primer: 5'-GACCTGTTTACATTTTCACGTCTTTATT-3' (SEQ ID NO: 29);

rice GRCWP (BF889438) reverse primer: 5'-GAGGCAACGGACACCACTAAG-3' (SEQ ID NO: 30);

rice GRCWP (BF889438) reverse primer: 5'-TGTAAAGCAGAGAGAGAGGCTCATT-3' (SEQ ID NO: 31);

rice PBZ1(D38170) forward primer: 5'-AAGCTCAAGTCACACTCGAC-3' (SEQ ID NO: 32);

rice PBZ1(D38170) reverse primer: 5'-GATGTCCTTCTCCTTCTCC-3' (SEQ ID NO: 33).

Actin primers used for normalization were:

rice OsAc1D (X15865) forward primer: 5'-CTTCATAGGAATGGAAGCTGCGGGTA-3' (SEQ ID NO: 34);

rice osaac 1D (X15865) reverse primer: 5'-GACCACCTTGATCTTCATGCTGCTA-3' (SEQ ID NO: 35).

Fig. 10A shows the detection of OsRHC1 overexpression in transgenic lines by real-time PCR. Western blot analysis performed as described in example 2 gave the results shown in figure 10B. Transformants generally show higher protein contents than the wild type.

FIG. 11 shows the induction of expression of 3 rice defense marker genes by overexpression of OsRHC 1. The degree of induction of these 3 defense marker genes is usually positively correlated with the expression level of OsRHC 1. For example, two transgenes R8 and R12 that showed higher levels of OsRHC1 also induced a greater expression of these 3 defense marker genes (compare fig. 10 and 11).

Example 9

OsRHC1 is E3 ubiquitin ligase

This example demonstrates the ability of OsRHC1 to self-ubiquitinate, a shared property of ubiquitin E3 ligase.

A partial fragment of OsRHC1 lacking the transmembrane region located at the N-terminus (OsRHC1p) was prepared. Only the RING-HC region located at the C-terminus was included, because the presence of the transmembrane region made extraction from E.coli cells difficult.

The appropriate C-terminal portion of the coding sequence was amplified with the following primer set flanking the C-terminal half containing the RING-HC region: HMOL5743 (5'-CCGGAATTCGTTGTTCTACTATTACGAAATGG-3') (SEQ ID NO: 36) and HMOL2625 (5'-CAGGTCGACGTTAAACATCATATACGGGCATG-3') (SEQ ID NO: 37). The PCR reaction was performed according to the following cycle: 94 ℃ for 5 minutes; 30 cycles of 94 ℃, 30 seconds, 55 ℃, 30 seconds and 72 ℃ for 1 minute; followed by extension at 72 ℃ for 5 minutes. The amplified product was subcloned into the pGex-4T-1 vector with EcoRI and XhoI restriction sites for in-frame fusion with the GST coding region. The fusion protein was then expressed in DE3 cells and induced with 1.5mM IPTG for 2 hours at 30 ℃ during growth. The GST-OsRHC1p protein was extracted by lysing the bacterial cells with 1mg/ml lysozyme for 1 hour at room temperature, followed by 5 rounds of freeze/thaw with liquid nitrogen and warm water bath. The extracted protein was purified using GST SpinTrap purification module from GE Healthcare (GH Healthcare).

In vitro ubiquitination assays were performed in ubiquitination buffer (40mM Tris-HCl (pH 7.5), 5mM MgCl, 2mM ATP, 2mM dithiothreitol, 300 ng/. mu.l ubiquitin, 25. mu.M MG132, 5. mu.l wheat germ extract (providing E1 and E2 enzymes) (Promega) plus 400ng GST-OsRHC1p or GST protein only. The same reaction buffer without ATP and ubiquitin was used as a negative control (as described in Bazirgan, O.A. et al, J.biol.chem. (2006) 281: 38989-39001; Matsuda, N. et al, J.cell.Sci. (2001) 114: 1949-1957). The reaction mixture was left at room temperature for 2 hours, followed by 10% SDS-PAGE gel electrophoresis, followed by Western blot analysis with an anti-OsRHC 1-specific antibody (FIG. 12).

Autoubiquitination of GST-OsRHC1 was observed in reactions containing ATP and ubiquitin (+ ve), but not in reactions without ATP and ubiquitin (-ve).

These results demonstrate that OsRHC1 undergoes self-ubiquitination similar to other E3 ligases.

Example 10

Identification of OsRHC1 binding partners

The protein encoded by the clone deposited under GenBank accession number ABA98865.1 was identified as a binding partner. This was determined using a yeast two-hybrid protocol and verified by co-precipitation. The protein encoded by this deposited clone is expressed in rice (Japanese Cultivar (Japonica Cultivar-Group)), but it does not have an identified function. However, it is estimated to modulate plant defense responses due to its interaction with OsRHC 1.

Yeast two-hybrid method using commercial kit, BD Matchmaker^TMLibrary construction and screening kit (cloning technologies K1516-1). OsRHC1 was first amplified with oligomers HMOL2624(5, -CCGAATTCATGCCAGCCCCTTCGCTTC-3 ') (SEQ ID NO: 38) and HMOL2625(5 ' CAGGTCGACGTTAAACATCATATACGGGCATG-3 ') (SEQ ID NO: 39), digested with EcoRI and SalI, subcloned into the reading frame of pGBKT7, and transformed into yeast strain Y187. Proteins were extracted from yeast clones transformed with pGBKT7-OsRHC1 and control pGBKT 7. Western blot analysis using an anti-c-Myc epitope tag antibody confirmed the presence of a DNA binding region fused to OsRHC1 protein.

An AD region fusion yeast library was constructed in yeast AH109 using RNA samples of several rice lines (each containing one of the following R genes: Xa2, Xa12, Xa14, Pita, Pib and Pik) inoculated with the corresponding incompatible pathogen (Xa2 is T2; Xa12 is P1; Xa14 is LN 44; Pita, Pib and Pik are Ken54-04) for 4 days as starting materials according to the manufacturer's manual. Two rounds of library screening were performed by pairing between the Y187 and AH109 yeast libraries transformed with pGBKT7-OsRHC 1. Yeast diploid mate was selected on SD-Trp, Leu and His (SD/-3) agar plates and incubated for 4 days at 30 ℃. Only colonies grown to 2-3mm in diameter were streaked onto SD-Trp, Leu, His, and Ade (SD/-4) agar plates. Colony transfer Filter assay by lacZ receptor Gene Activity (colony-lift filter assay) (Yeast protocol Manual)Yeast Protocols Handbook) Cloning technology Co. PT3024-1)To check the selected clones. A partial clone (accession number: ABA98865.1) (marked HML1797) encoding the expressed protein gave a positive result. Retransformation (transformation) of pGBKT7-OsRHC1 and pGADT7-HML1797 into AH109 confirmed that this was not due to mutation.

