A protein related to resistance to Xanthomonas disease and its encoding gene and application

Abstract

The present invention discloses a disease-resistant protein against Xanthomonas diseases, its encoding gene and application. The disease-resistant protein disclosed in the present invention is A1) or A2): A1) a protein having an amino acid sequence of sequence 2; A2) a protein having the same function as the protein shown in sequence 2, wherein one or more amino acid residues are substituted and / or deleted and / or added to the amino acid sequence shown in sequence 2 in the sequence table. Experiments have shown that the protein can enhance disease resistance to bacterial leaf streak pathogens when expressed in rice; as a Xanthomonas hydrolase, after being expressed in plants, it can decompose xanthomonasine synthesized by the Xanthomonas effector protein AvrBs2, reduce the accumulation of the compound in plant cells, thereby weakening the inhibitory effect of the compound on rice immunity and reducing the bacteria's uptake of nutrients through the compound, thereby inhibiting bacterial growth and lesion expansion.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a protein related to resistance to Xanthomonas diseases, a coding gene thereof, and an application thereof. Background Art

[0002] Xanthomonas pathogenic bacteria are important plant pathogens with a wide host range. They cause serious bacterial diseases on many important crops, such as rice white leaf blight, rice bacterial leaf streak, citrus canker, Solanaceae bacterial spot disease, etc., and their disease prevention and control is relatively difficult. Studies have shown that the three types of effector proteins secreted into host cells by this type of pathogenic bacteria are the main factors that constitute their pathogenicity. Among them, AvrBs2 is one of the most conserved effector proteins of Xanthomonas species. It is ubiquitous in Xanthomonas species and is a key virulence factor for multiple Xanthomonas pathogenic variants, such as bacterial leaf streak fungus.

[0003] Xanthomonas oryzae pv. oryzicola (Xoc), bacterial spot of pepper

[0004] Xanthomonas campestris pv. vesicatoria, etc. However, its molecular mechanism is still unclear.

[0005] The applicant of this patent has revealed for the first time the pathogenic molecular mechanism of the type III effector protein AvrBs2 of Xanthomonas through research. The study found that AvrBs2 is a cyclic phosphoglycolipid synthase, such as Figure 1 During the infection of plants by Xanthomonas, a new cyclic glycolipid phosphate compound can be synthesized in plant cells. We named it Xanthomonas alkaloid. The structural formula of this compound is bis-1',6'-cyclic dimericα-D-galactose-phosphate,c-di-galactose-phosphae. Figure 2 Our results show that AvrBs2 performs its toxic function by synthesizing xanthomonasine, helping bacteria cause pathogenicity. Summary of the Invention

[0006] like Figure 3 The applicant discovered that there is a gene xanP encoding phosphodiesterase in Xanthomonas bacteria, and confirmed that the protein product encoded by this gene can decompose xanthomonas alkaloids and eliminate their toxic function to plants.

[0007] Based on the above breakthrough research findings, the present invention aims to provide a protein related to resistance to Xanthomonas diseases, its encoding gene, and its application.

[0008] The anti-Xanthomonas disease-related protein provided by the present invention is derived from the rice bacterial leaf spot pathogen (Xoc) strain RS105, named Xanthomonas alkaline phosphodiesterase, XanP, and is as follows A1) or A2):

[0009] A1) a protein having the amino acid sequence of sequence 2;

[0010] A2) A protein having the same function as the protein shown in Sequence 2, wherein one or more amino acid residues are substituted and / or deleted and / or added.

[0011] Sequence 2 in the sequence listing consists of 261 amino acid residues.

[0012] In order to facilitate the purification of the protein in A1), a tag as shown in Table 1 can be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing.

[0013] Table 1. Sequences of tags

[0014] Label residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0015] The proteins in A1) or A2) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically. The encoding genes for the proteins in A2) above can be obtained by deleting one or more codons for amino acid residues from the DNA sequence represented by SEQ ID NO: 1-786 from the 5′ end, and / or performing missense mutations of one or more nucleotide pairs, and / or attaching the coding sequence of the tag shown in Table 1 to its 5′ and / or 3′ end.

[0016] The coding genes of the aforementioned Xanthomonas disease resistance-related proteins also fall within the scope of protection of the present invention.

[0017] The present invention provides an isolated polynucleotide comprising, from 5' to 3' direction, a nucleotide sequence encoding a xanP family of xanthomonas alkaline hydrolases from the genus Xanthomonas. In one embodiment, the nucleotide sequence encoding xanP is a sequence of a xanthomonas alkaline hydrolase xanP from Xanthomonas oryzae, as shown in SEQ ID NO: 1.

[0018] Alternatively, the nucleotide sequence encoding xanP may be modified, for example, by deletion and / or addition and / or substitution of one or more codons (i.e., nucleotide triplets). Such modifications may be silent, for example, such that the amino acid sequence encoding XanP after modification is identical to the native amino acid sequence of XanP. Alternatively, such modifications may be conservative, for example, such that the amino acid sequence encoding XanP after modification is different from the native amino acid sequence of XanP, and the XanP variant has comparable or identical xanthomonas alkali hydrolysis activity as native XanP. The present invention also encompasses non-silent or non-conservative modifications.

[0019] Alternatively, the nucleotide sequence encoding xanP can be modified, for example, by deletion and / or addition and / or substitution of one or more codons (i.e., nucleotide triplets). In a specific embodiment, the modified nucleotide sequence encoding xanP has about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% nucleotide sequence homology or identity with the native amino acid sequence of XanP, and the amino acid sequence encoded by the modified nucleotide sequence is identical to the native amino acid sequence of XanP. In another specific embodiment, the modified nucleotide sequence encodes a variant having an amino acid sequence different from the native amino acid sequence of XanP, and the XanP variant has the same or equivalent xanthomonas alkali hydrolysis activity as native XanP. In another embodiment, the nucleotide sequence encoding XanP is modified, and the modified nucleotide sequence encodes a XanP variant having an amino acid sequence different from the native amino acid sequence of XanP, wherein the amino acid sequence of the XanP variant has about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% amino acid sequence homology or identity with the native amino acid sequence of XanP, and the XanP variant has the same or comparable xanthomonas alkali hydrolysis activity as native XanP.

[0020] Alternatively, nucleotide sequences from bacteria other than the genus Xanthomonas encode protein sequences that are about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to XanP from the 5' to 3' direction, and have the same or equivalent chemical activity as XanP and are capable of hydrolyzing xanthomonasine.

