Breeding method of rhizoctonia cerealis-resistant solanaceae and gramineae plants

Abstract

The invention discloses a breeding method of rhizoctonia cerealis resistant solanaceae and gramineae plants. The research finds that the RcLPMO1 gene plays a role in photosynthesis and plant immune pathways. Therefore, the RcLPMO1 gene and the encoding protein of the RcLPMO1 gene are used as targets to prepare the rhizoctonia cerealis fungicide, so that the resistance of solanaceae and gramineous plants to the destructive wheat pathogen is improved, and a solid foundation is laid for preventing and treating wheat sharp eyespot.

Description

Technical Field

[0001] The present application relates to anti-Rhizoctonia graminearum technology, and specifically to the use of the RcLPMO1 gene or its encoded protein as a target for preparing anti-Rhizoctonia graminearum agents. Background Art

[0002] Rhizoctonia cerealis is a soil-borne fungus that causes wheat sharpeyespot, which leads to a significant reduction in wheat yield. Currently, due to the lack of disease-resistant varieties, fungicides are mainly used to control the pathogen. However, chemical control is not environmentally friendly and is costly. At the same time, the lack of stable genetic transformation tools has hindered the study of the function of toxic genes. Summary of the invention

[0003] The invention systematically establishes the RNAi silencing system of Rhizoctonia graminearum and studies the function of secretory proteins.

[0004] The present invention first verified that RcLPMO1 was significantly upregulated during the infection period by means of RT-qPCR; then, after knocking down RcLPMO1 by HIGS and stable RNAi, it was found that the infection ability of Rhizoctonia graminearum was greatly reduced. These results together prove that RcLPMO1 is an important pathogenic gene during the infection period of Rhizoctonia graminearum.

[0005] The applicant found that the RcLPMO1 protein has the function of lytic polysaccharide monooxygenases (LPMOs), which can oxidatively cleave glycosides to help increase the activity of other glycoside hydrolases. The enzyme functional site is the 179th amino acid residue; RcLPMO1 has typical LPMO enzyme activity, but the plant necrosis reaction it triggers may not be completely dependent on enzyme activity.

[0006] The applicant also found that the 1-20 amino acid residues of RcLPMO1 are signal peptide regions with secretion function. The signal peptide has a significant effect on the subcellular localization of RcLPMO1, which can make it distributed outside the cell or in the chloroplast. It is speculated that RcLPMO1 with different localizations has different functions and jointly regulates the pathogenic function of Rhizoctonia graminearum.

[0007] Furthermore, the inventors screened and analyzed the downstream receptor proteins of RcLPMO1. RcLPMO1 can trigger plant immune responses with BAK1 and SOBIR1 as co-receptors outside the cell. Similarly, we suspect that RcLPMO1 affects the biological processes of plant chloroplasts at the protein level. Therefore, we screened and analyzed the downstream targets of RcLPMO1. Through the combined analysis of transcriptome and proteome, we found that RcLPMO1 can target the oxidase activating enzyme TaRCA, which is very important for plant photosynthesis, in chloroplasts. By targeting TaRCA, we discovered a new pathogenic mechanism by which Rhizoctonia graminearum destroys plant photosynthesis, disrupts the photosynthetic electron transport chain of plants, and produces a large amount of reactive oxygen to destroy the plant immune system.

[0008] In addition, RNA-seq analysis of wheat infected with R. graminearum revealed a set of highly differentially expressed genes, 52 of which had signal peptides. Transient expression of these secreted proteins in tobacco revealed that RcLPMO1 could significantly cause tobacco necrosis and promote necrotic pathogenicity by inducing host cell death. Targeted knockdown of RcLPMO1 significantly reduced R. graminearum infection. In tobacco, RcLPMO1 exhibits different functions depending on its localization in different cell regions. After secretion into the apoplast, RcLPMO1 triggers BRI1-ASSOCIATED KINASE-1 (BAK1) and SOBIR1-dependent plant immune responses, while when localized in chloroplasts, RcLPMO1 targets wheat oxidase activating enzyme (TaRCA), which is essential for plant photosynthesis. RCA is the Rubisco activating enzyme, which enables Rubisco to reach maximum activation under in vivo conditions. Rubisco is a key enzyme in photosynthesis that determines the rate of carbon assimilation and is also a key enzyme in plant photorespiration. It can use solar energy to fix CO2 in the air to form carbohydrates, thereby maintaining plant life. When RcLPMO1 interacts with TaRCA, Rhizoctonia graminearum stimulates the accumulation of reactive oxygen species (ROS) in plants, disrupts the photosynthetic electron transport chain to destroy photosynthesis, induces plant cell death, and promotes pathogen infection.

[0009] Based on this, the present invention provides a breeding method for Solanaceae and Poaceae plants resistant to Rhizoctonia graminearum, the method comprising transforming an RNAi interference vector of the RcLPMO1 gene in the plant, the RcLPMO1 gene sequence is shown in SIQ ID NO.2 or its encoded protein is shown in SIQ ID NO.1. Optionally, the RNAi interference vector structure of the RcLPMO1 gene is pK7GWIWG2D-RcLPMO1.

[0010] The present invention also provides another method for breeding plants of Solanaceae and Poaceae that are resistant to Rhizoctonia graminearum, the method comprising transforming RcLPMO1 in the plant Δsp Gene RNAi interference vector, the RcLPMO1 Δsp The gene sequence is shown in SIQ ID NO.3 or the encoded protein is shown in SIQ ID NO.4.

[0011] The Gramineae plant in the present invention is wheat; the Solanaceae plant is tobacco, pepper or tomato.

[0012] Accordingly, the present invention also provides an application of the RcLPMO1 gene or its encoded protein as a target for preparing an anti-Rhizoctonia graminearum agent, wherein the RcLPMO1 gene sequence is shown in SIQ ID NO.2 or its encoded protein is shown in SIQ ID NO.1. An application of the RNAi interference vector of the RcLPMO1 gene for preparing an anti-Rhizoctonia graminearum agent, wherein the RcLPMO1 gene sequence is shown in SIQ ID NO.2 or its encoded protein is shown in SIQ ID NO.1. And RcLPMO1 Δsp Application of a gene or a protein encoded by the gene as a target for preparing an anti-Rhizoctonia graminearum agent, wherein the RcLPMO1 Δsp The gene sequence is shown in SIQ ID NO.3 or the encoded protein is shown in SIQ ID NO.4. Δsp Application of RNAi interference vector of gene for preparing anti-granitoids fungus agent, the RcLPMO1 Δsp The gene sequence is shown in SIQ ID NO.3 or the encoded protein is shown in SIQ ID NO.4.

[0013] In addition, the present invention also provides another method for breeding plants of Solanaceae and Poaceae resistant to Rhizoctonia graminearum, the related method comprising low expression of RCA gene in the original Rhizoctonia graminearum resistant plants. Further, the RCA gene sequence is shown in SIQ ID NO.5 or the encoded protein sequence is shown in SIQ ID NO.8. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1Identification and characterization of proteins induced during infection with Rhizoctonia graminearum; (a) Heat map of differentially expressed genes during infection, values ​​are log2 values ​​of normalized gene expression levels; (b) RcLPMO1 induces plant cell death. Tobacco leaves carrying PVX-RcLPMO1-Flag or PVX-GFP were infected with Agrobacterium. The photo was taken 5 days after Agrobacterium injection, and immunoblotting was used to analyze tobacco proteins transiently expressing RcLPMO1-Flag; from left to right, the three pictures are: the phenotype of tobacco injected with RcLPMO1-Flag Agrobacterium, the decolorization phenotype after trypan blue staining of injected tobacco, and the protein bands after transient expression of RcLPMO1-Flag; (c) Agrobacterium transiently expressing RcLPMO1-Flag infected tobacco, pepper and tomato leaves, inducing plant cell death, from left to right, the three pictures are: the phenotype of tobacco, pepper and tomato injected with RcLPMO1-Flag Agrobacterium; GFP in the picture indicates: PVX-GFP empty vector, negative control.

[0015] Figure 2Pathogenicity analysis of RcLPMO1; (a) Relative expression level of RcLPMO1 during the infection stage of R. graminearum, with wheat leaves inoculated with strain R0301 as samples and hyphae grown on PDA as controls (0 hpi); (b) The siFi21 tool was used to predict the off-target effects of different RNAi fragments on the reference genome of R. graminearum (siRNA = 21 bp). The gray box highlights the area used for RNAi. No effective siRNA was predicted in the reference genomes of wheat or humans; (c) After inoculation with BSMV, the fourth wheat leaf showed slight chlorosis at 10 dpi. Wheat leaves infected with BSMV:TaPDS as a positive control showed a photobleaching phenotype. The fourth leaf of MOCK had no obvious phenotype change. The symptoms of the fourth leaf after inoculation with strain R0301 are shown in the figure. Plants with silenced GFP were negative controls. Bar = 1 cm. The mean and standard deviation of the diseased area of ​​three biological replicates were estimated. Asterisks indicate (*P<0.05; **P<0.01), and the difference was statistically significant by unpaired two-tailed Student's t test. The total DNA of inoculated wheat leaves was extracted to determine the ratio of fungal biomass to wheat biomass, and the absolute quantitative internal reference genes RcActin and TaEF1a were used to determine the relative expression of RcLPMO1. The data were normalized to the expression level of RcActin, BSMV:GFP indicates: vector control, BSMV:GFP is set to 1, and the mean and standard deviation were calculated using the data of 3 independent repetitions. Asterisks indicate (*P<0.05; **P<0.01) significant differences; in the figure, as1 indicates RNAi fragment 1 of RcLPMO1: as2 indicates RNAi fragment 2 of RcLPMO1.