To verify the results of the yeast-2-hybrid experiment, a co-immunoprecipitation test was performed. The full-length coding region of ABA98865.1 was amplified with primers HMOL5311 (5'-AACCCGGGATGGCCGTGGGGTCAGAG-3') (SEQ ID NO: 40) and HMOL5312 (5'-TTCCCGGGTCAAAATAAAAACAAATAAAAAAACAC-3') (SEQ ID NO: 41), digested with SmaI, subcloned into SmaI linearized pGADT7-Rec vector to generate a fusion protein containing an in-frame HA tag (HA-ABA 98865.1); this is referred to as HML 1846. Combined RiboMAX^TMLarge-Scale RNA production System-T7 (Promega), wheat germ extract (Promega), and Transcend^TMThe biotin-lysyl-tRNA system (Promega corporation) transcribes and translates the constructs separately in vitro.

Total protein was extracted from a rice line overexpressing OsRHC1 (modified from Boyes, D.C. et al, Proc. Natl.Acad.Sci.USA (1998) 95: 15849-15854; Greve, K. et al, biochem. J. (2003) 371: 97-108). Using BD Matchmaker^TMCo-immunoprecipitation kit (clone technologies 630449) was prepared by mixing a rice sample containing 100. mu.g of protein with 40. mu.l of the HA-tag fusion protein described above in a mixture containing 50mM Tris/HCl (pH 7.5), 250mM NaCl, and 2mM MgCl₂、0.5mMCaCl₂10% (v/v) glycerol, 1.5% (v/v)X-100, 1mM PMSF, 2mg/L leupeptin co-immunoprecipitation buffer (modified from Boyes et al, 1998 (supra); Greve et al, 2003 (supra)). anti-HA epitope-tagged antibodies were used to precipitate protein complexes. Protein signals were detected using anti-OsRHC 1 antibody.

Western blot showed that HA-tagged fused ABA98865.1 precipitated OsRHC1, but no protein was detected on western blot when rice protein extract was treated with HA-tagged fused unrelated protein.

Sequence listing

<110> Lin Hamming (LAM, Hon-Ming)

Xingshiwen (SUN, Sai-Ming Samuel)

<120> method for enhancing resistance of plant to infection

<130>549072000740

<140>PCT/US2008/068189

<141>2008-06-25

<150>US 60/947,365

<151>2007-06-29

<150>US 60/947,590

<151>2007-07-02

<160>52

<170>FastSEQ for Windows Version 4.0

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<211>107

<212>PRT

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<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(107)

<223> RING Zinc finger HC subclass consensus

<220>

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<223> Xaa can be any naturally occurring amino acid

<220>

<221>Misc_feature

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<223> Xaa can be any naturally occurring amino acid, and up to 30 can be absent

<220>

<221>Misc_feature

<222>(45)...(47)

<223> Xaa can be any naturally occurring amino acid, and up to 2 can be absent

<220>

<221>Misc_feature

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<223> Xaa can be any naturally occurring amino acid, and up to 1 can be absent

<220>

<221>Misc_feature

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<223> Xaa can be any naturally occurring amino acid

<220>

<221>Misc_feature

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<223> Xaa can be any naturally occurring amino acid, and up to 44 can be absent

<220>

<221>Misc_feature

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<223> Xaa can be any naturally occurring amino acid

<400>1

Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa His

35 40 45

Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

65 70 75 80

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

85 90 95

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys

100 105

<210>2

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<212>PRT

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<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(107)

<223> RING Zinc finger H2 subclass consensus

<220>

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<222>(2)...(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221>Misc_feature

<222>(5)...(43)

<223> Xaa can be any naturally occurring amino acid, and up to 30 can be absent

<220>

<221>Misc_feature

<222>(45)...(47)

<223> Xaa can be any naturally occurring amino acid, and up to 2 can be absent

<220>

<221>Misc_feature

<222>(49)...(51)

<223> Xaa can be any naturally occurring amino acid, and up to 1 can be absent

<220>

<221>Misc_feature

<222>(53)...(54)

<223> Xaa can be any naturally occurring amino acid

<220>

<221>Misc_feature

<222>(56)...(103)

<223> Xaa can be any naturally occurring amino acid, and up to 44 can be absent

<220>

<221>Misc_feature

<222>(105)...(106)

<223> Xaa can be any naturally occurring amino acid

<400>2

Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa His

35 40 45

Xaa Xaa Xaa His Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

65 70 75 80

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

85 90 95

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys

100 105

<210>3

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>3

ttctccatgt tcggtaaacc tttc 24

<210>4

<211>25

<212>DNA

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<220>

<223> primer

<400>4

taaagttgtg attgagacta catgg 25

<210>5

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>5

acattgcaca accaacatgt ac 22

<210>6

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>6

cctcactttt gtctcccac 19

<210>7

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>7

cgacattgca caaccaac 18

<210>8

<211>35

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(35)

<223> RING-HC Domain consensus

<220>

<221>Misc_feature

<222>(1)...(35)

<223> Xaa can be any naturally occurring amino acid

<400>8

Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys

1 5 10 15

Xaa His Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys

20 25 30

Xaa Xaa Cys

35

<210>9

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>9

Asn Cys Gly Tyr Pro Pro Glu Val Val Arg Lys Met Pro Lys Arg Asp

1 5 10 15

Cys

<210>10

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>10

aaagaagagc aagcccggtt at 22

<210>11

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>11

gcctccatac ctcttctgca a 21

<210>12

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>12

cttcatagga atggaagctg cgggta 26

<210>13

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>13

gaccaccttg atcttcatgc tgcta 25

<210>14

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>14

tcaagatagc ccacaagatt atc 23

<210>15

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>15

cttctcgttc acataattcc cac 23

<210>16

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>16

accaccactg atacgtctcc tc 22

<210>17

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>17

aacttcatac ttagactgtc gatc 24

<210>18

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>18

cccttatctt cgctgctctt gt 22

<210>19

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>19

ccctgaccat gtcccacttg 20

<210>20

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>20

agcactgcaa gttagggtgt ga 22

<210>21

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>21

acattgttcc gacgctccat 20

<210>22

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>22

gaaggtgctg agttgattg 19

<210>23

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>23

ggacttgacg ttgtttgg 18

<210>24

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400>24

ctgatgcata tacatgatgg 20

<210>25

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400>25

acattgcaca accaacatgt ac 22

<210>26

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400>26

aaagaagagc aagcccggtt at 22

<210>27

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400>27

gcctccatac ctcttctgca a 21

<210>28

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400>28

cggacagagg ccttactaag ttattt 26

<210>29

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400>29

gacctgttta cattttcacg tctttatt 28

<210>30

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400>30

gaggcaacgg acaccactaa g 21

<210>31

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400>31

tgtaaagcag agagagaggc tcatt 25

<210>32

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400>32

aagctcaagt cacactcgac 20

<210>33

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400>33

gatgtccttc tccttctcc 19

<210>34

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400>34

cttcatagga atggaagctg cgggta 26

<210>35

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400>35

gaccaccttg atcttcatgc tgcta 25

<210>36

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>36

ccggaattcg ttgttctact attacgaaat gg 32

<210>37

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>37

caggtcgacg ttaaacatca tatacgggca tg 32

<210>38

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>38

ccgaattcat gccagcccct tcgcttc 27

<210>39

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>39

caggtcgacg ttaaacatca tatacgggca tg 32

<210>40

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>40

aacccgggat ggccgtgggg tcagag 26

<210>41

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>41

ttcccgggtc aaaataaaaa caaataaaaa aacac 35

<210>42

<211>1230

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(1230)