[0021] Alternatively, the nucleotide sequence encoding the XanP homologous protein is modified, for example, by deletion and / or addition and / or substitution of one or more codons (i.e., nucleotide triplets). The amino acid sequence encoded by the XanP variant has about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% amino acid sequence homology or identity with the native amino acid sequence of XanP, and the protein encoded by the variant nucleotide has the same or equivalent xanthomonas alkali hydrolysis activity as the native XanP.

[0022] Alternatively, the fusion polynucleotide sequence of the above-mentioned polynucleotide sequence and other polynucleotide sequences and a partial nucleotide sequence in the polynucleotide sequence, and the encoded protein has the same or equivalent xanthomonas alkali hydrolysis activity as natural XanP.

[0023] The protein expressed from the nucleotide has the activity of hydrolyzing xanthocyanine.

[0024] Preferably, the coding gene is as follows 1) or 2) or 3):

[0025] 1) cDNA molecule or DNA molecule shown in Sequence 1 in the sequence listing;

[0026] 2) a cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the protein associated with resistance to Xanthomonas disease;

[0027] 3) A cDNA molecule or DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the protein associated with resistance to Xanthomonas disease.

[0028] The nucleotides 1 to 786 from the 5′ end of Sequence 1 in the sequence listing are the coding sequence, encoding the amino acid sequence shown in Sequence 2 in the sequence listing.

[0029] The use of biological materials related to the anti-Xanthomonas disease-related protein in regulating plant disease resistance also falls within the scope of protection of the present invention; the biological material is any one of the following B1) to B9):

[0030] B1) a nucleic acid molecule encoding the anti-Xanthomonas disease-related protein;

[0031] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0032] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0033] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0034] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2);

[0035] B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2);

[0036] B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2);

[0037] B8) a nucleic acid molecule that inhibits the expression of the gene encoding the anti-Xanthomonas disease-associated protein;

[0038] B9) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

[0039] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0040] In the above application, the nucleic acid molecule in B1) may be the following 1) or 2) or 3):

[0041] 1) cDNA molecule or DNA molecule shown in Sequence 1 in the sequence listing;

[0042] 2) a cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the protein associated with resistance to Xanthomonas disease;

[0043] 3) A cDNA molecule or DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the protein associated with resistance to Xanthomonas disease.

[0044] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0045] The stringent conditions may be hybridization and membrane washing in a 0.1×SSPE (or 0.1×SSC) and 0.1% SDS solution at 65°C.

[0046] The microorganism can be yeast, bacteria, algae or fungi. The bacteria can be Agrobacterium, such as Agrobacterium EHA105.

[0047] The transgenic cell lines, transgenic plant tissues and transgenic plant organs do not include plant propagation materials.

[0048] In the above application, the plant may be a monocotyledonous plant or a dicotyledonous plant.

[0049] The disease resistance is resistance to diseases caused by Xanthomonas bacteria.

[0050] The invention relates to a "xanthomonas-resistant transgenic disease-resistant breeding method" for pathogenic bacteria of the genus Xanthomonas, namely, by transforming the xanP gene encoding the xanthomonas phosphodiesterase XanP in Xanthomonas into different host plants (including but not limited to rice (Oryza, including Oryza sativa L.), citrus (Citrus), pepper (Capsicum frutescens L.), tomato (Solanumlycopersicum L.), rapeseed (Brassica campestris L.), cabbage (Brassica oleracea L.)), so that different host plants acquire the ability to decompose xanthomonas synthesized by AvrBs2, thereby reducing the cytotoxicity of the effector protein AvrBs2 and enhancing the disease resistance of transgenic plants to different Xanthomonas diseases.

[0051] Specifically, the present invention relates to a transgenic construct for expressing the xanthomonasine phosphodiesterase XanP in plant cells, a recombinant vector comprising the transgenic construct, and a host cell, a transgenic plant cell and a transgenic plant transformed with the transgenic construct or the recombinant vector, and a preparation method thereof. The present invention also relates to a method for conferring on plants the ability to hydrolyze xanthomonasine (bis-1',6'-cycl ic dimeric

[0052] α-D-galactose-phosphate, c-di-galactose-phosphae) and methods for enhancing disease resistance to Xanthomonas diseases.

[0053] The present invention also provides a kind of nucleic acid construct, it comprises the polynucleotide of separation among the present invention.The present invention also provides a kind of recombinant vector, it comprises the polynucleotide of separation among the present invention, or comprises the nucleic acid construct of the present invention.In one embodiment, the recombinant vector is a recombinant cloning plasmid or a recombinant expression plasmid.The present invention also provides a kind of host cell, it comprises the polynucleotide of separation among the present invention, or comprises the nucleic acid construct of the present invention, or comprises the recombinant vector of the present invention.In one embodiment, the host cell is a prokaryotic host cell, for example, a bacterial host cell.In a specific embodiment, the bacterial host cell is an Escherichia coli (Escherichia col i) host cell.In another specific embodiment, the bacterial host cell is an Agrobacterium host cell, such as Agrobacterium tumefaciens (Agrobacterium tumefaciens) host cell.

[0054] The present invention also provides a transgenic plant cell and a transgenic plant, which comprise the isolated polynucleotide of the present invention, or comprise the nucleic acid construct of the present invention, or comprise the recombinant vector of the present invention, or comprise the host cell of the present invention. In one embodiment, the isolated polynucleotide of the present invention is integrated into the genome of the transgenic plant cell. In one embodiment, the plant or the plant cell is a crop, including food crops, fruit and vegetable crops, and economic crops. In one embodiment, the plant or the plant cell includes but is not limited to the plant or plant cell of the following group: rice (Oryza, including Oryza sativa L.), citrus (Citrus), pepper (Capsicum frutescens L.), tomato (Solanumlycopersicum L.), rapeseed (Brassica campestris L.), cabbage (Brassica oleracea L.). In one embodiment, the transgenic plant cell and transgenic plant have the ability to hydrolyze xanthomonas alkaloids and enhance disease resistance to Xanthomonas diseases.

[0055] The present invention also provides a method for preparing the transgenic plant cells and transgenic plants of the present invention, comprising the step of introducing the isolated polynucleotide, nucleic acid construct, recombinant vector, or recombinant host cell of the present invention into a plant cell. In one embodiment, the isolated polynucleotide of the present invention is integrated into the genome of the transgenic plant cell. In one embodiment, the transgenic plant cell and transgenic plant have the ability to hydrolyze xanthomonadine and enhance disease resistance to Xanthomonas diseases.

