Functional Studies Of Three Pathogenic Genes In Ustilaginoidea Virens And The Construction Of Disease-resistant Transgenic Rice Lines

Rice false smut(RFS)caused by Ustilaginoidea virens,is a grain disease in the majority of rice-growing areas of the world.Occurrence of RFS not only causes yield loss but also threatens human or animal health by producing mycotoxins.At present,there are few reports on the pathogenic mechanism of U.virens and the resource mining of RFS resistance.In this study,the pathogenic mechanism of the three virulence factors,UvPal1,Uv CGBP1 and Uv Sec117 were investigated,and a new rice resistance-material for RFS was created through host-induced gene silencing(HIGS).The main research results are as follows:(1)The pathogenic mechanism of the cell morphology protein UvPal1 was revealed.With a random T-DNA insertion mutant library was previously generated in our laboratory,we found a mutant,T184,which affects the cell morphology and virulence of U.virens,TDNA flanking sequences were inserted into the fourth exon of UvPal1 gene.Knockout and complementation of the UvPal1,we found that UvPal1 knockout mutants had reduced mycelial growth,abnormal cell morphology,altered stress response and a complete loss of virulence.The interacting Septin protein Uv Cdc11 was screened from the U.virens library using yeast two-hybrid,and used yeast two-hybrid,bimolecular fluorescence complementation(Bi FC)and co-immunoprecipitation(Co-IP)to prove that UvPal1 interacts with Uv Cdc11 in the cytoplasm.Uv Cdc11 was localized to the cytoplasm and septum in the wild-type strain HWD-2,while Uv Cdc11 was largely aggregated and localized to the vacuole in the UvPal1 knockout mutant,indicating that UvPal1 affects the localization of Uv Cdc11.Uv Cdc11 is the core protein in the Septin complex,and yeast twohybrid demonstrated that Uv Cdc11 interacts with other core Septin(Uv Cdc3,Uv Cdc10 and Uv Cdc12)to form a complex.These four genes of Septin were knocked out and complemented,and it was found that the Septin knockout mutants showed severe defects in mycelial growth,conidiation,stress response and virulence.The phenotype of the Septin knockout mutants was similar to that of the UvPal1 knockout mutants,indicating that UvPal1 interacts with the Septin protein Uv Cdc11,thereby regulating the Septin complex to affect cell morphology,stress response and virulence of U.virens.(2)The pathogenic mechanism of C2H2 transcription factor Uv CGBP1 was revealed.Knockout and complementation of Uv CGBP1,we found that Uv CGBP1 knockout mutants had reduced vegetative growth,biomass and conidiation,increased pigment deposition,changed in tolerance to environmental stress and a complete loss of virulence.Subcellular localization of Uv CGBP1 in the nucleus.A total of 865 downstream target genes of Uv CGBP1 was identified using Ch IP-seq,which was involved in various functional categories,such as transcription factor complexes,transcription factor activity,transcription,signal transduction and protein phosphorylation.KEGG enrichment analysis indicated that Uv CGBP1 target genes were involved in MAPK and benzoic acid metabolism pathways.Combined analysis of RNA-seq and Ch IP-seq data,we identified576 differentially expressed genes were regulated by Uv CGBP1.Approximately 36% of these target genes contain the G-box motif in their promoter,indicating that Uv CGBP1 binds G-box motif to regulate gene expression.Uv CGBP1 bound to the promoter regions of MAPK pathway kinases Uv Slt2 and Uv Pmk1 was demonstrated by Ch IP-PCR,yeast one-hybrid and electromobility shift assays(EMSA).Compared with the wild-type strain HWD-2,the expression levels of Uv Slt2 and Uv Pmk1 genes and proteins were significantly reduced in the ΔUv CGBP1-33 mutant,indicating that Uv CGBP1 regulates the expression of Uv Slt2 and Uv Pmk1.Overexpression of Uv Slt2 and Uv Pmk1 in the ΔUv CGBP1-33 mutant,we found that overexpression of Uv Pmk1 in the ΔUv CGBP1-33 mutant restored partially its mycelial growth and virulence,suggesting that Uv CGBP1 regulates Uv Pmk1 to affect the virulence and mycelial growth of U.virens.Therefore,we confirmed that Uv CGBP1 regulates mycelial growth and virulence of U.virens through mediating the Pmk1-MAPK pathway.