<223> RING zinc finger protein gene (OsRHCL)

<220>

<221>CDS

<222>(1)...(1230)

<400>42

atg cca gcc cct tcg ctt cct cat ggc cgt cat tgg gct cct tgc cat 48

Met Pro Ala Pro Ser Leu Pro His Gly Arg His Trp Ala Pro Cys His

1 5 10 15

tca att gtt gca gcg ccg ttg crt att gcg ttt gag ctg ctg ctt tgc 96

Ser Ile Val Ala Ala Pro Leu Leu Ile Ala Phe Glu Leu Leu Leu Cys

20 25 30

ata tat ctc gaa agt ttg aga gtt aaa agt aag ccg act gtt gat ttg 144

Ile Tyr Leu Glu Ser Leu Arg Val Lys Ser Lys Pro Thr Val Asp Leu

35 40 45

aag att gta ttc ctt cct ctt ctg gcc ttt gaa gtg att att ctt gtt 192

Lys Ile Val Phe Leu Pro Leu Leu Ala Phe Glu Val Ile Ile Leu Val

50 55 60

gac aat ttc aga atg tgt aga gct tta atg cca gga gat gaa gaa agt 240

Asp Asn Phe Arg Met Cys Arg Ala Leu Met Pro Gly Asp Glu Glu Ser

65 70 75 80

atg agc gat gaa gct att tgg gag aca ctt cct cac ttt tgg gtt gca 288

Met Ser Asp Glu Ala Ile Trp Glu Thr Leu Pro His Phe Trp Val Ala

85 90 95

att tct atg gtg ttt ctt ata gct gct aca acc ttc aca ctt ttg aag 336

Ile Ser Met Val Phe Leu Ile Ala Ala Thr Thr Phe Thr Leu Leu Lys

100 105 110

ctg tct ggt gat gtt ggt gct ttg gga tgg tgg gat ttg ttt ata aat 384

Leu Ser Gly Asp Val Gly Ala Leu Gly Trp Trp Asp Leu Phe Ile Asn

115 120 125

tat gga atc gcg gag tgt ttt gca ttt ctt gtt tgt act aga tgg ttt 432

Tyr Gly Ile Ala Glu Cys Phe Ala Phe Leu Val Cys Thr Arg Trp Phe

130 135 140

aat ccc atg att cat aaa tct cct aat cct ggg gag gct agc tca tca 480

Asn Pro Met Ile His Lys Ser Pro Asn Pro Gly Glu Ala Ser Ser Ser

145 150 155 160

tca gcg gca att aga tac cgt gat tgg gag agt ggt ctt ctc ctc cca 528

Ser Ala Ala Ile Arg Tyr Arg Asp Trp Glu Ser Gly Leu Leu Leu Pro

165 170 175

tca cta gaa gat cat gaa caa gag agg ctc tgt ggt ctt cct gac ata 576

Ser Leu Glu Asp His Glu Gln Glu Arg Leu Cys Gly Leu Pro Asp Ile

180 185 190

ggc ggt cac gta atg aaa ata cca ctg gtg att ttc caa gtt ttg ctt 624

Gly Gly His Val Met Lys Ile Pro Leu Val Ile Phe Gln Val Leu Leu

195 200 205

tgt atg cgc ttg gag ggt acg cct cct agt gct cag tat att ccg ata 672

Cys Met Arg Leu Glu Gly Thr Pro Pro Ser Ala Gln Tyr Ile Pro Ile

210 215 220

ttt gca ctg ttc tcc cca cta ttt att tta caa ggc gct ggt gtc ctt 720

Phe Ala Leu Phe Ser Pro Leu Phe Ile Leu Gln Gly Ala Gly Val Leu

225 230 235 240

ttc tct cta gca aga ttg ttg gag aag gtt gtt cta cta tta cga aat 768

Phe Ser Leu Ala Arg Leu Leu Glu Lys Val Val Leu Leu Leu Arg Asn

245 250 255

gga cca gtt agt cct aat tac ctt aca atc tca tca aaa gtc cgt gat 816

Gly Pro Val Ser Pro Asn Tyr Leu Thr Ile Ser Ser Lys Val Arg Asp

260 265 270

tgc ttt gct ttt ctt cat cgt ggt tca agg ctt ctt ggt tgg tgg tct 864

Cys Phe Ala Phe Leu His Arg Gly Ser Arg Leu Leu Gly Trp Trp Ser

275 280 285

att gat gaa ggc agc aaa gaa gag caa gcc cgg tta ttc tat act gaa 912

Ile Asp Glu Gly Ser Lys Glu Glu Gln Ala Arg Leu Phe Tyr Thr Glu

290 295 300

tct act ggg tac aac aca ttt tgt ggc tat cca cct gag gta gtc agg 960

Ser Thr Gly Tyr Asn Thr Phe Cys Gly Tyr Pro Pro Glu Val Val Arg

305 310 315 320

aaa atg cct aag agg gat ctt gca gaa gag gta tgg agg ctc caa gca 1008

Lys Met Pro Lys Arg Asp Leu Ala Glu Glu Val Trp Arg Leu Gln Ala

325 330 335

gct ttg gga gag caa tca gaa att acc aaa tgt acc aag cag gaa ttt 1056

Ala Leu Gly Glu Gln Ser Glu Ile Thr Lys Cys Thr Lys Gln Glu Phe

340 345 350

gaa agg ctt caa aat gag aag gtt ctt tgt agg att tgc tac gag ggg 1104

Glu Arg Leu Gln Asn Glu Lys Val Leu Cys Arg Ile Cys Tyr Glu Gly

355 360 365

gag ata tgc atg gtc tta ctt cct tgc cgg cac aga aca tta tgc aag 1152

Glu Ile Cys Met Val Leu Leu Pro Cys Arg His Arg Thr Leu Cys Lys

370 375 380

act tgt tct gat aag tgc aag aaa tgt cca atc tgc cgt gtg ccc att 1200

Thr Cys Ser Asp Lys Cys Lys Lys Cys Pro Ile Cys Arg Val Pro Ile

385 390 395 400

gaa gaa cgc atg ccc gta tat gat gtt taa 1230

Glu Glu Arg Met Pro Val Tyr Asp Val *

405

<210>43

<211>409

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(409)

<223> RING zinc finger protein gene (OsRHC1)