[0016] Figure 3Results of analysis of wheat materials with stable silencing of RcLPMO1; (a) Schematic diagram of the hairpin structure of RcLPMO1-RNAi; (b) PCR was performed using primers specific for the RcLPMO1 fragment, and wheat DNA from the second leaf of the transgenic plant was used to detect the RcLPMO1 fragment. The wild type (WT) was the wheat cultivar Fielder, and TaEF1a was used as a positive control; L1, L2, L4, L11, and L15 represent the 1st, 2nd, 4th, 11th, and 15th transgenic lines of the T3 generation of RcLPMO1-RNAi, respectively; (c) GUS staining was used to identify RNAi-silenced RcLPMO1 transgenic plants. The activity of the GUS reporter gene was detected by GUS staining of transgenic wheat glumes; (d) RcLPMO1-RNAi transgenic plants significantly improved the resistance of wheat to Rhizoctonia graminearum. The leaves of the third-generation transgenic wheat (RNAi#L11 and #L15) were inoculated with Rhizoctonia graminearum, and phenotypes were taken 5 days after inoculation. The total DNA of the inoculated wheat leaves was extracted, and the ratio of fungal biomass to wheat biomass was determined. The absolute quantitative reference genes RcActin and TaEF1a were used. Asterisks indicate that the samples with HIGS silencing gene were significantly different from the control group (*P<0.05; **P<0.01); (e) H2O2 content of different transgenic lines at 48hpi, the mean and standard deviation were calculated using the data of 3 independent replicates; (f) RT-qPCR analysis of the silencing efficiency of RcLPMO1 in different transgenic lines, the second leaf of RcLPMO1-RNAi line was taken at 36 and 48h after inoculation to extract RNA, the data were normalized to the expression level of RcActin, and the mean and standard deviation were calculated using the data of 3 independent replicates. Asterisks indicate that the difference is statistically significant compared with WT (P<0.05, **P<0.01).

[0017] Figure 4 Analysis of the lytic polysaccharide monooxygenase activity and signal peptide of RcLPMO1; (a) Polymorphism analysis of RcLPMO1. The RcLPMO1 protein sequence of all AA9 family proteins is consistent with CLUSTALW; (b) Functional verification of the RcLPMO1 signal peptide; The Avr1b signal peptide of soybean phytophthora was cloned into pSUC2 as a positive control, and the untransformed Saccharomyces cerevisiae YTK12 and YTK12 carrying an empty pSUC2 vector were used as negative controls; CMD / -W means: also known as SS / -Trp medium, used to screen YTK12 yeast transferred into the pSUC2 vector; YPRAA means: YPRAA medium contains two parts, YPR Agar medium and Antimycin A; (c) RcLPMO1 and RcLPMO1 Y179AProkaryotic expression of RcLPMO1 and electrophoretic analysis of the expression products separated by 15% SDS-PAGE gel; (d) Chromogenic enzymatic characterization of RcLPMO1; Under standard conditions, the optimal temperature or pH value can be determined by simply changing the assay temperature or pH value, while K + ions seemed to show opposite effects, and the mean and standard deviation were calculated using data from three independent replicates.

[0018] Figure 5 Analysis of RcLPMO1 encoding lytic polysaccharide monooxygenase; (a) Enzymatic characterization of RcLPMO1 by absorbance method. The mean and standard deviation were calculated using the data of three independent replicates. The asterisk indicates that the difference is statistically significant compared with RcLPMO1 (**P<0.01); (b) Carrying PVX-RcLPMO1-Flag, PVX-RcLPMO1 Y179A Tobacco leaves infected with RcLPMO1-Flag or PVX-GFP were photographed on the fifth day. Y179A -Flag tobacco protein immunoblot analysis. The degree of cell death was quantified by measuring electrolyte leakage, and the mean and standard deviation were calculated using the data of 3 independent replicates. Asterisks indicate (P>0.05; **P<0.01), which are statistically significant compared with the expression of GFP protein; BAX in the figure represents: bcl2-associated protein X, a pro-apoptotic protein, which is a positive control; (c) Distribution of RcLPMO1 homologous proteins, the number of homologous sequences (specifically 4985) in the selected species (specifically: Basidiomycetes and Ascomycetes) is shown in the figure, and the cutoff value is e-value = 1*e-10.

[0019] Figure 6 Classification and expression of AA9 family proteins in Rhizoctonia graminearum; (a) Pfam domain analysis; (b) Phylogenetic analysis of homologous genes of RcLPMO1 in Rhizoctonia graminearum using the maximum neighbor method; (c) Heat map of differentially expressed genes encoding AA9 family proteins during infection, with values ​​being log2 values ​​of normalized gene expression levels.

[0020] Figure 7 Subcellular localization analysis of RcLPMO1; (a) Confocal microscopy imaging of tobacco cells after plasmolysis; RcLPMO1-GFP can be observed in the exoplasm of tobacco cells, but RcLPMO1 cannot be observed ΔSP-GFP and GFP, arrows point to extracellular protein fluorescence, FM4-64 was used as a plasma membrane marker, cell membrane (red) and green fluorescent protein (green), Bars = 50 μm; Merged: overlapping images, a new color is obtained by recalculating the color data of the pixels at the same position of two (or more) equal-sized images according to a formula; (b) transient expression of RcLPMO1-GFP, RcLPMO1 ΔSP -Fluorescence images of tobacco cells expressing GFP and free GFP, the photos were taken after Agrobacterium infection and protein expression, chloroplast (Chl, red) and GFP fluorescence (green), Bars = 50 μm; (c) transient expression in wheat protoplast cells under the regulation of 35S promoter, chloroplast (Chl, red) and GFP fluorescence (green), Bars = 5 μm.

[0021] Figure 8 RcLPMO1 triggers immune response and its necrosis induction depends on BAK1 and SOBIR1; (a, b) RcLPMO1, RcLPMO1 Y179A and RcLPMO1 ΔSPPromote the transcriptional accumulation of PTI or immune response marker genes in tobacco. After 60 hours of infection, total RNA was extracted from tobacco leaves, and relative expression levels were analyzed by RT-qPCR. The data were normalized to the expression level of NbActin. GFP-inoculated plants were used as negative controls, and the means and standard deviations of three biological replicates were calculated. Asterisks indicate (*P<0.05; **P<0.01), which were statistically significant compared with the negative control; NbAcre31 is a putative calcium-binding protein and a PTI marker gene, NbPT15 is a disease resistance-related gene, NbWRKY7 is a PTI marker gene, NbWRKY8 is a PTI marker gene, NBHIN1 is a HR-specific marker gene, and NbPR 1a is: salicylic acid marker gene, NbPR4 is: jasmonic acid marker gene, NbLOX is: jasmonic acid marker gene, NbERF is: ethylene-dependent immune marker gene; pTRV2-PDS represents the positive control experimental group, pTRV2-GEP represents the green fluorescent protein as the negative control experimental group, pTRV2-BAK1 represents experimental group 1, and pTRV2-SOBIR1 represents experimental group 2; (c) VIGS test was carried out on tobacco by inoculating TRV vector, and leaves infected with TRV:NbPDS were used as positive control, showing photobleaching phenotype; (d) The relative expression levels of genes were determined, and the data were normalized to the expression level of NbActin, TRV:GFP was set as 1. The mean and standard deviation were calculated using data from three independent replicates. Asterisks indicate significant differences (*P<0.05; **P<0.01) between samples silenced by VIGS and the control group. (e) After VIGS, Agrobacterium carrying the above vector had infected tobacco leaves. The photo was taken on the fourth day after Agrobacterium infection. The experiment was repeated three times. Immunoblotting analysis was performed to analyze the protein transiently expressed in gene-silenced tobacco leaves fused with RcLPMO1 and a Flag tag. inf1 represents a kinin protein from Phytophthora infestans. Rubisco represents ribulose-1,5-bisphosphate carboxylase / oxygenase, which is the main enzyme for absorbing CO(2) in the biosphere. It is highly abundant in leaves and has a molecular weight of about 55. It was used as an internal reference after staining with Ponceau red.

[0022] Fig. 9 To overexpress RcLPMO1 Δsp Produces ROS and affects plant photosynthesis; (a) RcLPMO1 ΔSP Induced plant cell death in tobacco leaves carrying PVX-RcLPMO1 ΔSP -Flag or PVX-GFP. The photo was taken five days after infection. Two days after infection, the cells transiently expressing RcLPMO1 were extracted. ΔSP-Flag tobacco protein was immunoblotted for analysis; (b) H2O2 content in tobacco, the photo was taken three days after infection, the mean and standard deviation were calculated using data from three independent replicates, and the asterisk (**P<0.01) indicates that the difference is statistically significant compared with that expressing GFP; (c) The electron transfer rate (ETR) of tobacco leaves was measured three days after infection, and the asterisk indicates that the difference is statistically significant compared with that expressing GFP (**P<0.01); (d) PCR was performed using RcLPMO1-specific primers, and wheat DNA from the second leaf of transgenic plants was used to detect the full length of RcLPMO1. WT is the wheat variety Fielder, and TaEF1a is used as a positive control; (e) The third generation RcLPMO1 ΔSP -3*Immunoblotting analysis of HA transgenic wheat protein; L7, L8, L13, and L19 represent: the third generation RcLPMO1 ΔSP -OE transgenic plants of the 7th, 8th, 13th, and 19th lines; (f) Determination of electron transfer efficiency of wheat leaves. Asterisks indicate comparison with WT (**P<0.01); (g) H2O2 content in wheat. The mean and standard deviation were calculated using data from 3 independent replicates, and asterisks indicate significant differences (*P<0.05) compared with WT; (h) RcLPMO1 ΔSP -OE transgenic plants significantly reduced wheat resistance to sheath blight. The leaves of the third-generation transgenic wheat (OE#L7 and #L8) were inoculated with Rhizoctonia graminearum. Bar=1cm. The photos were taken four days after infection. The ratio of fungal biomass to wheat biomass was determined by the total DNA content of the inoculated wheat leaves. The absolute quantitative reference genes RcActin and TaEF1a were used to calculate the mean and standard deviation using the data of 3 independent replicates. Asterisks indicate statistically significant differences compared with WT (**P<0.01).