[0056] The present invention also provides a method for conferring the ability of plant cells or plants to hydrolyze xanthomonasine and enhance disease resistance to Xanthomonas diseases, which comprises the step of introducing the isolated polynucleotide, nucleic acid construct, recombinant vector or recombinant host cell of the present invention into plant cells.

[0057] In one embodiment, the isolated polynucleotide of the present invention is integrated into the genome of the transgenic plant cell.

[0058] Experiments have shown that expressing xanP in rice enhances resistance to bacterial leaf streak pathogens. XanP, a Xanthomonas hydrolase, degrades xanthomonadine, a compound synthesized by the Xanthomonas effector protein AvrBs2, when expressed in plants. This reduces the compound's accumulation in plant cells, thereby weakening its inhibitory effect on rice immunity and reducing bacterial uptake of nutrients through the compound, ultimately inhibiting bacterial growth and lesion expansion. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The Xanthomonas secretes effector protein AvrBs2 to synthesize xanthomonasine in plant cells. The left picture shows Xoc wild type (WT), avrBs2 gene knockout (ΔavrBs2), wild type avrBs2 gene complement (ΔavrBs2-C) and enzyme activity deficient avrBs2. H319A complement strain (ΔavrBs2-C H319A ) Statistics of lesions on leaves of wild-type Nipponbare rice after inoculation. The right panel shows thin-layer chromatography analysis of xanthocyanine accumulation in leaf lesions 14 days after inoculation. Significance analysis, t-test (*, P < 0.05).

[0060] Figure 2 Xanthomonasine promotes Xoc pathogenicity in rice. Rice leaves were sprayed with 1 mM xanthomonasine dissolved in 0.01% Silwet L-77, or a control (0.01% Silwet L-77). The avrBs2 knockout strain (ΔavrBs2) was then inoculated onto leaves of the wild-type rice variety Nipponbare. Disease development was assessed 14 days later. The upper figure shows a photograph of lesions, and the lower figure is a bar graph showing lesion length. Significance analysis was performed using a t-test (***, P < 0.001).

[0061] Figure 3 The xanP gene or its enzyme activity-deficient mutant xanP was analyzed by thin layer chromatography to hydrolyze xanthophylline. M1 / xanP M2After the polynucleotide fragment was recombined into the Escherichia coli prokaryotic expression vector pET28a, it was transformed into the Escherichia coli strain BL21 (DE3) for prokaryotic expression of the XanP protein. After purification by nickel column, it was incubated with xanthocyanine. The reaction products were then analyzed by thin-layer chromatography to determine the enzyme activity. From left to right, lane 1 is a negative control of xanthocyanine without the addition of purified protein; lane 2 is a lane after the reaction of purified wild-type XanP protein with xanthocyanine. XanP hydrolyzes xanthocyanine to produce two products (degradation product 1 and degradation product 2); lanes 3 and 4 are respectively XanP enzyme activity-deficient mutants xanP M1 / xanP M2 It cannot hydrolyze xanthocyanine; lane 5 is the incubation of phosphodiesterase RpfG, which specifically hydrolyzes c-di-GMP, with xanthocyanine, and the results show that rpfG cannot hydrolyze xanthocyanine.

[0062] Figure 4 Schematic diagram of the transgene expression cassette. LB, left border of the T-DNA; RB, right border of the T-DNA; 35S promoter, cauliflower mosaic virus 35S promoter; Ubipromoter, maize ubiquitin promoter; HptII, hygromycin phosphotransferase II gene; xanP, xanthomonas hydrolase gene from Xanthomonas oryzae pv. oryzicola (Xoc); 3FLAG, nucleotide sequence encoding a triple FLAG tag; transcription termination sequence of the nopaline synthase gene Nos.

[0063] Figure 5 Shown is a Western blot analysis of the XanP protein in transgenic rice. Western blot analysis of wild-type and XanP-expressing transgenic rice revealed XanP protein expression in both constitutively expressing transgenic lines, OE-xanP-4 and OE-xanP-5, while no band was detected in the wild-type rice. Ponceau S staining indicates total protein loading.

[0064] Figure 6 xanP transgenic rice exhibits enhanced disease resistance against different Xoc strains. Different Xoc strains (RS60, RS85, and SD21) were inoculated into wild-type or xanP transgenic rice plants, and disease incidence was assessed 14 days later. The left image shows a photograph of diseased lesions, and the right image shows a bar graph of lesion length. Letters a and b indicate significant differences after analysis of variance and Tukey's honest significance test.

[0065] Figure 7 Inoculation of xanP transgenic rice with the Xoc strain RS105 significantly reduced xanthocyanin accumulation in the plants. Xanthocyanin content in rice lesions was analyzed after inoculation of wild-type rice (Nipponbare) or xanP transgenic rice lines OE-xanP-4 and OE-xanP-5 with the Xoc strain RS105. The left figure shows xanthocyanin content measured by thin-layer chromatography, and the right figure shows a statistical bar graph. a, b, and c indicate significant differences after analysis of variance and Tukey's honest significance test. DETAILED DESCRIPTION

[0066] DETAILED DESCRIPTION OF THE INVENTION Definitions

[0067] As used herein, "% sequence homology or identity" refers to the percentage of amino acid residues in a candidate sequence that are identical to those in a target sequence by comparing the sequences and, if necessary, inserting gaps to obtain the maximum percentage of homology or identity (and, in calculating % sequence identity, not taking into account any conservative substitutions). The sequences described herein include amino acid sequences and nucleotide sequences. Sequence homology or identity percentage can be determined using various methods known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalogin (DNASTAR). Those skilled in the art can determine the specific parameters for sequence comparison, as well as any algorithm required for maximum comparison of full-length sequences.

[0068] As used herein, "nucleic acid construct" is synonymous with "transgenic construct" and refers to single-stranded or double-stranded nucleic acid molecules that are isolated from a native gene or altered to contain nucleic acid segments that are associated and juxtaposed in a manner not found in nature. When a nucleic acid construct contains all regulatory sequences required for expression of a XanP of the present invention, the terms "nucleic acid construct" and "expression cassette" are synonymous.

[0069] As used herein, a "host cell" includes any cell capable of receiving an isolated nucleic acid sequence of the present invention, or a construct or vector containing the sequence, and stably maintaining the sequence within the cell. A host cell containing an isolated nucleic acid sequence of the present invention is capable of acquiring the trait or characteristic encoded by the sequence.