<400>43

Met Pro Ala Pro Ser Leu Pro His Gly Arg His Trp Ala Pro Cys His

1 5 10 15

Ser Ile Val Ala Ala Pro Leu Leu Ile Ala Phe Glu Leu Leu Leu Cys

20 25 30

Ile Tyr Leu Glu Ser Leu Arg Val Lys Ser Lys Pro Thr Val Asp Leu

35 40 45

Lys Ile Val Phe Leu Pro Leu Leu Ala Phe Glu Val Ile Ile Leu Val

50 55 60

Asp Asn Phe Arg Met Cys Arg Ala Leu Met Pro Gly Asp Glu Glu Ser

65 70 75 80

Met Ser Asp Glu Ala Ile Trp Glu Thr Leu Pro His Phe Trp Val Ala

85 90 95

Ile Ser Met Val Phe Leu Ile Ala Ala Thr Thr Phe Thr Leu Leu Lys

100 105 110

Leu Ser Gly Asp Val Gly Ala Leu Gly Trp Trp Asp Leu Phe Ile Asn

115 120 125

Tyr Gly Ile Ala Glu Cys Phe Ala Phe Leu Val Cys Thr Arg Trp Phe

130 135 140

Asn Pro Met Ile His Lys Ser Pro Asn Pro Gly Glu Ala Ser Ser Ser

145 150 155 160

Ser Ala Ala Ile Arg Tyr Arg Asp Trp Glu Ser Gly Leu Leu Leu Pro

165 170 175

Ser Leu Glu Asp His Glu Gln Glu Arg Leu Cys Gly Leu Pro Asp Ile

180 185 190

Gly Gly His Val Met Lys Ile Pro Leu Val Ile Phe Gln Val Leu Leu

195 200 205

Cys Met Arg Leu Glu Gly Thr Pro Pro Ser Ala Gln Tyr Ile Pro Ile

210 215 220

Phe Ala Leu Phe Ser Pro Leu Phe Ile Leu Gln Gly Ala Gly Val Leu

225 230 235 240

Phe Ser Leu Ala Arg Leu Leu Glu Lys Val Val Leu Leu Leu Arg Asn

245 250 255

Gly Pro Val Ser Pro Asn Tyr Leu Thr Ile Ser Ser Lys Val Arg Asp

260 265 270

Cys Phe Ala Phe Leu His Arg Gly Ser Arg Leu Leu Gly Trp Trp Ser

275 280 285

Ile Asp Glu Gly Ser Lys Glu Glu Gln Ala Arg Leu Phe Tyr Thr Glu

290 295 300

Ser Thr Gly Tyr Asn Thr Phe Cys Gly Tyr Pro Pro Glu Val Val Arg

305 310 315 320

Lys Met Pro Lys Arg Asp Leu Ala Glu Glu Val Trp Arg Leu Gln Ala

325 330 335

Ala Leu Gly Glu Gln Ser Glu Ile Thr Lys Cys Thr Lys Gln Glu Phe

340 345 350

Glu Arg Leu Gln Asn Glu Lys Val Leu Cys Arg Ile Cys Tyr Glu Gly

355 360 365

Glu Ile Cys Met Val Leu Leu Pro Cys Arg His Arg Thr Leu Cys Lys

370 375 380

Thr Cys Ser Asp Lys Cys Lys Lys Cys Pro Ile Cys Arg Val Pro Ile

385 390 395 400

Glu Glu Arg Met Pro Val Tyr Asp Val

405

<210>44

<211>467

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(467)

<223> annotated protein NP-564052

<400>44

Met Ser Cys Arg Arg Val Leu Lys Ser Ile Gln Ala Leu Ala Ala His

1 5 10 15

Ser Leu Leu Phe Cys Phe Thr Leu Leu Leu Val Leu Lys Leu Asp His

20 25 30

Thr Val Ser Ser Ser Trp Trp Met Val Phe Phe Pro Leu Trp Ala Phe

35 40 45

His Ala Val Val Ala Arg Gly Arg Phe Ser Leu Pro Ala Pro Val Ala

50 55 60

Pro Arg Asn Arg His Trp Ala Pro Cys His Ala Val Val Ala Thr Pro

65 70 75 80

Leu Leu Val Ala Phe Glu Leu Leu Leu Cys Ile Tyr Leu Glu Ser Ser

85 90 95

Tyr Ala Arg Trp Pro Pro Ala Val Ser Leu Lys Ile Ala Phe Leu Pro

100 105 110

Leu Leu Ala Phe Glu Leu Thr Ile Leu Val Asp Asn Leu Arg Met Cys

115 120 125

Arg Ala Leu Met Pro Gly Asp Asp Asp Ser Ile Thr Asp Asp Ala Ile

130 135 140

Trp Glu Ala Leu Pro Hi s Phe Trp Val Ala Ile Ser Met Val Phe Thr

145 150 155 160

Leu Ala Ala Thr Phe Phe Thr Leu Leu Lys Leu Ser Gly Asp Val Val

165 170 175

Ala Leu Gly Trp Trp Asp Leu Phe Ile Asn Phe Gly Ile Ala Glu Cys

180 185 190

Phe Ala Phe Leu Val Cys Thr Lys Trp Ser Asn Pro Val Ile His Arg

195 200 205

Ser Ser Arg Ala Arg Glu Thr Gly Ser Ser Ser Thr Ser Ile Arg Tyr

210 215 220

Leu Asp Trp Asn Ser Gly Leu Val Val Ala Pro Glu Glu Asp Arg His

225 230 235 240

Gln Asp Arg Trp Cys Gly Leu Gln Asp Ile Gly Gly His Met Leu Lys

245 250 255

Ile Pro Val Ile Leu Phe Gln Val Val Leu Cys Met Tyr Leu Glu Gly

260 265 270

Thr Pro Glu Arg Ala Lys Asp Ile Ser Ile Pro Val Leu Phe Ser Pro

275 280 285

Leu Phe Leu Leu Gln Gly Leu Gly Val Leu Phe Ala Ala Ser Lys Leu

290 295 300

Leu Glu Lys Ile Val Leu Leu Leu Arg Gly Glu Ala Gly Pro Gly Leu

305 310 315 320

Tyr Phe Arg Phe Ser Ser Ser Ala His Asp Cys Leu Gly Phe Leu His

325 330 335

His Gly Ser Arg Leu Leu Gly Trp Trp Ser Ile Asp Glu Gly Ser Arg

340 345 350

Glu Glu Gln Ala Arg Leu Tyr Phe Asp Gln Glu Ser Gly Tyr Asn Thr

355 360 365

Phe Ser Gly His Pro Pro Glu Ile Val Lys Lys Met Pro Lys Glu Asp

370 375 380

Leu Ala Glu Glu Val Trp Arg Leu Gln Ala Ala Leu Gly Glu Gln Thr

385 390 395 400

Glu Ile Thr Lys Phe Ser Gln Gln Glu Tyr Glu Arg Leu Gln Asn Glu

405 410 415

Lys Val Leu Cys Arg Val Cys Phe Glu Lys Asp Ile Ser Leu Val Leu

420 425 430

Leu Pro Cys Arg His Arg Val Leu Cys Arg Thr Cys Ala Asp Lys Cys

435 440 445

Thr Thr Cys Pro Ile Cys Arg Ile Asp Ile Glu Lys Arg Leu Ser Val

450 455 460

Tyr Asp Val

465

<210>45

<211>466

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(466)