[0023] Fig.10 To overexpress RcLPMO1 in tobacco Δsp Affects photosynthesis; (a) Based on the GO (Gene Ontology) enrichment analysis of differentially expressed genes in tobacco transcriptome, the vertical axis in the figure is: differentially expressed genes; (b, c) respectively determine the overexpression of RcLPMO1 in tobacco ΔSP The photosynthetic efficiency (Pn) and intercellular carbon dioxide concentration (Ci) of 3 biological replicates were calculated, and the asterisks indicate (*P<0.05; **P<0.01), which were statistically significant compared with those expressing GFP.

[0024] Fig.11 Analysis of the interaction between RcLPMO1 and RCA; (a) Luciferase reporter gene detection of RcLPMO1 ΔSP co-expression of RcLPMO1 in tobacco leaves ΔSP-Cluc and TaRCA-Nluc, with expression of Cluc (indicates: C-terminal functional fragment of luciferase protein) or Nluc (indicates: N-terminal functional fragment of luciferase protein) as negative control; (b) co-expression of TaRCA-GFP and RcLPMO1 ΔSP -RFP-Flag tobacco leaf protein extraction for Co-IP, anti-GFP antibody western blot detection of GFP protein; anti-Flag antibody western blot detection of Flag protein; expression of GFP and RcLPMO1 ΔSP -RFP-Flag is a negative control; (c) BiFC detection of RcLPMO1 ΔSP and TaRCA; negative control was expressed nYFP (indicated: bimolecular fluorescent protein n-terminal molecular fragment) or cYFP (indicated: bimolecular fluorescent protein c-terminal molecular fragment), expressed TaRCA-nYFP or RcLPMO1 ΔSP- cYFP; Bright field, chloroplast (Chl, red) and YFP fluorescence (green) images were taken by confocal microscopy; Bar = 50 μm; (d) RcLPMO1 in yeast ΔSP Interaction with TaRCA only occurs when co-expressing RcLPMO1 ΔSP Yeast expressing RcLPMO1 was grown in SD-Trp-Leu-His-Ade medium. ΔSP Or TaRCA cells cannot grow in SD-Trp-Leu-His-Ade medium. In the figure, AD represents: transcription activation domain, and BD represents: DNA binding domain.

[0025] Fig.12 The results of silencing TaRCA in the susceptible material Yangmai 158; after inoculation with BSMV, the fourth leaf of wheat showed slight chlorosis at 10 dpi. The symptoms of the fourth leaf inoculated with R0301 strain are shown in the figure. The BSMV:GFP inoculated plants were used as negative controls. Bar = 1 cm. The total DNA of the inoculated wheat leaves was extracted to determine the ratio of fungal biomass to wheat biomass. The absolute quantitative internal reference genes RcActin and TaEF1a were used. The asterisks indicate that the samples with VIGS silenced genes were significantly different from the control group (*P<0.05; **P<0.01). The relative expression level of RcLPMO1 transcripts was measured, and the data were normalized to the expression level of RcActin. BSMV:γ was set to 1. The mean and standard deviation were calculated using the data of 3 independent replicates. The asterisks indicate that the samples with VIGS silenced genes were significantly different from the control group (*P<0.05; **P<0.01). as1 and as2 represent: RNAi fragment 1 and RNAi fragment 2 of TaRCA, respectively.

[0026] Fig.13 The results of TaRCA positively regulating plant immunity; (a) The relative expression of TaRCA during the infection period of Rhizoctonia graminearum. Wheat leaves inoculated with R0301 were used as samples, and uninoculated wheat leaves were used as controls (0 hpi); (b) Tobacco leaves expressed RcLPMO1 ΔSP After inoculation with BSMV, the H2O2 content was determined. The photo was taken three days after Agrobacterium infection. The mean and standard deviation of the data from three independent experiments were calculated. Asterisks indicate (*P<0.05; **P<0.01), and the difference was statistically significant compared with the expression of GFP; (c) TaRCA expression in the silent disease-resistant material (cv.CI12633). After inoculation with BSMV, the fourth leaf of wheat showed slight chlorosis symptoms at 10dpi. The wheat leaves infected with BSMV:TaPDS as the positive control showed a photobleaching phenotype. After inoculation with R0301 strain, the symptoms of the fourth leaf were shown in the figure. The BSMV:GFP inoculated plants were used as Negative control, Bar=1cm. The ratio of fungal biomass to wheat biomass was determined by extracting total DNA from inoculated wheat leaves. The relative expression levels of the reference genes RcActin, TaEF1a and RcLPMO1 transcripts were used for absolute quantification. The data were normalized to the expression level of RcActin. BSMV:γ was set to 1. The mean and standard deviation were calculated using data from three independent replicates. Asterisks indicate (*P<0.05; **P<0.01) that the samples with VIGS-silenced genes were significantly different from those in the control group.

[0027] Fig.14 RcLPMO1 can participate in the normal interaction between TaRCA and RubisCo to regulate plant immunity; (a) VIGS test was carried out on tobacco by inoculating TRV vector, and the relative expression level of genes was determined. The data were normalized to the expression level of NbActin, TRV:GFP was set as 1, and the mean and standard deviation of three repeated independent experimental data were calculated. Asterisks indicate (*P<0.05; **P<0.01). Compared with TRV:GFP, the samples of VIGS silenced genes were statistically different from TRV:GFP. The leaves infected with TRV:GFP were used as negative control. The disease symptom area (cm) was shown 2 days after inoculation with Sclerotinia sclerotiorum. 2 ), Bar=1 cm; (b) Co-expression of TaRCA-GFP and RcLPMO1 ΔSP -RFP-Flag tobacco leaf protein extraction was processed for Co-IP, and GFP protein was detected by western blot using anti-GFP antibody, Flag protein was detected by western blot using anti-GFP antibody, and western blot was detected by anti-RubisCo antibody to express GFP and RcLPMO1 ΔSP-RFP-Flag is a negative control; (c) RubisCo (cv.CI12633) was knocked out in the disease-resistant material. After inoculation with BSMV, the fourth leaf of wheat showed slight chlorosis symptoms at 10 dpi. The R0301 strain was inoculated, and the symptoms of the fourth leaf were shown in the figure. The BSMV:GFP inoculated plants were used as negative controls. Bar = 1 cm. The total DNA of the inoculated wheat leaves was extracted, the ratio of fungal biomass to wheat biomass was determined, and the relative expression levels of the absolute quantitative internal reference genes RcActin and TaEF1a, RcLPMO1 transcripts were normalized to the expression level of RcActin, BSMV:γ was set to 1, and the mean and standard deviation were calculated using the data of 3 independent replicates. The asterisk indicates (*P<0.05; **P<0.01) that the samples with VIGS silenced genes were significantly different from the control group.

[0028] Fig.15 Analysis of the relationship between photosynthesis and wheat resistance to sheath blight; the base of wheat stems was inoculated, and the phenotype was taken 14 days after inoculation. Asterisks indicate significant differences (P<0.01, **P<0.01). DETAILED DESCRIPTION

[0029] Unless otherwise specified, the scientific and technical terms in this article are understood according to the knowledge of ordinary technicians in the relevant fields. Similarly, unless otherwise specified, the abbreviations of genes or proteins described in this article are all abbreviations of related genes or proteins known in the art.

[0030] The inventors previously analyzed the key secretory protein RcOSP1 and learned that secretory proteins play an important role during the infection period of Rhizoctonia graminearum. Based on the existing HIGS / VIGS silencing technology and transgenic stable silencing technology, the inventors systematically analyzed the secretory proteins induced during the infection period. Through transcriptome analysis of Rhizoctonia graminearum infection, the inventors identified a series of genes that were upregulated during the infection period and found that 52 of them were predicted to encode secretory proteins with signal peptides. The expression levels of these genes are as follows (see Figure 1 a).

[0031] Based on the above-mentioned preliminary research conclusions of the inventors, the present invention screened these genes and verified their functions. First, the full-length 52 secretory proteins with signal peptides obtained by transcriptome analysis were cloned into the PVX vector and transiently expressed in tobacco. It was found that the protein encoded by the gene with ID RhcerR0301Chr12A01440 can induce strong plant cell necrosis ( Figure 1b) The study found that the protein encoded by the gene with ID RhcerR0301Chr12A01440 belongs to the lytic polysaccharide monooxygenase family (LPMO), named RcLPMO1 gene encoding protein, the corresponding protein sequence is shown in SIQ ID NO.1, the RcLPMO1 gene sequence is shown in SIQ ID NO.1, the cLPMO1 gene is 702bp long, encoding a total of 233 amino acids, using online analysis software to predict that the protein has a molecular weight of 24.24kDa and an isoelectric point of 9.15. RcLPMO1 without the signal peptide fragment RcLPMO1 Δsp The gene sequence is shown in SIQ ID NO.3, RcLPMO1 Δsp The encoded protein sequence is shown in SIQ ID NO.4.

[0032] The Rhizoctonia graminearum R0301 isolate used in the following examples was collected in Nanjing, Jiangsu, and was donated by Associate Researcher Li Wei of Jiangsu Academy of Agricultural Sciences.

[0033] Example 1: Functional analysis of Rhizoctonia graminearum lytic polysaccharide monooxygenase RcLPMO1

[0034] 1.1 Materials, reagents and instruments

[0035] 1.1.1 Materials

[0036] Escherichia coli DH5α strain was used for vector construction and cultured on LB (lysogeny broth) medium at 37°C; Agrobacterium tumefaciens GV3101 strain carrying pSoup companion factor was used to infect plants; yeast mutant strain YTK12 was used to verify the signal peptide function.

[0037] This embodiment uses three wheat varieties: CI12633, a material resistant to wheat sheath blight, Yangmai 158, a material susceptible to wheat sheath blight, and Fielder, a transgenic material; all wheats were cultured in a greenhouse with a photoperiod of 22°C, 14 h light / 20°C, 8 h dark; N. benthamiana, peppers, and tomatoes were cultured in a greenhouse with a photoperiod of 22°C, 14 h light / 20°C, 8 h dark.