[0070] Xanthopine, a cyclic glycophospholipid compound discovered and named by the applicant, has the English name xanthopine and the structural formula bis-1',6'-cyclic dimeric α-D-galactose-phosphate. Xanthopine is a metabolite synthesized by the type III effector protein AvrBs2 secreted into plant cells by pathogenic bacteria of the genus Xanthomonas and is a key toxic factor involved in the pathogenicity of Xanthomonas.

[0071] The present invention also relates to variants or mutants of the above-mentioned proteins, wherein the amino acid sequence of the variant or mutant has one or more amino acid substitutions, deletions, and / or additions relative to the amino acid sequence described herein, but the variant or mutant still has xanthocyanine hydrolysis activity. The amino acid substitutions, deletions, and / or additions can be accomplished using conventional techniques in the art.

[0072] Examples of conservative substitutions are substitutions within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small amino acids (such as glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter a specific activity are well known in the art and have been described, for example, by N. Neurath and RL Hill in Protein, published by Academic Press, New York, 1979. The most common interchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Iys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, as well as their reverse interchanges.

[0073] It will be apparent to those skilled in the art that such substitutions can be made outside of regions critical for the function of the molecule and still produce an active polypeptide. For the polypeptides encoded by the isolated nucleic acid sequences of the present invention, amino acid residues that are essential for their activity and therefore selected not to be substituted can be identified using methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (e.g., see Cunningham and Wells, 1989, Science 244:1081-1085). The latter technique introduces mutations at every positively charged residue in the molecule and tests the resulting mutant molecules for xanthomonasine hydrolysis activity, thereby identifying amino acid residues that are important for the activity of the molecule. The substrate-enzyme interaction site can also be determined by analysis of its three-dimensional structure, which can be determined by techniques such as nuclear magnetic resonance analysis, crystallography, or photoaffinity labeling (see, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol 224:899-904; Wlodaver et al., 1992, FEBS Letters 309:59-64).

[0074] The present invention also relates to amino acid sequences having a certain homology or identity to the amino acid sequences described herein (e.g., the amino acid sequence set forth in SEQ ID NO: 2), such as an amino acid sequence having a homology or identity of at least about 50%, preferably at least about 60%, preferably at least about 65%, preferably at least about 66%, preferably at least about 67%, preferably at least about 68%, preferably at least about 69%, preferably at least about 70%, preferably at least about 71%, preferably at least about 72%, preferably at least about 73%, preferably at least about 74%, preferably at least about 75%, preferably at least about 76%, preferably at least about 77%, preferably at least about 78%, preferably at least about 79%, more preferably at least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, As long as the protein having the amino acid sequence has the activity of hydrolyzing xanthomonasine, the sequence belongs to the scope of the present invention.

[0075] The present invention also relates to proteins having at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 100% of the xanthocyanine hydrolyzing activity of a protein comprising the amino acid sequence described herein (e.g., the amino acid sequence shown in SEQ ID NO: 2).

[0076] The present invention also relates to variants or mutants of the aforementioned polynucleotides, wherein the nucleotide sequence of the variant or mutant comprises one or more triplet codon substitutions, deletions, and / or additions relative to the nucleotide sequence described herein, but the variant or mutant still produces a protein having xanthomonasine hydrolysis activity. The triplet codon substitutions, deletions, and / or additions can be accomplished using conventional techniques in the art.

[0077] The nucleic acid sequence of the present invention can be a genomic sequence, a cDNA sequence, an RNA sequence, a semisynthetic sequence, a completely artificially synthesized sequence or any combination thereof.

[0078] Where appropriate, the nucleotide sequence encoding XanP can be optimized to enhance expression in plants. In other words, the nucleotide sequence encoding XanP can be synthesized using plant-preferred codons to enhance expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11. Methods for synthesizing plant-preferred genes are well known in the art. See, for example, U.S. Patent Nos. 5,380,831; 5,436,391; and Murray et al. (1989) Nucleic Acids Res. 17:477-498. The nucleotide sequence encoding XanP can also be optimized to reduce secondary nucleic acid structure, thereby enhancing gene expression.

[0079] Nucleic acid construct or transgenic construct

[0080] Another aspect of the present invention relates to nucleic acid constructs.

[0081] In one embodiment, the nucleic acid construct is used to express xanthomonas alkali hydrolase XanP in plant cells. Methods for transforming callus induced from mature embryos of rice are known. See, for example, Wang et al., Nat. Commun. 12, 5479.

[0082] In one embodiment, the nucleic acid construct of the present invention comprises a nucleotide sequence encoding a xanthomonas alkali hydrolase in a 5' to 3' direction. For example, the nucleotide sequence encoding a xanthomonas alkali hydrolase can be a nucleotide sequence encoding Xanthomonas oryzae pv. oryzae, as shown in SEQ ID NO: 1 in the sequence listing.

[0083] The nucleic acid construct of the present invention may further contain a regulatory sequence necessary for expressing the above sequence in a selected host cell, wherein the regulatory sequence is operably linked to the above isolated nucleic acid sequence in the nucleic acid construct.

[0084] The regulatory sequence can be a promoter sequence, including transcriptional regulatory sequences that mediate the expression of a polypeptide. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a cell, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides, which may or may not be homologous to the cell. Numerous promoters for use in prokaryotes are well known in the art.

[0085] The regulatory sequence can also be an appropriate transcription termination sequence, i.e., a sequence that is recognized by the host cells described herein to terminate transcription. The termination sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the host cell can be used in the present invention.

[0086] The regulatory sequence can be an appropriate leader sequence, i.e., a non-translated region on the mRNA that is important for translation in the cell. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell can be used in the present invention.

[0087] The regulatory sequence can also be a polyadenylation sequence. This sequence is operably linked to the 3' end of the nucleic acid sequence and, when transcribed, is recognized by the cell as a signal to add polyadenylic acid residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the cell can be used in the present invention.

[0088] The nucleic acid construct may also include one or more nucleic acid sequences encoding one or more factors that facilitate the expression of a heterologous polypeptide, such as transcriptional activators (e.g., trans-acting factors), chaperones, and processing proteases. Any factor that is effective in host cells, particularly bacterial cells and plant cells, may be used in the present invention. Nucleic acids encoding one or more of these factors are not necessarily tandem with the nucleic acid sequence encoding the heterologous polypeptide.