<223> annotated protein NP-177535

<400>45

Met Asn Cys Trp Arg Val Leu Lys Ser Val Gln Ala Ser Val Ala His

1 5 10 15

Cys Phe Leu Phe Ser Phe Thr Leu Ala Leu Val Leu Lys Leu Asp His

20 25 30

Ser Ile Thr Tyr Ser Trp Trp Val Val Cys Leu Pro Leu Trp Ala Phe

35 40 45

His Ala Val Val Ala Arg Gly Arg Phe Ser Leu Pro Ala Pro Ile Ala

50 55 60

Pro Arg Asn Arg His Trp Ala Pro Cys His Ala Ile Val Ser Thr Pro

65 70 75 80

Leu Leu Ile Ala Phe Glu Leu Leu Leu Cys Val Tyr Leu Glu Thr Ala

85 90 95

Tyr Ala Asp Ser Pro Pro Ala Val Ser Leu Lys Ile Val Phe Leu Pro

100 105 110

Leu Leu Ala Phe Glu Val Ile Ile Leu Val Asp Asn Ala Arg Met Cys

115 120 125

Arg Ala Leu Met Pro Gly Asp Glu Glu Ser Val Asn Asp Glu Ala Val

130 135 140

Trp Glu Ala Leu Pro His Phe Trp Val Ala Ile Ser Met Val Phe Phe

145 150 155 160

Leu Ala Ala Thr Val Phe Thr Leu Leu Lys Leu Ser Gly Asp Val Ala

165 170 175

Ala Leu Gly Trp Trp Asp Leu Phe Ile Asn Phe Gly Ile Ala Glu Cys

180 185 190

Phe Ala Phe Leu Val Cys Thr Lys Trp Ser Asn Pro Val Ile His Arg

195 200 205

Ser Ser Arg Asp Arg Glu Thr Gly Ser Ser Ser Thr Asn Ile Arg Tyr

210 215 220

Leu Asp Trp Asn Ser Gly Leu Gly Val Phe Ser Glu Asp Asp Arg Asn

225 230 235 240

Gln Asp Thr Cys Gly Leu Gln Asp Ile Gly Gly His Ile Met Lys Ile

245 250 255

Pro Leu Ile Val Phe Gln Val Val Leu Cys Met His Leu Glu Gly Thr

260 265 270

Pro Glu Ala Ala Lys Ser Ile Ser Val Pro Val Leu Phe Ser Pro Leu

275 280 285

Phe Leu Leu Gln Gly Val Gly Val Leu Phe Ala Ala Ser Lys Leu Ile

290 295 300

Glu Lys Val Val Leu Leu Leu Arg Gly Glu Asp Asp Thr Gly Leu Tyr

305 310 315 320

Phe Arg Phe Leu Ser Arg Ala His Asp Cys Leu Gly Phe Leu His His

325 330 335

Gly Ser Arg Leu Leu Gly Trp Trp Ser Ile Asp Glu Gly Ser Arg Glu

340 345 350

Glu Glu Ala Arg Leu Tyr Phe Asp Gln Glu Ser Gly Tyr Asn Thr Phe

355 360 365

Cys Gly His Pro Pro Glu Ile Val Lys Lys Met Pro Lys Lys Glu Leu

370 375 380

Ala Glu Glu Val Trp Arg Leu Gln Ala Ala Leu Gly Glu Gln Thr Glu

385 390 395 400

Ile Thr Lys Phe Ser Gln Gln Glu Tyr Glu Arg Leu Gln Asn Glu Lys

405 410 415

Val Leu Cys Arg Val Cys Phe Glu Arg Glu Ile Ser Val Val Leu Leu

420 425 430

Pro Cys Arg His Arg Val Leu Cys Arg Asn Cys Ser Asp Lys Cys Lys

435 440 445

Lys Cys Pro Phe Cys Arg Ile Thr Ile Glu Glu Arg Leu Pro Val Tyr

450 455 460

Asp Val

465

<210>46

<211>467

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(467)

<223> annotated protein AAW81737

<400>46

Met Ser Cys Arg Arg Val Leu Lys Ser Ile Gln Ala Leu Ala Ala His

1 5 10 15

Ser Leu Leu Phe Ser Phe Thr Leu Phe Leu Val Phe Lys Leu Asp His

20 25 30

Thr Leu Ser Cys Ser Trp Trp Met Val Phe Phe Pro Leu Trp Ala Phe

35 40 45

His Ala Val Val Ala Arg Gly Arg Phe Ser Leu Pro Ala Pro Ile Ala

50 55 60

Pro Arg Asn Arg His Trp Ala Pro Cys His Ala Val Val Ala Thr Pro

65 70 75 80

Leu Leu Val Ser Phe Glu Leu Leu Leu Cys Ile Tyr Leu Glu Ser Ser

85 90 95

Tyr Ala Ser Trp Pro Pro Ala Val Ser Leu Arg Ile Ala Ser Leu Pro

100 105 110

Leu Leu Ala Phe Glu Val Thr Ile Leu Ile Asp Asn Leu Arg Met Cys

115 120 125

Arg Ala Leu Met Pro Gly Asp Asp Asp Ser Ile Asn Asp Glu Ala Ile

130 135 140

Trp Glu Ala Leu Pro His Phe Trp Val Ala Ile Ser Met Val Phe Thr

145 150 155 160

Leu Ala Ala Thr Phe Phe Ala Leu Leu Lys Leu Thr Gly Asp Val Ala

165 170 175

Ala Leu Ser Trp Trp Asp Leu PheIle Asn yal Gly Ile Ala Glu Cys

180 185 190

Phe Ala Phe Leu Val Cys Thr Lys Trp Ser Asn Pro Val Ile His Arg

195 200 205

Ser Ser Arg Pro Arg Glu Thr Gly Ser Ser Ser Thr Pro Val Arg Tyr

210 215 220

Leu Asp Trp Asn Ser Gly Leu Val Val Thr Pro Glu Gln Asp Asn His

225 230 235 240

Gln Asp Arg Tyr Cys Gly Leu Gln Asp Ile Gly Gly His Leu Leu Lys

245 250 255

Ile Pro Val Ile Val Phe Gln Val Val Leu Cys Met His Leu Glu Gly

260 265 270

Thr Pro Glu Arg Ala Lys Asp Ile Ser Ile Pro Val Leu Phe Ser Pro

275 280 285

Ile Phe Leu Leu Gln Gly Leu Gly Val Leu Phe Ala Thr Ser Lys Leu

290 295 300

Ile Glu Lys Ile Val Asp Leu Leu Gln Gly Glu Ala Gly Thr Gly Leu

305 310 315 320

Tyr Phe Arg Val Ser Ser Arg Ala His Asp Cys Leu Gly Phe Leu His

325 330 335

His Gly Ser Arg Leu Leu Gly Trp Trp Ser Ile Asp Glu Gly Ser Arg

340 345 350

Glu Glu Gln Ala Arg Leu Tyr Phe Asp Gln Glu Ser Gly Tyr Asn Thr

355 360 365

Phe Ser Gly His Pro Pro Glu Ile Val Lys Lys Met Pro Lys Glu Asp

370 375 380

Leu Ala Glu Glu Val Trp Arg Leu Gln Ala Ala Leu Gly Glu Gln Thr

385 390 395 400

Glu Ile Thr Lys Phe Ser Gln Gln Glu Tyr Glu Arg Leu Gln Asn Glu

405 410 415

Lys Val Leu Cys Arg Val Cys Phe Glu Lys Glu Ile Ser Leu Val Leu

420 425 430

Leu Pro Cys Arg His Arg Val Leu Cys Arg Ile Cys Ser Asp Lys Cys

435 440 445

Thr Lys Cys Pro Ile Cys Arg Val Ala Ile Glu Glu Arg Leu Leu Val

450 455 460

Tyr Asp Val

465

<210>47

<211>466

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(466)