[0038] The main vectors used in this example are pDONR222; pK7GWIWG2D; pCub; pTRV system vector; pGR106; pSUC2; BSMV-HIGS system vector; pDR195, 1302::GFP\1302::flag; PVX106::Flag; PET15b::His; pGADT7; pGBKT7; YNE\YCE; NLUC\CLUC.

[0039] The above-mentioned bacteria, wheat, Nicotiana benthamiana (N. benthamiana), pepper and tomato carrier experimental materials are all commercially available products.

[0040] 1.1.2 Reagents

[0041] Reverse transcription kit (Fermentas, USA), Ex TaqTM (Takara, Japan), restriction endonuclease (NEB, UK), plasmid microextraction kit (OMEGA, USA), Biozol (BioFluxTM), DNAase (Fermentas, USA), RNase inhibitor (Promege, USA), Ribo m7G Cap Analog (Promege, USA), Cellulase-R-10 (Yakult, Japan), Mecerozyme-R-10 (Yakult, Japan), β-mercaptoethanol (Amresco, USA), BSA (Amresco, USA), PEG4000 (Wako, Japan), MES (MP, France), SYBR Green (Invitrogen, USA), and others are commonly used reagents in biochemistry and molecular biology laboratories.

[0042] 1.1.3 Instruments

[0043] Gel imager (Bio-Rad, USA), fluorescence quantitative analyzer (Bio-Rad, USA), nucleic acid protein concentration detector NanoDrop-2000 (Thermo, USA), PCR instrument (Bio-Rad, USA), ultrapure water generator (Milli-Q, USA), HV-50 fully automatic high pressure sterilizer (HIRAYAMA, Japan), shaker NRY-200 (Shanghai, China), blood cell counter MC-02270113 (MC, China), metal bath (eppendorf, Germany), AKTA protein chromatograph (GE, USA).

[0044] 1.2 Solution

[0045] 1.2.1 Bioinformatics analysis

[0046] In order to determine whether the protein RcLPMO1 has secretory function, SignalP-5.0 (https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ) was first used to identify the signal peptide; then the protein RcLPMO1 predicted to have a secretory signal was input into TMHMM (Krogh et al., 2001) to determine whether it contains a transmembrane domain. Proteins containing signal peptides but no transmembrane domains have been identified as candidate secretory proteins.

[0047] The molecular size of RcLPMO1 was predicted using the Compute pI / Mw tool, and the localization of RcLPMO1 was determined by WoLFPSORT II (https: / / www.genscript.com / wolf-psort.html). All candidate R.cerealis CAZymes described by Wibberg (2016) were annotated using the dbCAN database (Yin et al., 2012), and the protein domains were analyzed using the HMMER software (http: / / www.ebi.ac.uk / Tools / hmmer / ).

[0048] 1.2.2 Gene function verification

[0049] 1.2.2.1 Transient silencing and overexpression of RcLPMO1 gene in tobacco (pepper / tomato)

[0050] The pGR107-RcLPMO1 vector (transient overexpression) and pTRV2-RcLPMO1 vector (transient silencing) were constructed, and the pGR107-RcLPMO1 and pTRV2-RcLPMO1 reconstruction vectors were respectively transferred into Agrobacterium GV3101 (pSoup), and cultured at 28°C for about 48 hours; the Agrobacterium culture was treated with 10mL 10mM MgCl2 solution (Dou et al., 2008), diluted to OD600 of 0.8, and injected. The transient expression vectors in pepper and tomato are both: PVX106::Flag.

[0051] 1.2.2.2 Transient gene silencing of RcLPMO1 in wheat

[0052] (1) Using the si-fi tool to align the RcLPMO1 similar sequence, and select the specific fragments of RcLPMO1 100-300bp as the target sequence for RNAi silencing, construct a HIGS / VIGS vector system mediated by BSMV (Barley stripe mosaic virus), the vector structure is: γ-PDS, restriction endonuclease NotI, PacI for sequencing.

[0053] (2) The three subunits α, β, and γ of barley streak mosaic virus were linearized by constructing plasmids. The constructed plasmids were: BSMV-α, BSMV-β, BSMV-γ, BSMV-γ-PDS, BSMV-γ-PDS-as1, and BSMV-γ-PDS-as2.

[0054] (3) The linearized DNA fragments were then transcribed into RNA in vitro using the RiboMAXTM Large Scale Production System-T7 Kit (Promega, USA) according to the instruction manual; all consumables must be RNase / DNase free.

[0055] (4) The virus inoculation method was as described in (Holzberg et al., 2002). The control group consisted of linearized α, β, and γ (10 μL each) added to 300 μL of FES buffer. The liquid was inoculated onto the second leaf of wheat by friction inoculation and rubbed three times.

[0056] 1.2.2.3 Transgenic wheat materials

[0057] (1) Construction of silencing (RNAi) or overexpression vector: For silencing, the PCR product was constructed into the intermediate vector pDONR222 using the Gateway technology and the BP enzyme; the constructed intermediate vector was linearized using the restriction endonuclease NRUI; the LR enzyme was used to construct it into the destination vector pK7GWIWG2D (the silencing vector is pK7GWIWG2D-RcLPMO1); for overexpression, the RcLPMO1 gene was constructed into the pCub vector (Huai et al., 2022) to obtain the pCub-RcLPMO1 reconstructed vector.

[0058] (2) The sequencing-verified plasmid was transformed into Agrobacterium EHA105 and spread on LB agar plates containing kanamycin and rifampicin, and a single clone was picked for PCR detection.

[0059] (3) The Agrobacterium plates were sent to the Transgenic Platform of the State Key Laboratory of Stress Biology of Arid Crops.

[0060] (4) Transgenic wheat seedlings were transplanted to the greenhouse. When the T0 generation wheat plants grew to the one-leaf and one-heart stage, DNA samples were collected, wheat DNA was extracted, and positive plants were identified. They were inoculated with Rhizoctonia graminearum, sprayed with water to keep moisture, and the phenotype was observed for about a week.

[0061] (5) Extract transgenic wheat DNA\RNA samples.

[0062] (6) Seeds from individual ears of PCR-positive plants were collected, marked, and then dried. After sowing, PCR testing of T1 and T2 generation wheat was performed using the same method as that of T0 generation plants, and finally T3 generation wheat seeds were harvested.

[0063] (7) Total RNA was extracted from wheat-rhizoctonia solani and reverse transcribed into cDNA by RT-PCR. The relative expression level of the target gene was detected by qRT-PCR. The experiment should be repeated three times.

[0064] (8) Transcriptome sequencing to further analyze the changes in gene expression levels in RNAi or overexpression transgenic wheat materials.

[0065] For transient silencing, overexpression and related transgenic material creation in tobacco, pepper and tomato, please refer to 1.2.2.1 and 1.2.2.3 above.

[0066] 1.2.2.4 Verification of signal peptide secretion function

[0067] (1) The target gene RcLPMO1 sp (Signal peptide fragment of RcLPMO1) was connected to the pSUC2 vector to obtain the recombinant plasmid pSUC2-RcLPMO1 sp , and prepare relevant reagents: YPD solid and liquid culture medium, CMD-W solid and liquid culture medium, YPRAA solid culture medium, 10mM acetic acid-sodium acetate (pH=4.7), 1M NaOH, 10% / 40% sucrose solution.

[0068] (2) Streak the yeast strain YTK12 stored at -80°C on a YPD plate and culture at 30°C for 2 days.

[0069] (3) Pick out the activated bacteria or directly pipette 200uL of the preserved bacterial solution into 50mL YPD liquid medium. Incubate at 30℃ and 250rpm with shaking overnight (16-18h).

[0070] (4) Collect the yeast culture obtained by shaking overnight into 2 mL centrifuge tubes, with 5 mL of culture in each tube. Centrifuge at 4500 rpm at room temperature for 5 min and discard the supernatant.

[0071] (5) Wash twice with 2 mL ddH2O and resuspend in 100 uL PEG / LiAc transformation solution to obtain competent state.

[0072] (6) Add 0.1 μg of recombinant plasmid and 10 μl of 10 mg / mL salmon sperm to each tube and vortex to mix.

[0073] (7) Add 600uL PEG / LiAc conversion solution, shake vigorously, and incubate in a 30°C water bath for 30 min.

[0074] (8) Add 70uL DMSO, gently invert to mix, place in a 42°C water bath for 15 min, and quickly cool in ice for 1-2 min.

[0075] (9) Centrifuge at 4500 rpm for 5 min at room temperature, discard the supernatant, resuspend in 500 uL ddH2O, spread 100 uL on a CMD-W medium plate, and culture at 30°C for 2-3 days.

[0076] (10) Select appropriate bacterial colonies on the CMD-W solid medium and the control preserved bacterial solution and add them to 3-5 mL of CMD-W liquid medium. Incubate at 30 °C and 250 rpm with shaking for at least overnight. When the bacterial solution becomes turbid, subsequent experiments can be performed.

[0077] (11) The obtained bacterial solution was collected into a 2 mL centrifuge tube and centrifuged at 8000 rpm for 1 min. 600 ≈1.0.

[0078] (12) After washing twice with ddH2O, the cells were plated on CMD-W solid medium or YPRAA solid medium and cultured at 30°C for 2-3 days.

[0079] 1.2.2.5 Enzyme activity assay

[0080] The purified RcLPMO1 protein was induced by prokaryotic expression, and the LPMO enzymatic properties of RcLPMO1 were tested according to the literature (Breslmayr et al., 2018).