[0089] Recombinant vector or recombinant expression vector

[0090] The various nucleic acids and regulatory sequences described above can be connected to conventional vectors such as plasmids or viruses to produce the "recombinant expression vector" of the present invention, and the methods used are well known to those skilled in the art (for example, see J. Sambrook, E.F. Fritsch and T. Maniatus, 1989, Molecular Cloning Laboratory Manual, 2nd edition, Cold Spring, NY). The choice of vector usually depends on the compatibility of the vector with the host cell used. The vector can be a linear or closed circular plasmid. Such a vector can be an autonomously replicating vector, that is, a vector that exists in the form of an extrachromosomal entity, and its replication is independent of chromosomal replication, such as a plasmid (an extrachromosomal element), a minichromosome, or an artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can also be integrated into the genome and replicated along with the chromosome after introduction into the cell. The vector system can be a single vector or plasmid or two or more vectors or plasmids (which together contain the target nucleic acid sequence), or a transposon.

[0091] host cells

[0092] The present invention also relates to recombinant "host cells" containing the nucleic acid sequences of the present invention. A nucleic acid construct or vector containing the nucleic acid sequence of the present invention can be introduced into a host cell, causing the nucleic acid sequence of the present invention to integrate into the chromosome or causing the vector to replicate autonomously, thereby allowing the nucleic acid sequence of the present invention to be stably expressed or secreted in the host cell, thereby conferring xanthomonasine hydrolyzing activity on the host cell.

[0093] Host cells can be prokaryotes such as bacterial cells, or eukaryotic cells such as plant cells. Commonly used bacterial cells include Gram-positive bacteria such as Bacillus cells, or Gram-negative bacteria such as Escherichia coli and Pseudomonas cells.

[0094] Introduction of expression vectors into bacterial host cells can be achieved by protoplast transformation (e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), by using competent cells (e.g., Young and Spizizin, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation (e.g., Shigekawa and Dower, 1988, Biotech. 6: 742-751), or by conjugation (e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5771-5278).

[0095] genetically modified plants

[0096] After the heterologous DNA is introduced into plant cells, the integration of the heterologous gene into the plant genome can be confirmed by various methods known in the art.

[0097] PCR assay: PCR can be performed using oligonucleotide primers specific for the target gene or the Agrobacterium vector background to screen for the presence of the integrated gene in plant cells.

[0098] Southern Assay: Plant transformation can be confirmed by Southern blot analysis of genomic DNA.

[0099] In general, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, separated on an agarose gel, and transferred to a nitrocellulose or nylon membrane. The membrane or blot is then probed with, for example, a radioactively labeled target DNA fragment according to standard techniques to confirm integration of the introduced foreign gene into the plant genome.

[0100] RT-PCR assay: Transcription of heterologous genes in transgenic plants can be confirmed by RT-PCR assay of total RNA.

[0101] Western blot assay: Transgenic plants can be subjected to Western blots using antibodies that bind to one or several epitopes on the XanP protein.

[0102] The methods in the following examples are all conventional methods unless otherwise specified.

[0103] Xoc strains RS105, RS60, and RS85: described in the literature Wang et al. 2015. Rice OsFLS2-mediated perception of bacterial flagellins is evaded by

[0104] Xanthomonas oryzae pvs. oryzae and oryzicola. Molecular Plant 8, 1024-1037. Xoc strain SD21 was isolated from the field by the applicant. The above strains have undergone routine characterization and are available to the public from China Agricultural University.

[0105] Example 1: Obtaining the disease-resistant gene against Xanthomonas diseases of the present invention

[0106] Through research, the applicant of this patent has revealed for the first time the pathogenic molecular mechanism of the type III effector protein AvrBs2 of Xanthomonas.

[0107] Research has found that AvrBs2 is a cyclic phosphoglycolipid synthase. The applicant used homologous recombination technology to construct an avrBs2 gene knockout strain and its plasmid complement strain in the background of rice bacterial leaf streak pathogen (Xoc) RS105. For specific steps, see Li et al., "The Type III Effector AvrBs2 in Xanthomonas oryzae pv. oryzicola Suppresses Rice Immunity and Promotes Disease Development. Mol. Plant Microbe Interact. 2015, 28, 869-880." After culturing each strain overnight in NA medium, the cells were collected and injected into leaves of wild-type rice Nipponbare plants. Two weeks later, the length of the diseased leaf lesions was counted (Li et al., The Type III Effector AvrBs2 in Xanthomonas oryzaepv.oryzicola Suppresses Rice Immunity and Promotes Disease Development. Mol. Plant Microbe Interact. 2015, 28, 869-880). In addition, the lesion area of ​​the diseased leaf was cut and collected, frozen in liquid nitrogen, and then crushed into powder. The powder was weighed, and 100 μl of distilled water and 100 μl of phenol solution were added to every 100 mg of powder. After mixing, the mixture was treated at 65°C for 5 minutes, and then centrifuged at 13,400 g for 10 minutes. The supernatant was collected and analyzed on a TLC silica gel 60 thin layer chromatography plate (Merck Millipore, Darmstadt, Germany), chromatographed in a developing solvent (isopropanol / distilled water / acetic acid = 3 / 1 / 1; v / v / v) for 20 min, then treated with diphenylamine-aniline-phosphoric acid color developer and allowed to stand at 100°C for 10 min before color development. Figure 1 As shown, during the infection of plants by Xanthomonas, avrBs2 can synthesize a new phosphoglycolipid compound in plant cells, which we named xanthomonasine, and its structural formula is shown in Formula I. We further isolated and purified the compound and identified its structure as bis-1',6'-cyclicdimericα-D-galactose-phosphate.

[0108]

[0109] The method for obtaining the xanthomonasine is as follows: first, the coding region of avrBs2 is constructed into the dexamethasone (Dex)-inducible expression vector pTA7001, and then the pTA7001-avrBs2 plasmid DNA verified to be correct by sequencing is transformed into the Agrobacterium strain EHA105, which is used to transform rice to obtain pTA7001-avrBs2-transformed rice (Li et al., The Type III Effector AvrBs2 in Xanthomonas oryzae pv. oryzicola Suppresses Rice Immunity and Promotes Disease Development. Mol. Plant Microbe Interact. 2015, 28, 869-880.).