<223> annotated protein BAE71207

<400>47

Met Ser Trp Ser Arg Val Leu Lys Ser Ala Gln Ala Phe Ala Ala His

1 5 10 15

Thr Phe Leu Leu Cys Phe Thr Leu Leu Leu Leu Leu Lys Leu Asp His

20 25 30

Gln Ile Ser Ser Ser Trp Trp Ile Ile Phe Ser Pro Leu Trp Met Phe

35 40 45

His Gly Val Val Ala Arg Gly Arg Phe Ser Leu Pro Ala Pro Ser Ala

50 55 60

Pro Arg Asn Arg His Trp Ala Pro Cys His Ala Val Val Ala Met Pro

65 70 75 80

Leu Leu Ile Ala Phe Glu Leu Leu Leu Cys Ile Tyr Leu Glu Ser Leu

85 90 95

Tyr Val Arg Gly Phe Pro Ala Val Asp Leu Lys Ile Val Phe Leu Pro

100 105 110

Leu Leu Thr Phe Glu Val Ile Ile Leu Ile Asp Asn Phe Arg Met Cys

115 120 125

Lys Ala Leu Met Pro Gly Asp Glu Glu Arg Met Ser Asp Glu Ala Ile

130 135 140

Trp Glu Thr Leu Pro His Phe Trp Val Ala Ile Ser Met Val Phe Phe

145 150 155 160

Val Ala Ala Thr Val Phe Thr Leu Leu Lys Leu Ser Gly Ser Val Ala

165 170 175

Ser Leu Gly Trp Trp Asp Leu Phe Ile Asn Phe Thr Ile Ala Glu Cys

180 185 190

Phe Ala Phe Leu Val Cys Thr Lys Trp Ser Asn Pro Val Ile His Arg

195 200 205

Ser Ser Arg Glu Pro Ser Ser Ser Ser Ser Thr Thr Ile Arg Tyr Leu

210 215 220

Asp Trp Asn Asn Gly Leu Leu Val Ser Ser Glu Glu Asp Gln Arg Gln

225 230 235 240

Ala Arg Ile Cys Thr Leu Gln Asp Ile Gly Gly His Phe Met Lys Val

245 250 255

Pro Ile Ile Val Phe Gln Val Leu Leu Cys Met His Leu Glu Gly Thr

260 265 270

Pro Ala Phe Ala Ala Gln Leu Pro Leu Ala Val Leu Phe Ser Pro Leu

275 280 285

Phe Val Leu Gln Gly Val Gly Val Ile Leu Ser Ala Ser Lys Phe Val

290 295 300

Glu Lys Leu Val Leu Leu Leu Arg Ser Gly Ala Gly Gly Gly Leu Tyr

305 310 315 320

Phe Arg Val Ser Ser Ile Ala His Asp Cys Leu Gly Phe Leu His His

325 330 335

Gly Ser Arg Leu Leu Gly Trp Trp Ser Ile Asp Glu Gly Ser Arg Glu

340 345 350

Glu Gln Ala Arg Leu Tyr His Glu Gly Ala Ser Gly Tyr Asn Thr Phe

355 360 365

Ser Gly Tyr Pro Pro Glu Ile Val Lys Lys Met Pro Lys Arg Asp Leu

370 375 380

Ala Glu Glu Val Trp Arg Leu Gln Ala Ala Leu Gly Glu Gln Thr Glu

385 390 395 400

Ile Thr Lys Tyr Ser Gln Gln Glu Tyr Glu Arg Leu Lys Asn Glu Lys

405 410 415

Val Leu Cys Arg Ile Cys Phe Glu Gly Glu Ile Ser Val Val Leu Leu

420 425 430

Pro Cys Arg His Arg Val Leu Cys Ser Leu Cys Ser Glu Lys Cys Lys

435 440 445

Met Cys Pro Ile Cys Arg Asn Tyr Ile Ala Glu Arg Leu Pro Val Tyr

450 455 460

Asp Val

465

<210>48

<211>468

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(468)