[0081] 1.2.2.6 Photosynthetic electron transfer rate (ETR) determination

[0082] The photosynthetic electron transfer rate (ETR) was measured using Dual Pam100 (Walz Germany). RcLPMO1-FLAG, TaRCA-GFP and PVX-GFP were transiently expressed on 3-4 week-old tobacco leaves by Agrobacterium injection. When measuring, first clamp the tobacco leaves with leaf clips, keep them in the dark for 30 minutes, and then adjust the instrument parameters according to the instrument manual to measure the photosynthetic electron transfer rate ETR of the leaves. Select Fluo as the measurement mode and DR as the detector Detector1. Measure more than 5 leaves for each sample, and the measurement is carried out at 22°C.

[0083] 1.2.3 Protein interaction analysis

[0084] 1.2.3.1 Co-immunoprecipitation mass spectrometry analysis

[0085] After obtaining the immunoprecipitation sample, it was separated by electrophoresis and transferred to the membrane. Western blot detection showed that the target protein had been well transferred to the membrane and successfully recognized and captured by the GFP antibody. The sample was not longer than 1 cm when running on the separation gel (the gel strip was as short as possible to facilitate the recovery of protein by sol). Coomassie brilliant blue staining was performed, and the strips were cut out and placed in tubes and sent to Hangzhou Jingjie Biotechnology Co., Ltd. for mass spectrometry sequencing.

[0086] 1.2.3.2 Yeast two-hybrid

[0087] (1) Construction of AD\BD recombinant plasmid.

[0088] (2) The target DNA constructed in the BD vector was transformed into yeast AH109.

[0089] (3) Detect the self-activation ability of the bait protein.

[0090] (4) The AD vector was used to transform the yeast AH109 containing the BD vector.

[0091] (5) Positive clones were screened on four-deficient plates.

[0092] 1.2.3.3 Firefly luciferase (LUC) complementation

[0093] (1) Construct nLUC / cLUC vector and transform into Agrobacterium GV3101 (pSoup),

[0094] (2) The Agrobacterium culture solution was cultured at 28°C and 200 rpm for approximately 48 h.

[0095] (3) Centrifuge the bacterial solution at 8000 rpm for 10 min. Discard the supernatant, add 10 mM MgCl2, and shake gently. Add an appropriate amount of infection solution (AS), mix well by pipetting, and then place in the dark for 2 h. Take the Agrobacterium infection solution and mix it evenly until the final concentration OD600 value reaches 0.6-1.0, and inject it into tobacco.

[0096] (4) After culturing the tobacco in the dark for 24 hours, place it in normal light conditions for 24 hours.

[0097] (5) Apply firefly luciferase substrate away from light, let stand in the dark for 5 minutes, and then expose for 10 minutes on a plant living molecular labeling imaging instrument to obtain the interaction results.

[0098] 1.2.3.4 Co-immunoprecipitation (co-IP)

[0099] (1) The candidate gene RcLPMO1 was constructed with a 1302 framework (1302-RFP-Flag; 1302-GFP) vector and then mixed and injected into tobacco. The fluorescence was observed after 48 hours. If the corresponding fluorescence appeared, the protein was extracted for immunoprecipitation.

[0100] (2) Take 10 μL of Anti-GFP Magnetic Beads (ThermoScientific, USA) (mix before use and pipette) into a 1.5 mL centrifuge tube. Add 0.8 mL of non-denaturing tissue / cell lysis buffer (Solabo, China), centrifuge at 700 g for 10 min at 4°C, discard the supernatant, and repeat once. Balance the column.

[0101] (3) Add all the protein extracts and incubate in a 4°C refrigerator on a rotator for 4-8 hours.

[0102] (4) Wash the Anti-GFP Magnetic Beads to remove impurities, centrifuge at 2500 g for 2 min at 4°C, discard the supernatant, add 0.5 mL of non-denaturing tissue / cell lysis buffer, and repeat four to five times.

[0103] (5) Add protein loading buffer, boil for 10 min, and perform immunoassay.

[0104] 1.3 Results

[0105] 1.3.1 Identification and characterization of secretory proteins induced during Rhizoctonia graminearum infection

[0106] This example confirms the successful expression of RcLPMO1 in tobacco by Western blot experiments, and the expression of negative control GFP protein does not cause necrosis ( Figure 1 b) To verify its broad spectrum of necrosis induction, RcLPMO1 was transiently expressed in pepper and tomato ( Figure 1 c) (transient expression vectors in pepper and tomato are: PVX106::Flag), and found that RcLPMO1 can also cause necrosis. The inventors speculate that RcLPMO1 may trigger PTI\DTI (pathogen-associated molecular pattern-triggered immunity\damage-associated molecular pattern-triggered immunity) or may be due to other toxic effects.

[0107] 1.3.2 Silencing RcLPMO1 can inhibit pathogen invasion

[0108] In order to explore whether RcLPMO1 is an important pathogenic gene, we first verified that RcLPMO1 was significantly upregulated during the infection period by RT-qPCR. Figure 2 a), this result verifies the reliability of the transcriptome data.

[0109] Furthermore, to explore whether RcLPMO1 can participate in the pathogenicity of Rhizoctonia graminearum, two independent RNAi fragments, as1 and as2 ( Figure 2 b) (as1 (57-257bp) sequence: GGGTCACTACCGCTTCAACAAATATATCAATCCGTCCGGTGCGGTGACTGGCGAGTACCAATATGTCCGTGCGAACACTAACATGAATTCGCCGGTGACGGACGTTAACTCCAACGACTTCCGCTGCAACGTCGGTGGTCTCGCATCTGGAGGCAAGACCTCAACCACAACGGTTGCGGCCGGCTCAACTGTGGGATTCCA; as2(4 42-628bp) sequence: AAGATTCCCGCTGCTACACCCGCAGGAAATTATCTTCTCCGTATTGAGCACATTGGCTTACATGTTGCTTCCAGTGCCGGTGGAGCACAATACTACATAAGCTGCGCACAGCTAAGTATTACGGGTAACGgaaacggaaATCCAGCCAAAGTTTCAATCCCTGGTATTTACAAAGGGAACGAGCCCG), followed by host-induced gene silencing (Host-induced Gene silencing (HIGS) technology was used to transiently silence RcLPMO1. After verifying that HIGS could knock down RcLPMO1 through RT-qPCR, it was found that the infection area of ​​Rhizoctonia graminearum was greatly reduced, and the biomass of Rhizoctonia graminearum relative to wheat was greatly reduced ( Figure 2 c). This suggests that RcLPMO1 may be an important virulence gene during the infection period of Rhizoctonia graminearum.

[0110] Since RcLPMO1 may be beneficial to the pathogenesis of Rhizoctonia graminearum, a transgenic wheat material (RcLPMO1-RNAi) expressing a specific RNAi fragment of the RcLPMO1 gene was further constructed, and the variety was Fielder, in order to study the potential application of RcLPMO1 in improving wheat resistance. The transgenic positive plants were detected by using RcLPMO1 fragment-specific primers (the primer sequences used were RcLPMO1-F: CAAGATTCCCGCTGCTACACC; RcLPMO1-R: GGGCTCGTTCCCTTTGTAA) and GUS reporter gene activity assay, and the plants were successfully confirmed ( Figure 3 bc).

[0111] Next, we inoculated wheat fielder with Rhizoctonia graminearum strain R0301, and through statistical analysis of the diseased area and biomass, we found that the resistance of the T3 transgenic lines RcLPMO1-RNAi#L11 and #L15 was significantly enhanced ( Figure 3 d). At the same time, it was observed that the accumulation of reactive oxygen species (ROS) in RcLPMO1-RNAi plants was significantly reduced 5 days after inoculation ( Figure 3 e).

[0112] To verify whether the above results were due to the silencing effect of RcLPMO1, RT-qPCR was performed at 36 and 48 h after inoculation (hpi), and it was found that the relative expression of RcLPMO1 in RcLPMO1-RNAi#L11 and #L15 decreased by 70%-90% ( Figure 3 f) These results indicate that knocking down RcLPMO1 created stable transgenic material resistant to Rhizoctonia graminearum and that ROS accumulation was reduced in RcLPMO1-silenced plants after inoculation with the pathogen.

[0113] 1.3.3 RcLPMO1 encodes a lytic polysaccharide monooxygenase

[0114] In order to explore the potential function of RcLPMO1, the 233 amino acid sequence of RcLPMO1 was used as a query to search the HMMER protein database (http: / / fungi.ensembl.org / hmmer / index.html). It was found that RcLPMO1 has a typical signal peptide (1-20) and an Auxiliary Activity family 9 (AA9) domain (Pfam number: PF03443, 21-223), belonging to the lytic polysaccharide monooxygenase (LPMO) family (Forsberg et al., 2019).

[0115] The nine reported AA9 family protein sequences in the Carbohydrate Active Enzyme Database (CAZy, http: / / www.cazy.org / ) were used to perform multiple sequence alignment with RcLPMO1. Figure 4 a). The results showed that they have a high degree of homology. Combined with the position of each amino acid in the active site of AA9 family proteins, the inventors further explored the structure and function of these proteins (Kjaergaard et al., 2014), and speculated that the histidine residue at position 21 (H21), the histidine residue at position 93 (H93), and the tyrosine residue at position 179 (Y179) of RcLPMO1 together constitute the active center of RcLPMO1 ( Figure 4a). Using homology modeling (Chang et al., 2017), it was found that the RcLPMO1 protein has 10 β-folds, 8 of which form a tight β-sandwich structure, which is consistent with the reported characteristics of the AA9 LPMO protein (Kjaergaard et al., 2014). The two histidine residues in the active center are copper ion binding sites, and the tyrosine residues play a role in substrate binding.

[0116] In order to verify that RcLPMO1 has the function of a lytic polysaccharide monooxygenase, the inventors tested the activity of RcLPMO1. Purified RcLPMO1 protein was obtained through prokaryotic expression ( Figure 4 c), and the enzymatic properties were tested (Breslmayr et al., 2018). In the in vitro experiment, 2,6-DMP was oxidized to generate colored substances, indicating that RcLPMO1 has LPMO enzyme activity. When the pH is 9 and the temperature is 40 degrees Celsius, the activity of RcLPMO1 is the best and the K + Can promote the catalytic activity of RcLPMO1 ( Figure 4 d).