[0110] After dehulling, rice seeds transformed with pTA7001-avrBs2 were soaked in 75% ethanol for 2 minutes, then surface-sterilized by soaking in 50% sodium hypochlorite solution for 40 minutes. The seeds were then washed six times with sterile water. After drying on sterile filter paper, the seeds were placed on 1 / 2 MS solid medium and grown for one week. The grown seedlings were treated with 1 / 2 MS liquid medium supplemented with 30 μM DEX (400 ml per bottle of 1 / 2 MS liquid medium supplemented with 30 μM DEX) for 1-2 weeks. 400 ml of culture medium (or 200 g of seedlings ground into powder and added with 200 ml of ddH2O) and 400 ml of phenol were mixed and then heated at 65°C for 30 minutes. The mixture was stirred every 10 minutes and filtered to remove solid impurities. The mixture was centrifuged at 10,000 g for 10 minutes at 25°C. The aqueous layer was collected and the phenol layer discarded. The aqueous phase was extracted with ether (volume ratio, aqueous phase: ether = 10:1) and then concentrated by rotary evaporation at 65°C to a final volume of 20 ml. The column was passed through a Sephadex G-25 molecular sieve column, 20 ml of the concentrated extract was added, and elution was performed using 0.01 M NH4HCO3 as the mobile phase with 500 ml of 0.01 M NH4HCO3 solution. The first 100 ml was discarded, and the subsequent 10 ml fractions were collected in separate tubes. The compound was then analyzed by thin-layer chromatography. Fractions containing the novel compound of Formula I at a higher concentration were combined and concentrated by rotary evaporation at 65°C to a final volume of 5 ml. The molecular sieve column was repeated once. The concentrated compound was passed through a 1 ml anion exchange chromatography column (GE Healthcare), pre-equilibrated with deionized water, and then linearly eluted with 0-100% 1 M NH4HCO3 solution over 30 column volumes. The eluent was continuously sampled at 1 ml per column for thin-layer chromatography analysis. The eluates containing the new compound were combined and analyzed by thin layer chromatography. The eluted sample containing the new compound was rotary evaporated at 65°C to form a powder. The anion exchange chromatography was repeated three times and the powder was dried by rotary evaporation to obtain the compound xanthomonasine.

[0111] The applicant further analyzed the toxicity of xanthomonasine in infection. First, the applicant dissolved the purified xanthomonasine in a 0.001% silwet solution to prepare a 1mM suspension. Then, the applicant sprayed the xanthomonasine suspension or mock (0.001% silwet aqueous solution) on six-week-old rice leaves. After 24 hours, the leaves were treated according to the Figure 1 The ΔavrBs2 knockout strain was used to inoculate rice leaves, and the length of leaf lesions was counted two weeks later. Figure 2 As shown in the figure, the length of the lesions on rice leaves pre-treated with xanthomonasine solution was significantly greater than that on leaves treated with mock, indicating that AvrBs2 performs its toxic function by synthesizing xanthomonasine, helping bacteria to cause disease.

[0112] The applicant, through genome sequence comparison and analysis of different bacteria of the genus Xanthomonas, found that there is a highly conserved gene xanP near the avrBs2 gene. This gene is predicted to encode a 2H type phosphodiesterase of unknown function. We speculate that XanP may be a hydrolase that can hydrolyze xanthomonasine. The applicant then amplified the coding region of xanP by PCR amplification. It was amplified from the bacterial leaf streak pathogen RS105 by PCR. The genomic gene of the bacterial leaf streak pathogen RS105 was used as a template and pET28a-xanP-F AAAGCATATGatgtcgtcgtttctggacac; pET28a-xanP-RaaaGAATTCTCAGACCTCATGCATCGTGC were used as primers for PCR amplification. After the amplified product was recovered and purified, it was digested with restriction endonucleases NdeI and EcoRI and ligated into the pET28a vector with T4 ligase to obtain the recombinant plasmid pET28a-xanP. The recombinant plasmid was then transformed into competent cells of the Escherichia coli strain BL21 (DE3), and the monoclonal colony was expanded and cultured. The plasmid was extracted and sequenced for verification. The results showed that the phosphodiesterase gene xanP amplified was the disease-resistant gene against Xanthomonas diseases of the present invention, and its sequence was shown in Sequence 1 in the sequence listing. The nucleotides 1-786 from the 5′ end of Sequence 1 in the sequence listing were the coding sequence, and the amino acid sequence shown in Sequence 2 in the coding sequence listing was the anti-Xanthomonas disease-related protein XanP of the present invention.

[0113] The nucleotide sequence of sequence 1 in the sequence table is:

[0114]

[0115] The amino acid sequence of sequence 2 in the sequence table is:

[0116]

[0117] Example 2: Verification of the degradation effect of the anti-Xanthomonas disease-related protein of the present invention on xanthomonas alkaloids

[0118] Based on the pET28a-xanP plasmid, the catalytic active site mutant plasmid pET28a-xanP was obtained by PCR site-directed mutagenesis. M1 (H99A / T101A) and pET28a-xanP M2 (H204A / S206A), of which pET28a-xanP M1 (H99A / T101A) expressed protein xanP M1Compared with XanP, the 99th H at the amino terminal was mutated to A, and the 101st T at the amino terminal was mutated to A; pET28a-xanP M2 (H204A / S206A)) expressed protein xanP M2 Compared with XanP, the 204th amino terminal H was mutated to A, and the 206th amino terminal S was mutated to A. M1 (H99A / T101A) and pET28a-xanP M2 (H204A / S206A) and pET28a-xanP were transformed into BL21(DE3) competent cells respectively. Then the correct transformants were cultured in LB liquid medium containing 50 mg / L kanamycin sulfate at 37°C overnight, and then transferred to new LB liquid containing 50 mg / L kanamycin sulfate at a ratio of 1:100 and cultured at 37°C until the OD 600 = 0.6, then 100μM IPTG was added and cultured overnight at 16°C. The cells were then collected by centrifugation and 20ml of lysis buffer (50mM Tris-Cl, pH 8.0, 150mM NaCl and 10mM imidazole) was added. The cells were ultrasonically disrupted on ice and the lysate was centrifuged at 13,400g for 10 minutes at 4°C. The supernatant was added to a nickel column and passed through the column twice with the lysis buffer. The column was then passed through twice with the wash buffer (50mM Tris-Cl, pH 8.0, 150mM NaCl and 20mM imidazole). Finally, the protein was eluted with the elution buffer (50mMTris-Cl, pH 8.0, 150mM NaCl and 150mM imidazole). pET28a-xanP M1 (H99A / T101A) and pET28a-xanP M2 (H204A / S206A) and purified proteins after expression in pET28a-xanP (XanP M1 、XanP M2 After quantification, 1 μg protein XanP was taken. M1 、XanP M2and XanP and phosphodiesterase rpfG (specifically hydrolyzes c-di-GMP, the protein was obtained by the applicant through prokaryotic expression and purification, see Wei, et al. (2021). The PdeK-PdeR two-component system promotes unipolar localization of FimXand pilus extension in Xanthomonasoryzaepv.oryzicola. Science Signaling 14, i9589.) were added to a 1 mM concentration of xanthomonasine aqueous solution, reacted at 28 ° C for 1 hour, and then analyzed by thin layer chromatography. The results are as follows Figure 3 As shown, Figure 3 , purified XanP can efficiently degrade xanthophylline, while mutant XanP M1 、XanP M2 The phosphodiesterase rpfG (which specifically hydrolyzes c-di-GMP) could not degrade xanthan gum, indicating that XanP could be a xanthan gum hydrolase.