<223> annotated protein NP-564945

<400>48

Met Leu Val Gln Arg Arg Val Met Ser Trp Arg Arg Val Trp Lys Ser

1 5 10 15

Phe Gln Ala Ala Ser Ala His Cys Leu Leu Phe Ser Phe Thr Leu Leu

20 25 30

Leu Ala Leu Lys Leu Asp His Val Val Ser His Ser Trp Trp Phe Val

35 40 45

Phe Ala Pro Leu Trp Leu Phe His Ala Val Ile Ala Arg Gly Arg Phe

50 55 60

Ser Leu Pro Ala Pro Ser Met Pro His Asp Arg His Trp Ala Pro Phe

65 70 75 80

His Ser Val Met Ala Thr Pro Leu Leu Val Ala Phe Glu Ile Leu Leu

85 90 95

Cys Val His Leu Glu Asp Lys Tyr Val Val Asp Leu Lys Ile Val Phe

100 105 110

Leu Pro Leu Leu Ala Phe Glu Val Ala Ile Leu Ile Asp Asn Val Arg

115 120 125

Met Cys Arg Thr Leu Met Pro Gly Asp Glu Glu Thr Met Ser Asp Glu

130 135 140

Ala Ile Trp Glu Thr Leu Pro His Phe Trp Val Ser Ile Ser Met Val

145 150 155 160

Phe Phe Ile Ala Ala Thr Thr Phe Thr Leu Leu Lys Leu Cys Gly Asp

165 170 175

Val Ala Ala Leu Gly Trp Trp Asp Leu Phe Ile Asn Phe Gly Ile Ala

180 185 190

Glu Cys Phe Ala Phe Leu Val Cys Thr Lys Trp Ser Asn Gln Ser Ile

195 200 205

His Arg Tyr Ser His Ile Pro Glu Pro Ser Ser Ser Ser Met Val Val

210 215 220

Arg Tyr Leu Asp Trp Asn Arg Gly Leu Val Val Thr Ala Asp Asp Glu

225 230 235 240

His Gln Gln Ser Asn Arg Ile Cys Gly Leu Gln Asp Ile Gly Gly His

245 250 255

Val Met Lys Ile Pro Phe Val Thr Phe Gln Ile Ile Leu Phe Met Arg

260 265 270

Leu Glu Gly Thr Pro Ala Ser Ala Lys Asn Ile Pro Ile Leu Val Leu

275 280 285

Phe Val Pro Leu Phe Leu Leu Gln Gly Ala Gly Val Leu Phe Ala Met

290 295 300

Tyr Arg Leu Val Glu Lys Ser Val Leu Leu Ile Asn Ser Gly Ser Gly

305 310 315 320

Ser Tyr Gly Arg Tyr Phe Thr Ala Thr Ser Ser Ala Arg Glu Phe Leu

325 330 335

Gly Phe Phe Gln His Gly Ala Arg Leu Leu Gly Trp Trp Ser Ile Asp

340 345 350

Glu Gly Ser Arg Glu Glu Gln Ala Arg Leu Tyr Ser Gly Glu Ala Thr

355 360 365

Gly Tyr Asn Thr Phe Ser Pro Glu Val Val Lys Lys Met Pro Lys Ser

370 375 380

Asp Leu Val Glu Glu Ile Trp Arg Leu Gln Ala Ala Leu Ser Glu Gln

385 390 395 400

Thr Asp Ile Thr Ser Tyr Ser Gln Gln Glu Tyr Glu Arg Leu Gln Asn

405 410 415

Glu Lys Ile Leu Cys Arg Val Cys Phe Glu Asp Pro Ile Asn Val Val

420 425 430

Leu Leu Pro Cys Arg His His Val Leu Cys Ser Thr Cys Cys Glu Lys

435 440 445

Cys Lys Lys Cys Pro Ile Cys Arg Val Leu Ile Glu Glu Arg Met Pro

450 455 460

Val Tyr Asp Val

465

<210>49

<211>497

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(497)

<223> annotated protein ABE90658

<400>49

Met Leu Val Arg Arg Arg Val Met Ser Trp Arg Arg Val Phe Lys Ser

1 5 10 15

Leu Gln Ala Met Leu Ala His Ala Phe Leu Phe Ser Phe Ser Leu Leu

20 25 30

Leu Val Leu Lys Leu Asp Arg Phe Phe Leu Phe Ser Trp Trp Thr Val

35 40 45

Phe Phe Pro Leu Trp Leu Phe His Val Val Ile Ala Arg Gly Arg Phe

50 55 60

Ser Leu Pro Ala Pro Ser Met Pro His Gly Arg Gln Trp Ala Pro Cys

65 70 75 80

His Ser Val Ile Ala Thr Pro Leu Leu Val Ala Phe Glu Leu Leu Leu

85 90 95

Cys Ile His Leu Gly Ser Ser Tyr Val Val Asn Leu Lys Ile Val Phe

100 105 110

Ile Pro Leu Ile Ala Phe Glu Leu Ala Ile Leu Ile Asp Asn Ile Arg

115 120 125

Met Cys Arg Ala Leu Met Pro Gly Asp Glu Glu Asn Met Thr Asp Glu

130 135 140

Ala Val Trp Glu Thr Leu Pro His Phe Trp Ile Ser Ile Ser MetVal

145 150 155 160

Phe Phe Val Ala Ala Thr Val Phe Thr Leu Leu Lys Ile Cys Gly Asp

165 170 175

Val Ala Ala Leu Gly Trp Trp Asp Leu Phe Ile Asn Tyr Gly Tyr Asn

180 185 190

Gln Tyr Leu Leu Val Asp Cys Phe Lys His Phe Ile Leu Ile Leu Tyr

195 200 205

Phe Phe His His Lys Leu Ile Leu Ser Phe Cys Ser Ile Ala Gln Cys

210 215 220

Phe Ala Phe Leu Val Cys Thr Lys Trp His Asn Pro Thr Ile His Gly

225 230 235 240

Asn Gly His Ile Thr Glu Pro Cys Ser Ser Ser Asn Thr Val Arg Tyr

245 250 255

Leu Glu Trp Ser Arg Glu Gly Ile Val Ile Ser Thr Glu Glu Asp Glu

260 265 270

Gln Gln Asn Val Phe Cys Ser Leu Gln Asp Ile Gly Gly His Ile Met

275 280 285

Lys Ile Pro Phe Ile Ala Phe Gln Ile Leu Leu Phe Met His Leu Glu

290 295 300

Gly Thr Pro Ser Gly Ala Lys Asp Ile Pro Ile Trp Val Ile Phe Ser

305 310 315 320

Pro Leu Leu Leu Leu Gln Gly Ala Gly Val Leu Phe Ala Ala Tyr Arg

325 330 335

Leu Ile Glu Lys Ile Ile Leu Leu Leu Tyr Asn Gly Asp Ile Pro Arg

340 345 350

Ser Tyr Ser Ser Ile Ser Ser Lys Ser Arg Asp Cys Phe Gly Phe Phe

355 360 365

Asn His Gly Ser Arg Leu Leu Gly Trp Trp Ser Ile Asp Glu Gly Ser

370 375 380

Arg Glu Glu Glu Ala Arg Leu Phe Cys Ala Gly Ser Ser Gly Tyr Asn

385 390 395 400

Thr Phe Ser Pro Asp Thr Val Lys Lys Met Pro Arg Gly Glu Leu Val

405 410 415

Glu Glu Ile Trp Arg Leu Gln Ala Ala Leu Gly Glu Gln Thr Glu Val

420 425 430

Thr Lys Tyr Ser Gln Glu Glu Tyr Glu Arg Leu Gln Asn Glu Lys Ile

435 440 445

Leu Cys Arg Val Cys Phe Glu Glu Gln Ile Asn Val Val Leu Leu Pro

450 455 460

Cys Lys His His Val Leu Cys Ser Thr Cys Cys Glu Lys Cys Lys Lys

465 470 475 480

Cys Pro Ile Cys Arg Gly Thr Ile Glu Glu Arg Met Pro Ile Tyr Asp

485 490 495

Val

<210>50

<211>498

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(498)