[0117] In order to further verify the association between the enzyme function of RcLPMO1 and necrosis induction, the inventors mutated the tyrosine residue at position 179 of RcLPMO1 to an alanine residue (Y179A) to obtain RcLPMO1 Y179A , found that RcLPMO1 Y179A The LPMO enzyme activity was significantly reduced ( Figure 5 a) but RcLPMO1 Y179A Can still cause plant cell necrosis ( Figure 5 b).

[0118] In order to understand the importance of proteins containing the AA9 domain to Rhizoctonia graminearum and its importance to fungi, HMMER was used to identify AA9 family proteins (Lozano et al., 2015). The study found that there are 48 genes encoding secreted proteins containing the AA9 domain in the Rhizoctonia solani genome. Among them, 40 proteins only contain the AA9 domain, and the length is significantly smaller than that of proteins containing two domains. By establishing a phylogenetic tree, it was found that AA9 family proteins can be clearly classified into two types of subfamilies ( Figure 6 ab). Transcriptome analysis revealed that 36 genes were expressed during the infection period, among which RcLPMO1 was abundantly expressed during the infection period ( Figure 6c). In nature, AA9 family proteins are found only in the subphyla Ascomycota and Basidiomycota, and are most widely distributed in the classes Agaricomycetes and Sordariomycetes, respectively. Typical plant pathogenic fungi are widely present in these two classes ( Figure 5 c).

[0119] Furthermore, it was found that biotrophic pathogens (such as rust, powdery mildew, and smut) rarely have genes encoding AA9, while semi-biotrophic or necrotrophic pathogen genomes (such as Rhizoctonia graminearum, Sclerotinia sclerotiorum, and Erythromyces erythrocytes) have more genes encoding AA9, which is similar to previous studies (Jagadeeswaran et al., 2021). Therefore, with such a rich distribution, AA9 family proteins may be very important weapons for Rhizoctonia graminearum and even most necrotrophic fungi during the period of infecting plants.

[0120] In summary, RcLPMO1 belongs to the AA9 family, and the encoded lytic polysaccharide monooxygenase is an important pathogenic factor in necrotrophic and semi-necrotrophic fungi. RcLPMO1 has typical LPMO enzyme activity, but the plant necrosis reaction it triggers may not be completely dependent on enzyme activity and may not trigger typical DTI (damage-associated molecular pattern-triggered immunity).

[0121] 1.3.4 The signal peptide of RcLPMO1 is required for its secretion

[0122] According to the prediction, amino acid residues 1 to 20 are the signal peptide region of RcLPMO1, which was named RcLPMO1. sp To verify that RcLPMO1 sp To determine whether the secretory function was exerted, its secretory function was tested.

[0123] First, RcLPMO1 sp The sequence was cloned into the pSUC2 vector and transformed into the yeast YTK12 strain; all transformants grew on CMD-W agar, and the experimental results showed that RcLPMO1 was transformed sp The transformant can be Avr1b sp This suggests that the candidate signal peptide residues of RcLPMO1 can play a role in secreting proteins outside the membrane ( Figure 4 b), thus a similar secretion function may also exist in Rhizoctonia graminearum.

[0124] Next, we further validated RcLPMO1 in tobacco spHas secretory function. Transient overexpression of RcLPMO1 and RcLPMO1 Δsp The subcellular localization of these proteins after tobacco plasmolysis was observed by confocal microscopy. It was found that the fusion protein RcLPMO1 with the candidate signal peptide residues could be secreted into the apoplast, but RcLPMO1 Δsp No, RcLPMO1 may be localized in chloroplasts ( Figure 7 a).

[0125] Therefore, RcLPMO1 was further verified Δsp Subcellular localization of RcLPMO1 in tobacco and wheat protoplasms observed by confocal microscopy Δsp Clearly localized in chloroplasts ( Figure 7 abc).

[0126] In summary, the signal peptide of RcLPMO1 has a secretory function and can be secreted into the plant body by plant pathogens. There are two distribution patterns in the plant body. One is that it is located in the apoplast during the hydrolysis of the cell wall, and the other is distributed around the chloroplast cells of the plant. We know that the subcellular localization of secretory proteins may affect their functions. Therefore, it is worth exploring the different functions of RcLPMO1 given by different locations.

[0127] 1.3.5 RcLPMO1 induces PTI that is dependent on BAK1 and SOBIR1

[0128] TtAA9E (Zarattini et al., 2021) is a homologous protein of RcLPMO1, which can hydrolyze cellulose into cos_AA9 and induce immune necrosis response DTI. Therefore, combined with previous results, we hope to understand whether AA9 family proteins can trigger PTI independently of enzyme function in addition to triggering DTI. The above study found that intact RcLPMO1 can induce plant cell necrosis. Y179A Can still induce necrosis ( Figure 5 b).

[0129] Therefore, we further examined the expression levels of PTI marker genes in tobacco. The PTI marker genes NbWRKY7, NbWRKY8, NbPTI5, and NbAcre31 (Wang et al., 2010, Kim et al., 2014) were expressed in RcLPMO1 and RcLPMO1 Y179A The expression of RcLPMO1 and RcLPMO1 was significantly increased in Y179A However, RcLPMO1 Δsp The increase was not significant ( Figure 8a). In summary, RcLPMO1 can induce PTI in tobacco and is not completely dependent on enzyme function.

[0130] Then, the expression of HR-specific marker genes (NbHSR203J and NbHIN1) and salicylic acid (NbPR1a), jasmonic acid (NbPR4 and NbLOX) ethylene-dependent immunity (NbERF1) marker genes was detected (Nie et al., 2019). RT-qPCR revealed that plant immune responses were expressed in RcLPMO1, RcLPMO1 Y179A The expression of RcLPMO1 was upregulated at Δsp The immune response triggered by the Figure 8 b).

[0131] Furthermore, by overexpressing RcLPMO1 in the presence of silenced BAK1 and SOBIR1 receptors, Figure 8 cde), found RcLPMO1 and RcLPMO1 Y179A The triggered necrosis response depends on BAK1 and SOBIR1 receptors.

[0132] In summary, when RcLPMO1 is distributed in the apoplast, it can induce plant cell death that depends on BAK1 and SOBIR1, and the necrosis is not completely dependent on its AA9 enzyme activity. Therefore, it is inferred that for the apoplast of Nicotiana benthamiana, RcLPMO1 may directly act as a PAMP to induce PTI. And the induction of this PTI depends on the triggering of BAK1, and BAK1 plays the function of a co-receptor. However, it is not yet known whether there is some direct receptor RLK / RLP that regulates the function of BAK1. At the same time, if RcLPMO1 does not enter the apoplast, it can still induce plant cell necrosis, and its necrosis is not entirely a typical PTI reaction.

[0133] 1.3.6 RcLPMO1 induces plant cell death in the cell, promotes pathogen invasion and affects photosynthesis

[0134] The above study confirmed that during the infection process, RcLPMO1 is mainly distributed in two locations: around the cell wall and around the chloroplast. Δsp , found that it can induce a weaker necrotic response ( Fig. 9 a) DAB staining revealed that overexpression of RcLPMO1 Δsp Can induce the production of reactive oxygen species ( Fig. 9 b).

[0135] In order to explore the reasons why RcLPMO1 induces plant cell death in cells and further explore its pathogenesis. ΔspRNA-seq was performed on tobacco, and GO (Gene Ontology) enrichment analysis was performed on the differentially expressed genes. It was found that these genes were mainly concentrated in photosynthesis, thylakoids, photosystems, and the production of leaf precursor metabolites and energy. Therefore, it was learned that RcLPMO1 Δsp It may affect the photosynthesis of plants. Fig.10 a).

[0136] Furthermore, the photosynthetic indexes of tobacco transient overexpression materials were determined. The experiment showed that the photosynthetic rate (Pn) was significantly reduced, and the intercellular carbon dioxide concentration (Ci) of plants was significantly increased ( Fig.10 bc). LPMO family proteins have redox functions, and their enzyme activity is affected by the rate of electron transfer in photosynthesis. Therefore, the overexpression of RcLPMO1 was measured. Δsp Effects on the electron transport rate (ETR) of photosynthesis Fig. 9 c) These results together indicate that overexpression of RcLPMO1 in tobacco Δsp It will negatively regulate plant photosynthesis.

[0137] To further study RcLPMO1 Δsp The inventors constructed RcLPMO1 of the wheat variety Fielder to investigate its role in the pathogenesis of wheat sheath blight. Δsp Overexpression (OE) material.

[0138] First, RcLPMO1 was identified and verified at the RNA level. Δsp Overexpression lines ( Fig. 9 d).

[0139] Next, due to RcLPMO1 Δsp The HA tag was overexpressed and Western blot was performed using HA antibody to successfully verify the expression of the protein at the protein level ( Fig. 9 e) Compared with the control wheat variety Fielder, the third generation RcLPMO1 Δsp The photosynthetic electron transport chain of the overexpressed material was inhibited ( Fig. 9 f), and ROS burst is enhanced under natural conditions ( Fig. 9 g). After inoculation with wheat sheath blight, it was found that its pathogenicity increased ( Fig. 9 h). In summary, overexpression of RcLPMO1 in wheat ΔspRegulates plant growth, development, photosynthesis and immunity. The inhibition of the photosynthetic electron transport chain also suggests that RcLPMO1 functions as a LPMO enzyme (Kracher et al., 2016).

[0140] 1.3.7 RcLPMO1 can interact with RCA

[0141] The inventors speculate that after RcLPMO1 enters the plant cell, it regulates the plant immune function at the protein level. In order to better understand the potential virulence function of RcLPMO1 during the infection of Rhizoctonia graminearum, a co-immunoprecipitation coupled with mass spectroscopy (CoIP-MS) scheme was used to identify the proteins that interact with RcLPMO1. Δsp As bait, GFP antibody was used to recognize cleavage from GFP and overexpressed RcLPMO1 Δsp of tobacco and performed immunoprecipitation to obtain candidate interacting proteins.