[0119] Based on the above breakthrough research findings, the applicant proposed in the present invention a "xanthomonas-resistant transgenic disease-resistant breeding method" for Xanthomonas pathogenic bacteria, namely, by transforming the xanP gene encoding the phosphodiesterase XanP in Xanthomonas into different host plants (including but not limited to rice (Oryza, including Oryza sativa L.), citrus (Citrus), pepper (Capsicum frutescens L.), tomato (Solanumlycopersicum L.), rapeseed (Brassica campestris L.), cabbage (Brassica oleracea L.)), so that different host plants acquire the ability to decompose xanthomonas synthesized by AvrBs2, thereby reducing the cytotoxicity of the effector protein AvrBs2 and enhancing the disease resistance of transgenic plants to different Xanthomonas diseases.

[0120] Example 2: Acquisition of transgenic rice and verification of its disease resistance

[0121] 1. Construction of transgenic constructs

[0122] To express the xanP gene in plants, a transgenic construct ( Figure 4To detect the expression of XanP protein, the full-length coding sequence xanP (SEQ ID NO: 1) amplified by PCR was digested with restriction endonucleases KpnI and HindIII and then ligated into the plant expression vector pC1305 to obtain a recombinant vector expressing the xanP gene. This recombinant vector was named pXanP01.

[0123] In the pXanP01 vector, the 3' end of the polynucleotide sequence encoding the xanP gene is connected to the polynucleotide sequence encoding 3×Flag (SEQ ID NO: 3), and the xanP-3×Flag fusion polynucleotide fragment is transcribed by the maize Ubi promoter.

[0124] 2. Construction of transgenic plants

[0125] 2.1 Transformation of recombinant plasmid into Agrobacterium EHA105 competent cells

[0126] Add 5 μl of pXanP01 plasmid to 50 μl of Agrobacterium competent cells (EHA105); place on ice for 15 minutes, then quickly place in liquid nitrogen for 90 seconds, and then place in a 37°C water bath for 1 minute; then quickly place on ice for 3 minutes; add 500 μl of LB liquid culture medium; place in a horizontal shaker and recover at 28°C, 200 rpm for 3 hours; in a clean bench, take 50 ul of bacterial liquid and spread it on LB culture medium containing kanamycin (Ka) + rifampicin (Rif) antibiotics; place the inverted spread plate at 28°C and culture for 48 hours. Then pick multiple single spots and mix them, inoculate them into 5 ml of LB liquid culture medium containing Ka + Rif antibiotics, and culture them on a shaker at 28°C, 200 rpm for 24 hours. 2.2 Transformation of callus induced by mature embryos of rice Nipponbare using Agrobacterium

[0127] 2.2.1. Callus Induction from Mature Rice Embryos

[0128] Hulled mature seeds of the wild-type rice variety Nipponbare (Oryza sativa L. cv. Nipponbare) were first soaked in 75% ethanol for 1-2 minutes, then in 50% sodium hypochlorite solution for 40 minutes for surface sterilization (preferably on a shaker). The seeds were then rinsed 3-4 times with sterile water. After drying on sterile filter paper, the seeds were placed on mature embryo callus induction medium NBi at 28°C with 16 hours of light and 8 hours of darkness. After approximately 7, 14, and 30 days, the callus growing from the mature embryo scutellum was removed and transferred to mature embryo subculture medium NBi for 7 days under the same conditions.

[0129] 2.2.2. Preparation of Agrobacterium culture solution

[0130] Agrobacterium EHA105 containing the target gene vector was cultured in a liquid medium containing 50 μg / mL Ka and 25 μg / mL RifYEP at 200 rpm and 28°C for 16-24 hours. The culture was spread on a new solid medium containing 50 μg / mL Ka and 25 μg / mL RifYEP, cultured in the dark at 28°C for 2-3 days, and then scraped and transferred to a co-culture liquid medium. Acetosyringone (AS) was added to a final AS concentration of 100 μM. The bacterial concentration was adjusted to OD 600 =0.1, which is the Agrobacterium suspension used for co-cultivation and transformation of rice.

[0131] 2.2.3. Agrobacterium infection of rice callus

[0132] Place the subcultured callus in a 100 mL sterile Erlenmeyer flask and add an appropriate amount of Agrobacterium suspension (ensure sufficient suspension is in contact with the material). Incubate at room temperature for 15 minutes with occasional gentle agitation. Discard the suspension and place the infected rice callus on sterile filter paper for 30 minutes. Remove any excess suspension from the surface of the rice callus and place on solid co-culture medium at 25°C in the dark for 3 days.

[0133] 2.2.4. Screening of Hygromycin-Resistant Calli and Transgenic Rice Seedling Differentiation

[0134] Place the co-cultivated callus into a 100 mL sterile Erlenmeyer flask, wash nine times with sterile water, then once with sterile water containing 400 mg / L Timentin. Blot dry the callus surface with sterile filter paper. Place the callus on resistant callus screening medium NBs containing 40 mg / L Hygromycin and incubate at 28°C for 30 days. Most calli will turn brown around 10 days after screening, followed by the re-growth of bright yellow resistant calli around the edges of the browned tissue. Select the newly emerged resistant calli and transfer them to freshly prepared screening medium for another 7-10 days.

[0135] From the resistant calli that emerged after two rounds of screening, bright yellow, dense resistant calli were selected and transferred to a regeneration medium containing 40 mg / L hygromycin (NBr). After approximately 15-25 days of light incubation, green spots appeared. After 30-40 days, seedlings further differentiated. When the buds from the resistant calli reached approximately 2 cm in height, the seedlings were transferred to a rooting medium and cultured for approximately two weeks. Seedlings approximately 10-15 cm tall with well-developed root systems were selected, the culture medium was washed off, and the seedlings were cultured in a tissue culture room for approximately one week in sterile water. Once the stems of the seedlings had grown robust, they were transplanted into soil. This resulted in the xanP transgenic rice line.