<223> annotated protein AAF25982

<400>50

Met Val Phe Phe Pro Leu Trp Ala Phe His Ala Val Val Ala Arg Gly

1 5 10 15

Arg Phe Ser Leu Pro Ala Pro Val Ala Pro Arg Asn Arg His Trp Ala

20 25 30

Pro Cys His Ala Val Val Ala Thr Pro Leu Leu Val Ala Phe Glu Leu

35 40 45

Leu Leu Cys Ile Tyr Leu Glu Ser Ser Tyr Ala Arg Trp Pro Pro Ala

50 55 60

Val Ser Leu Lys Ile Ala Phe Leu Pro Leu Leu Ala Phe Glu Leu Thr

65 70 75 80

Ile Leu Val Asp Asn Leu Arg Met Cys Arg Ala Leu Met Pro Gly Asp

85 90 95

Asp Asp Ser Ile Thr Asp Asp Ala Ile Trp Glu Ala Leu Pro Val Ser

100 105 110

Pro Leu Leu Leu His Lys Ile Phe Glu Gly Leu Ser Leu Arg Leu Gly

115 120 125

Lys Ile Asn Leu Leu Asn Met Asn Glu Asn Leu Ser Leu Ile Phe Gln

130 135 140

Leu His Asn Ser Gly Leu Arg Arg Glu Lys Thr Leu Thr Asn His Phe

145 150 155 160

Trp Val Ala Ile Ser Met Val Phe Thr Leu Ala Ala Thr Phe Phe Thr

165 170 175

Leu Leu Lys Leu Ser Val Phe Glu Lys Tyr Leu Pro Phe Leu Trp Leu

180 185 190

Leu Val Lys Asn Met Lys Val Ile Tyr Met Lys Cys Ser Ala Cys Arg

195 200 205

Ile Ala Glu Cys Phe Ala Phe Leu Val Cys Thr Lys Trp Ser Asn Pro

210 215 220

Val Ile His Arg Ser Ser Arg Ala Arg Glu Thr Gly Ser Ser Ser Thr

225 230 235 240

Ser Ile Arg Tyr Leu Asp Trp Asn Ser Gly Leu Val Val Ala Pro Glu

245 250 255

Glu Asp Arg His Gln Asp Arg Trp Cys Gly Leu Gln Asp Ile Gly Gly

260 265 270

His Met Leu Lys Ile Pro Val Ile Leu Phe Gln Val Val Leu Cys Met

275 280 285

Tyr Leu Glu Gly Thr Pro Glu Arg Ala Lys Asp Ile Ser Ile Pro Val

290 295 300

Leu Phe Ser Pro Leu Phe Leu Leu Gln Gly Leu Gly Val Leu Phe Ala

305 310 315 320

Ala Ser Lys Leu Leu Glu Lys Ile Val Leu Leu Leu Arg Gly Glu Ala

325 330 335

Gly Pro Gly Leu Tyr Phe Arg Phe Ser Ser Ser Ala His Asp Cys Leu

340 345 350

Gly Phe Leu His His Gly Ser Arg Leu Leu Gly Trp Trp Ser Ile Asp

355 360 365

Glu Gly Ser Arg Glu Glu Gln Ala Arg Leu Tyr Phe Asp Gln Glu Ser

370 375 380

Gly Leu Val Trp Arg Leu Gln Ala Ala Leu Gly Glu Gln Thr Glu Ile

385 390 395 400

Thr Lys Phe Ser Gln Gln Glu Tyr Glu Arg Leu Gln Asn Val Tyr Ser

405 410 415

Phe Ile Ser His Asp Val Phe Val Thr Phe Leu Phe Arg Phe Tyr Phe

420 425 430

Phe Pro Leu Leu Asn Pro Val Ser Met Cys Leu Leu Leu Gln Glu Lys

435 440 445

Val Leu Cys Arg Val Cys Phe Glu Lys Asp Ile Ser Leu Val Leu Leu

450 455 460

Pro Cys Arg His Arg Val Leu Cys Arg Thr Cys Ala Asp Lys Cys Thr

465 470 475 480

Thr Cys Pro Ile Cys Arg Ile Asp Ile Glu Lys Arg Leu Ser Val Tyr

485 490 495

Asp Val

<210>51

<211>354

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(354)

<223> OsRHC1 binding partners

<220>

<221>CDS

<222>(1)...(354)

<400>51

atg gcc gtg ggg tca gag cgg ctc ggc gag gag gcc gcc cgg cgg cag 48

Met Ala Val Gly Ser Glu Arg Leu Gly Glu Glu Ala Ala Arg Arg Gln

1 5 10 15

ctc ggc gag gca agg aag gcc aga ggc ggc tgc tcg gcg acg agg gac 96

Leu Gly Glu Ala Arg Lys Ala Arg Gly Gly Cys Ser Ala Thr Arg Asp

20 25 30

ggc gcc gat gat gag ggc cgg cgg cag ata aac cct ccc tcc ccg gtg 144

Gly Ala Asp Asp Glu Gly Arg Arg Gln Ile Asn Pro Pro Ser Pro Val

35 40 45

tgg tcg tcc cct ccc tca ctc cct ctt cct ctc aga tct gcc cgg agg 192

Trp Ser Ser Pro Pro Ser Leu Pro Leu Pro Leu Arg Ser Ala Arg Arg

50 55 60

ggg acg ggt gga ggc cgg cgg cct ccc ttc cct ctt tcc tct cag atc 240

Gly Thr Gly Gly Gly Arg Arg Pro Pro Phe Pro Leu Ser Ser Gln Ile

65 70 75 80

cgc ccg gtg ggg aga ggc cac cgg cgg cag cgg cat ggc cct ccc ctc 288

Arg Pro Val Gly Arg Gly His Arg Arg Gln Arg His Gly Pro Pro Leu

85 90 95

tgc agc agt aga ggg cgg cag gga gga ggc cac aga gct gtg ttt ttt 336

Cys Ser Ser Arg Gly Arg Gln Gly Gly Gly His Arg Ala Val Phe Phe

100 105 110

tat ttg ttt tta ttt tga 354

Tyr Leu Phe Leu Phe *

115

<210>52

<211>117

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic constructs

<220>

<221>Misc_feature

<222>(1)...(117)

<223> OsRHC1 binding partners

<400>52

Met Ala Val Gly Ser Glu Arg Leu Gly Glu Glu Ala Ala Arg Arg Gln

1 5 10 15

Leu Gly Glu Ala Arg Lys Ala Arg Gly Gly Cys Ser Ala Thr Arg Asp

20 25 30

Gly Ala Asp Asp Glu Gly Arg Arg Gln Ile Asn Pro Pro Ser Pro Val

35 40 45

Trp Ser Ser Pro Pro Ser Leu Pro Leu Pro Leu Arg Ser Ala Arg Arg

50 55 60

Gly Thr Gly Gly Gly Arg Arg Pro Pro Phe Pro Leu Ser Ser Gln Ile

65 70 75 80

Arg Pro Val Gly Arg Gly His Arg Arg Gln Arg His Gly Pro Pro Leu

85 90 95

Cys Ser Ser Arg Gly Arg Gln Gly Gly Gly His Arg Ala Val Phe Phe

100 105 110

Tyr Leu Phe Leu Phe

115

Claims (6)

1. A recombinant expression system comprising a nucleotide sequence encoding a protein whose amino acid sequence is SEQ ID NO: 43, wherein the nucleotide sequence is operably linked to a regulatory system that effects expression in a plant cell.

2. A method of enhancing resistance of rice to infection or wounding, the method comprising modifying said rice to comprise an expression system according to claim 1.

3. A method of making a protein having an amino acid sequence of SEQ ID NO: a method comprising culturing a cell containing the expression system of claim 1 under conditions wherein the protein is produced and recovering the protein from the culture.

4. A protein having the amino acid sequence of SEQ ID NO: 43.

5. a method of identifying a compound or combination of compounds that modulates resistance of rice to infection or wounding, the method comprising

Contacting a protein according to claim 4 as a test protein with a candidate compound or a combination of candidate compounds, and

determining the ability of said compound or combination of compounds to bind to said protein, the compound or combination of compounds binding to said protein being a candidate for modulating the ability of rice to combat infection.

6. A method of identifying a compound or combination of compounds that modulates resistance of rice to infection or wounding, the method comprising

Contacting a protein according to claim 4 as a test protein with a candidate compound or a combination of candidate compounds, and

assaying the ability of the compound or combination of compounds to enhance or reduce self-ubiquitination of the protein.