[0142] In order to further confirm the interaction between the candidate target and RcLPMO1 protein, the candidate target was first transiently expressed in tobacco using the LUC technology. Through screening, it was found that the protein with the ID TraesCS4B02G140300 (a component of the oxygenase activating enzyme, named TaRCA) could bind to RcLPMO1 and produce strong fluorescence ( Fig.11 a). To further confirm the interaction between the target TaRCA and the effector protein RcLPMO1, RcLPMO1-RFP-Flag and TaRCA-GFP vectors based on 1302 were constructed for co-immunoprecipitation experiments. Western blot results showed that TaRCA-GFP protein attached to GFP magnetic beads could pull down the RcLPMO1-Flag protein ( Fig.11 b). The negative control group GFP cannot pull down the RcLPMO1-Flag protein ( Fig.13 b).

[0143] At the same time, transient expression experiments were carried out in tobacco using bimolecular fluorescence complementation technology. The results showed that after co-injection of TaRCA and RcLPMO1, YFP fluorescence appeared specifically in chloroplasts ( Fig.11 c). Similarly, when tobacco leaves were injected with Agrobacterium carrying an empty vector as a negative control, no fluorescence was observed ( Fig.11 c). This experiment shows that TaRCA and RcLPMO1 have a specific interaction in chloroplasts.

[0144] To further verify whether they can interact in vitro, we then used yeast two-hybrid technology to perform in vitro verification. TaRCA-pGADT7 and RcLPMO1-pGBKT7 were transformed into the AH109 strain, and it was found that the transformants could grow on SD-LWHA plates, while the controls could not ( Fig.11 d), the results showed that there was an in vitro interaction between the two. The above results confirmed that RcLPMO1 interacted with TaRCA, a component of the oxygenase activating enzyme, in vivo and in vitro, and the interaction was spatially located in the chloroplast.

[0145] 1.3.8 RCA positively regulates plant immunity

[0146] The TaRCA gene encodes 464 amino acids, and the total length of the TaRCA gene is 1395 bp, belonging to AAA + (ATPases Associated with diverse cellular Activities) family protein, is a subunit of oxygenase activating enzyme. The protein has a molecular weight of 50.96kDa and an isoelectric point of 6.91. In order to further explore the function of TaRCA in anti-graminearum immunity, RT-qPCR was first used, and the results showed that TaRCA expression was significantly downregulated during the infection period ( Fig.13 a), which suggests that there is a negative regulatory mechanism at the transcriptional level in wheat that affects the gene expression of TaRCA. In tobacco, RcLPMO1 and TaRCA were transiently overexpressed, and DAB staining and H2O2 content determination revealed that co-expression of RcLPMO1 and TaRCA produced more reactive oxygen species ( Fig.13 b) The large amount of reactive oxygen species produced may also disrupt the plant's disease resistance system, facilitating the pathogenicity of Rhizoctonia graminearum.

[0147] Furthermore, we explored whether TaRCA could participate in directly regulating the pathogenicity of Rhizoctonia graminearum and silenced TaRCA through virus-induced gene silencing (VIGS) technology (silencing vector: BSMV-γ vector).

[0148] First, TaRCA may be involved in negative regulation of the disease resistance of Rhizoctonia graminearum, so TaRCA was silenced in the Rhizoctonia graminearum susceptible material (Yangmai 158), but the results showed that there was no significant change after successful silencing ( Fig.12 ). The situation is different in the Rhizoctonia graminearum resistant material (CI12633). After RT-qPCR verification that VIGS can knock down TaRCA, it was found that the infection area of ​​Rhizoctonia graminearum was greatly reduced, and the biomass of Rhizoctonia graminearum relative to wheat was greatly reduced ( Fig.13c). This suggests that TaRCA may be involved in the positive regulation of Rhizoctonia graminearum disease resistance.

[0149] In summary, we know that TaRCA can help RcLPMO1 produce more reactive oxygen, which is not conducive to the plant's own immunity to necrotrophic fungi. However, when TaRCA is silenced, the plant's own disease resistance decreases, which shows that TaRCA is regulating the plant's disease resistance process. It is not only an enzyme necessary for plant growth and development, but also plays an important role in the disease resistance process.

[0150] 1.3.9 Normal photosynthesis is beneficial to wheat resistance to sheath blight

[0151] In order to study whether RCA can positively regulate the disease resistance of various plants, the effect on Rhizoctonia graminearum was tested on tobacco. The homologous gene NbRCA of TaRCA was silenced by pTRV system vector, and GFP was silenced as negative control. After 2 days of inoculation, the infection area of ​​Rhizoctonia graminearum in tobacco with silenced NbRCA was significantly larger than that of the negative control ( Fig.14 a). The results showed that similar NbRCA positively regulated tobacco's resistance to Rhizoctonia graminearum. This suggests that in the plant-necrotrophic fungus interaction system, the normal function of RCA plays a positive role in regulating plant immunity and is an indispensable and important component.

[0152] RubisCo is one of the most abundant enzymes in plants. It accounts for about half of the total chloroplast proteins and is mainly used to fix carbon dioxide (Zhang et al., 2002). In vivo, RCA is essential for the activation of RubisCo (Rundle & Zielinski, 1991). RcLPMO1-Flag and TaRCA-GFP were constructed for co-immunoprecipitation experiments, and RubisCo-specific antibodies were used to detect the presence of RubisCo during the interaction between RcLPMO1 and TaRCA. Western blot results showed that TaRCA-GFP attached to GFP magnetic beads could pull down RubisCo regardless of the presence of RcLPMO1-Flag, while the negative control group GFP could not ( Fig.13 b). Therefore, it is known that RcLPMO1 can participate in the interaction between TaRCA and RubisCo, but the specific interaction conformation is still unclear. In order to understand the function of RubisCo in wheat resistance to sheath blight, RubisCo was transiently silenced in the Rhizoctonia graminearum resistant material (CI12633) by VIGS technology. After RT-qPCR verification that VIGS can knock down RubisCo, it was found that the infection area of ​​Rhizoctonia graminearum was greatly reduced, and the biomass of Rhizoctonia graminearum relative to wheat was greatly reduced ( Fig.14c) This suggests that RubisCo may also be involved in the positive regulation of Rhizoctonia graminearum disease resistance.

[0153] RCA and RubisCo are both important enzymes in photosynthesis, and they jointly regulate photosynthesis. Therefore, the inventors speculate that if plants cannot perform photosynthesis normally, it will be more conducive to the infection of Rhizoctonia graminearum. When inoculated with Rhizoctonia graminearum in a dark environment, the study found that the disease index of Rhizoctonia graminearum was significantly increased compared with that under normal light ( Fig.15 ). Therefore, under natural conditions, normal photosynthesis may be more conducive to wheat resistance to sheath blight.

[0154] The above-mentioned example studies confirmed that after the RNAi interference vector targeting the Rhizoctonia graminearum gene was transferred into wheat, it could confer stable genetic resistance to Rhizoctonia graminearum; and analyzed the function of RcLPMO1, an important pathogenic gene of Rhizoctonia graminearum, in chloroplasts.

[0155] The following examples systematically screened all the induced expression and secretory proteins from the transcriptome during the infection period through a tobacco transient expression system, among which the protein encoded by RcLPMO1 can induce cell necrosis.

[0156] Furthermore, the pathogenic function of RcLPMO1 was preliminarily explored through the analysis of its expression level and BSMV-mediated transient gene silencing. After that, the RNAi interference vector was transformed into wheat through Agrobacterium, and stable genetic positive plants were successfully obtained. For these positive plants, disease resistance identification and various molecular biological tests were carried out, and it was proved that RNAi technology can be used to improve wheat's resistance to Rhizoctonia graminearum. This technology enables transgenic plants carrying RNAi vectors to produce siRNA interference target gene RcLPMO1, achieving the effect of knocking down RcLPMO1 gene expression, thereby limiting the growth of fungi and reducing its pathogenicity.

[0157] It was further found that the accumulation of reactive oxygen species was significantly reduced after knocking down RcLPMO1. Therefore, in view of the powerful pathogenic function of RcLPMO1, the present invention further explored how it exerts its pathogenic function and found that:

[0158] (1) RcLPMO1 is a typical secretory protein, which is not a typical effector protein because it contains a known structural domain. On the contrary, RcLPMO1 encodes a lytic polysaccharide monooxygenase, which is a typical cell wall degrading enzyme or coenzyme. Therefore, its function may only be to assist plant pathogenic fungi in causing disease by degrading plant cell walls. Therefore, the function of its lytic polysaccharide monooxygenase was verified by prokaryotic expression, and its taxonomic status in nature and in the pathogen itself was analyzed by family analysis.

[0159] (2) The subcellular localization of RcLPMO1 was analyzed, and the results showed that the signal peptide of RcLPMO1 can play a role. If the signal peptide is present, RcLPMO1 will be localized in the apoplast, where it may play an enzymatic role and participate in the degradation of the cell wall.

[0160] (3) Similar to proteins such as XEG1, VmEO2, and EIX2 (Ma et al., 2015, Nie et al., 2019, Yin et al., 2020), RcLPMO1 may function as a PAMP. It may also be consistent with previous studies (Zarattin et al., 2021, Yang et al., 2021), in which its cell wall degradation products function as DAMPs. When expressed extracellularly, RcLPMO1-induced necrosis also depends on BAK1 and SOBIR1.

[0161] (4) When RcLPMO1 is expressed intracellularly, it can still induce plant cell necrosis. After expressing it in tobacco and performing transcriptome sequencing, it was found that it can significantly affect the photosynthesis of plant cells. Furthermore, by creating RcLPMO1 Δsp Overexpression materials were found to overexpress RcLPMO1 Δsp This can reduce wheat's resistance to pathogens. Therefore, if RcLPMO1 is present inside the cell, rather than in the apoplast or cell wall, it may play a different role.