[0136] Callus induction medium Nbi: KNO3 2830mg / L, (NH4)2SO4 463mg / L, KH2PO4

[0137] 400mg / L, MgSO4.7H2O 185mg / L, CaCl2.2H2O 166mg / L, FeSO4.7H2O

[0138] 27.8mg / L, Na2EDTA 37.3mg / L, MnSO4.4H2O 10.0mg / L, H3BO3

[0139] 3.00mg / L, ZnSO4.7H2O 2.00mg / L, Na2MoO4.2H2O 0.25mg / L, CuSO4.5H2O 0.025mg / L, CoCl2.6H2O 0.025mg / L, KI 0.75mg / L, thiamine hydrochloride 10.0mg / L, pyridoxine hydrochloride 1.00mg / L, niacin 1.00mg / L, inositol 100mg / L, hydrolyzed casein 300mg / L, glutamine 500mg / L, proline 500mg / L, sucrose 30,000mg / L, 2,4-D2mg / L, plant gum 4g / L, pH adjusted to 5.8.

[0140] Co-culture medium Nbco:NBi medium was supplemented with 100 μM acetosyringone and the pH was adjusted to 5.5.

[0141] Resistant callus screening medium: NBi medium supplemented with 50 mg / L hygromycin and 400 μg / ml timentin.

[0142] Differentiation medium NBr: NBi medium supplemented with 0.5 mg / L α-naphthyl acetic acid, 3 mg / L 6-BA, and 200 μg / ml timentin.

[0143] 3. Western detection of transgenic rice

[0144] SDS-PAGE gel was prepared according to conventional techniques, wherein the concentration of the upper concentrated gel was 5% and the concentration of the lower separation gel was 12%. Leaf tissues of the xanP transgenic rice line were taken, and after being frozen in liquid nitrogen and crushed into powder, the total protein was extracted with 2×SDS loading buffer, subjected to SDS-PAGE, and then transferred to a nitrocellulose membrane. After the membrane was blocked for 1 hour in TBS / T buffer containing 5% milk, it was incubated with a 1:5000 diluted anti-Flag monoclonal antibody (sigma-Aldrich) at room temperature for 1 hour, and then washed 3 times with TBS / T buffer. Then, it was incubated with a 1:3000 diluted goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (CWBio) for 1 hour and washed three times with TBS / T buffer. Then, a chemiluminescent colorimetric solution was added for chemiluminescent imaging. As Figure 5 As shown, XanP expression bands were detected in the xanP transgenic rice lines OE-xanP-4 and OE-xanP-5, while no target bands were detected at the corresponding positions in the wild-type Nipponbare.

[0145] Example 4: Determination of resistance of transgenic rice to bacterial leaf streak fungus

[0146] Different Xoc strains (RS60, RS85, and SD21) were streaked on NA medium plates and cultured in a 28°C incubator for 48 hours. Single spots were then picked and cultured in NA liquid medium for 24 hours. After the cells were collected, they were washed three times with 10 mM MgCl2. The cell concentration was then determined and the bacterial solution concentration was adjusted to OD 600 =0.3. Six-week-old xanP transgenic rice lines OE-xanP-4 and OE-xanP-5 were selected and inoculated with the same upper leaves by pressure injection. The leaves were then placed at 28°C and kept moist. The length of the lesions was measured after about 14 days. Wild-type Nipponbare rice was used as a control. Figure 6 As shown, after inoculation with X. oryzae (Xoc) strains RS60, RS85, and SD21 (see Wang et al. 2015. Rice OsFLS2-mediated perception of bacterial flagellins is evaded by Xanthomonas oryzae pvs. oryzae and oryzicola. Molecular Plant 8, 1024-1037. Strain SD21 was isolated and identified from diseased rice in the field in 2021), the lesion lengths of xanP transgenic rice plants OE-xanP-4 and OE-xanP-5 were significantly shorter than those of wild-type rice Nipponbare. This suggests that expressing XanP in rice can enhance resistance to Xoc.

[0147] Example 5: Detection of xanthocyanine accumulation in transgenic rice after inoculation with leaf streak pathogen

[0148] Xanthomonas (Xoc) strain RS105 was used to inoculate xanP transgenic rice OE-xanP-4 and OE-xanP-5 and wild-type rice Nipponbare plants according to the method in Example 4. 14 days after inoculation, the diseased tissue of the leaves was taken, weighed, frozen with liquid nitrogen, and shaken into powder. Then, equal volumes of phenol and water were added, mixed, and incubated at 65°C for 5 minutes. Then, centrifuged at 13,400g and 4°C for 10 minutes, and the aqueous phase was taken. The sample was then subjected to thin layer chromatography analysis, and xanthomonasine (as shown in Formula I) was developed using aniline-diphenylamine colorimetric solution. Thin layer chromatography analysis was performed simultaneously using xanthomonasine at concentrations of 2mM, 1mM, 0.8mM, 0.6mM and 0.4mM as standard products. The grayscale value of the xanthomonasine band was counted using Photoshop software, a standard curve was drawn, and the xanthomonasine content in the lesions was calculated. As shown in FIG. Figure 7 As shown in the results, after Xoc infection, the accumulation of xanthocyanine in the leaf lesions of the xanP transgenic rice lines OE-xanP-4 and OE-xanP-5 was significantly lower than that in the leaves of the wild-type Nipponbare.

Claims

1. An application of an anti-Xanthomonas disease-related protein in enhancing plant disease resistance; the amino acid sequence of the anti-Xanthomonas disease-related protein is shown in Sequence 2 in the sequence listing; the disease resistance is resistance to rice bacterial leaf streak fungus; the plant is rice.

2. Use of a biomaterial related to the Xanthomonas disease-related protein shown in Sequence 2 in the sequence listing for enhancing plant disease resistance; the disease resistance is resistance to bacterial leaf streak pathogen of rice; the plant is rice; The biological material is any one of the following B1) to B7): B1) a nucleic acid molecule encoding the Xanthomonas disease resistance-related protein; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2); B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2).

3. The use according to claim 2, characterized in that: B1) The nucleic acid molecule is a cDNA molecule or a DNA molecule as shown in Sequence 1 in the sequence listing.

4. A method for cultivating disease-resistant transgenic plants, comprising: The gene encoding the protein related to Xanthomonas disease resistance shown in Sequence 2 in the sequence table is transferred into the target plant for expression to obtain a transgenic plant having higher disease resistance than the target plant; the disease resistance is resistance to bacterial leaf streak pathogen of rice; and the plant is rice.

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