[0162] (5) RcLPMO1 can interact with TaRCA at the protein level. A large number of secreted proteins RcLPMO1 can participate in the normal interaction between TaRCA and RubisCo to form heterotrimers. RubisCo plays an important role in photosynthesis, and is also affected by a variety of factors that regulate its efficient photosynthesis function (Rundle & Zielinski, 1991, Zhang et al., 2002). The presence of RcLPMO1 affects the normal function of RubisCo, affects the Calvin cycle, and thus affects photosynthesis.

[0163] SIQ ID NO.1

[0164] The protein sequence encoded by the RcLPMO1 gene (GenBank No. (OR593748)) is:

[0165] MKSVFTIVALAAAAVQPALGHYRFNKYINPSGAVTGEYQYVRANTNMNSPVTDVNSNDFRCNVGGLASGGKTSTTTVAAGSTVGFQSDIPIGHPGPLQVYLGKVPAGQTAATWDGSGANWFKIYTLGADFSSGTLAWPSSDKQNFNFKIPAATPAGNYLLRIEHIGLHVASSAGGAQYYISCAQLSITGNGNGNPAKVSIPGIYKGNEPGVLINIYYPPVTSYKAPGPAVWSG

[0166] SIQ ID NO.2

[0167] RcLPMO1 gene sequence:

[0168] ATGAAGTCAGTCTTTACCATTGTCGCACTTGCTGCTGCTGCAGTCCAGCCTGCATTG

[0169] GGTCACTACCGCTTCAACAAATATATCAATCCGTCCGGTGCGGTGACTGGCGAGTAC

[0170] CAATATGTCCGTGCGAACACTAACATGAATTCGCCGGTGACGGACGTTAACTCCAAC

[0171] GACTTCCGCTGCAACGTCGGTGGTCTCGCATCTGGAGGCAAGACCTCAACCACAAC

[0172] GGTTGCGGCCGGCTCAACTGTGGGATTCCAGTCTGATATCCCGATCGGCCATCCAGG

[0173] GCCTTTGCAGGTTTATCTTGGAAAGGTGCCTGCAGGTCAAACGGCTGCAACATGGG

[0174] ACGGAAGCGGCGCGAATTGGTTCAAGATATACACACTGGGTGCTGACTTCAGTTCG

[0175] GGTACTTTGGCGTGGCCTTCGAGCGACAAACAGAATTTCAATTTCAAGATTCCCGCT

[0176] GCTACACCCGCAGGAAATTATCTTCTCCGTATTGAGCACATTGGCTTACATGTTGCTT

[0177] CCAGTGCCGGTGGAGCACAATACTACATAAGCTGCGCACAGCTAAGTATTACGGGT

[0178] AACGGAAACGGAAATCCAGCCAAAGTTTCAATCCCTGGTATTTACAAAGGGAACGA

[0179] GCCCGGTGTCTTGATCAACATCTACTATCCACCAGTCACGAGCTACAAAGCGCCTGG

[0180] TCCCGCCGTATGGTCGGGTTGA

[0181] SIQ ID NO.3

[0182] RcLPMO1 Δsp Gene sequence:

[0183] CACTACCGCTTCAACAAATATATCAATCCGTCCGGTGCGGTGACTGGCGAGTACCAATATGTCCGTGCGAACACTAACATGAATTCGCCGGTGACGGACGTTAACTCCAACGACTTCCGCTGCAACGTCGGTGGTCTCGCATCTGGAGGCAAGACCTCAACCACAACGGTTGCGGCCGGCTCAACTGTGGGATTCCAGTCTGATATCCCGATCGGCCATCCAGGGCCTTTGCAGGTTTATCTTGGAAAGGTGCCTGCAGGTCAAACGGCTGCAACATGGGACGGAAGCGGCGCGAATTGGTTCAAGATATACACACTGGGTGCTGACTTCAGTTCGGGTACTTTGGCGTGGCCTTCGAGCGACAAACAGAATTTCAATTTCAAGATTCCCGCTGCTACACCCGCAGGAAATTATCTTCTCCGTATTGAGCACATTGGCTTACATGTTGCTTCCAGTGCCGGTGGAGCACAATACTACATAAGCTGCGCACAGCTAAGTATTACGGGTAACGGAAACGGAAATCCAGCCAAAGTTTCAATCCCTGGTATTTACAAAGGGAACGAGCCCGGTGTCTTGATCAACATCTACTATCCACCAGTCACGAGCTACAAAGCGCCTGGTCCCGCCGTATGGTCGGGTTGA

[0184] SIQ ID NO.4

[0185] RcLPMO1 Δsp Protein sequence:

[0186] HYRFNKYINPSGAVTGEYQYVRANTNMNSPVTDVNSNDFRCNVGGLASGGKTSTTTVAAGSTVGFQSDIPIGHPGPLQVYLGKVPAGQTAATWDGSGANWFKIYTLGADFSSGTLAWPSSDKQNFNFKIPAATPAGNYLLRIEHIGLHVASSAGGAQYYISCAQLSITGNGNGNPAKVSIPGIYKGNEPGVLINIYYPPVTSYKAPGPAVWSG

[0187] SIQ ID NO.5

[0188] TaRCA gene sequence:

[0189]

[0190] SIQ ID NO.6

[0191] TaRCA-as1: 1147bp-1374bp

[0192] CGAGAACATTGGCAAGAGGCTGGTGAACTCGCGGGACGGACCCGTGACCTTTGAGCAGCCAAAGATGACAGTGGAAGAAGCTGCTAGAGTACGGGCACATGCTTGTCCAGGAGCAGGACAATGTCAAGCGTGTGCAGCTTGCTGACACCACATGAGCCAGGCAGCTCTGGGTGATGCTAACCAGGATGCGATGGAAGACTGGTTCCTTCTACGGTAAAGGGGCACAGC

[0193] SIQ ID NO.7

[0194] TaRCA-as2: 1201bp-1375bp

[0195] GCAGCCAAAGATGACAGTGGAAGAAGCTGCTAGAGTACGGGCACATGCTTGTCCAGGAGCAGGACAATGTCAAGCGTGTGCAGCTTGCTGACACCACATGAGCCAGGCAGCTCTGGGTGATGCTAACCAGGATGCGATGGAAGACTGGTTCCTTCTACGGTAAAGGGCACAGCA

[0196] SIQ ID NO.8

[0197] TaRCADànbáixùliè:

[0198] MAAAFSSTVGAPASTPTNFLGKKLKKQVTSAVNYHGKSSKANRFTVMAAENLDEKRNTDKWKGLAYDISDDQQDITRGKGIVDSLFQAPTGDGTHEAVLSSYEYVSQGLKKYDFDNTMGGGFYIAPAFMDKLVVHLSKNFMTLPNIKIPLILGIWGGKGQGKSFQCELVFAKMGINPIMMSAGELESGNAGEPAKLIRQRYREAADMIKKGKMCCLFINDLDAGRMGGTTQYTVNNQMVNATLMNIADAPTNVQLPGMYNKEENPRVPIVVTGNDFSTLYAPLIRDGRMEKFYWAPTRDDRIGVCKGIFQTDNVSDESVVKIVDTFPGQSIDFFGALRARVYDDEVRKWVTSTGIENIGKRLVNSRDGPVTFEQPKMTVEKLLEYGHMLVQEQDNVKRVQLADTYMSQAALGDANQDAMKTGSFYGKGAQQGTLPVPAGCTDQTAKNFDPTARSDDGSCLYTF

Claims

1. A method for breeding Solanaceae and Poaceae plants resistant to Rhizoctonia graminearum, characterized in that: The method comprises transforming an RNAi interference vector of the RcLPMO1 gene in a plant, wherein the RcLPMO1 gene sequence is shown as SIQ ID NO.2 or the encoded protein is shown as SIQ ID NO.

1.

2. The breeding method according to claim 2 or 3, characterized in that: The RNAi interference vector structure of RcLPMO1 gene is pK7GWIWG2D-RcLPMO1.

3. A method for breeding Solanaceae and Poaceae plants resistant to Rhizoctonia graminearum, characterized in that: Transformation of RcLPMO1 in plants Δsp Gene RNAi interference vector, the RcLPMO1 Δsp The gene sequence is shown in SIQ ID NO.3 or the encoded protein is shown in SIQ ID NO.

4.

4. The breeding method according to claim 1 or 3, characterized in that: The grass plant is wheat; the solanaceous plant is tobacco, pepper or tomato.

5. The use of the RcLPMO1 gene or the protein it encodes as a target for preparing an anti-Rhizoctonia graminearum agent, wherein the RcLPMO1 gene sequence is shown in SIQ ID NO.2 or the protein it encodes is shown in SIQ ID NO.

1.

6. The use of the RNAi interference vector of the RcLPMO1 gene for preparing an anti-Rhizoctonia graminearum agent, wherein the RcLPMO1 gene sequence is as shown in SIQ ID NO.2 or the encoded protein is as shown in SIQ ID NO.

1. 7.RcLPMO1 Δsp Application of a gene or a protein encoded by the gene as a target for preparing an anti-Rhizoctonia graminearum agent, wherein the RcLPMO1 Δsp The gene sequence is shown in SIQ ID NO.3 or the encoded protein is shown in SIQ ID NO.

4. 8.RcLPMO1 Δsp Application of RNAi interference vector of gene for preparing anti-granitoids fungus agent, the RcLPMO1 Δsp The gene sequence is shown in SIQ ID NO.3 or the encoded protein is shown in SIQ ID NO.

4.

9. A method for breeding Solanaceae and Poaceae plants resistant to Rhizoctonia graminearum, characterized in that: The method includes under-expressing the RCA gene in a plant resistant to Rhizoctonia graminearum.

10. The breeding method according to claim 9, characterized in that: The RCA gene sequence is shown in SIQ ID NO.5 or the encoded protein sequence is shown in SIQ ID NO.8.

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