(3)The pathogenic mechanism of effector Uv Sec117 was revealed.A secreted protein Uv Sec117 was identified from the shaking culture liquid of U.virens,which contained a signal peptide(SP),the yeast secretory activity test proved that the SP of Uv Sec117 had secretory activity.Abundant expression of Uv Sec117 to inhibit Bax-induced programmed cell death,indicating that the effector Uv Sec117 could suppress the immune response of plants.Knockout and complementation of Uv Sec117,we found that the virulence of Uv Sec117 knockout mutants was significantly reduced.Subcellular localization of Uv Sec117 in the nucleus and cytoplasm of tobacco cells.The interacting histone deacetylase Os HDA701 was screened from the rice library using yeast two-hybrid,and further used yeast two-hybrid,Co-IP,Bi FC and GST pull-down to prove that Uv Sec117 and Os HDA701 interact in the nucleus and cytoplasm.Gene silencing of Os HDA701 enhances the resistance to RFS,rice blast and bacterial blight,indicating that Os HDA701 negatively regulates broad-spectrum resistance against rice pathogens.Os HDA701 was subcellularly localized to the nucleus and cytoplasm,and a large number of brightly stained puncta were localized in the vacuole.When Uv Sec117 and Os HDA701 were co-expressed in N.benthamiana leaves,the fluorescent signal of Os HDA701 which aggregated in vacuoles was disappeared,and the content of Os HDA701 in the nucleus was significantly increased,indicating that Uv Sec117 affects the localization of Os HDA701 and recruits more Os HDA701 into the nucleus.After prokaryotic purification of Os HDA701 and Uv Sec117,in vitro incubation experiments proved that Os HDA701 could remove histone H3K9 acetylation,and Uv Sec117 enhanced the ability of Os HDA701 to remove histone H3K9 acetylation.The enzyme activity of Os HDA701 was measured using the HDAC histone deacetylase activity fluorescence assay kit,and it was found that Uv Sec117 enhanced the activity of Os HDA701 deacetylase.Heterologous overexpression of Uv Sec117 transgenic rice plants(35S-Uv Sec117)significantly reduced the resistance to RFS,rice blast and bacterial blight,and the enhanced susceptibility of 35S-Uv Sec117 transgenic rice plants was associated with H3K9 acetylation,Ch IP-seq was used to identify the target genes regulated by H3K9 acetylation in 35S-EV(empty control)and 35 SUv Sec117 transgenic rice plants.28394 target genes were identified in 35S-EV transgenic rice plants,while 831 target genes were identified in 35S-Uv Sec117 transgenic rice plants,the number of target genes regulated by H3K9 acetylation was significantly reduced in 35 SUv Sec117 transgenic rice plants,RT-q PCR and Ch IP-PCR suggested that the expression of H3K9 acetylation-regulated defense response-related genes in 35S-Uv Sec117 transgenic rice plants was significantly reduced,indicating that Uv Sec117 affected rice immunity by affecting the expression of histone H3K9 acetylation-regulated defense response-related genes.This study indicate that Uv Sec117 interacts with Os HDA701,and Uv Sec117 recruits more Os HDA701 to localize in the nucleus,thereby removing histone H3K9 acetylation,affecting histone H3K9 acetylation to regulate the expression of defense response-related genes,which in turn affects the immunity of rice.(4)HIGS technology to create new materials resistant to rice false smut.The fungal specific virulence factors Uv Asp E,Uv Com1,Uv Pro1 and Uv Sec117 were selected as HIGS target genes in the genome of U.virens by comparative genomics method,and Uv Asp ERNAi,Uv Com1 RNAi,Uv Pro1 RNAi and Uv Sec117 RNAi transgenic rice plants were generated.After inoculation of T3 transgenic rice plants,a small amount of rice smut balls was produced,indicating that the resistance of transgenic rice plants to RFS was significantly enhanced and lasting and stable.RT-q PCR detection results showed that the expression levels of Uv Asp E,Uv Com1,Uv Pro1 and Uv Sec117 were significantly reduced during the infection of RNAi transgenic rice plants.Small RNA-seq data analysis showed that RNAi transgenic rice plants produced a large number of si RNAs targeting Uv Asp E,Uv Com1,Uv Pro1 and Uv Sec117.Fluorescence in situ hybridization(FISH)demonstrated that si RNA molecules produced by RNAi transgenic rice plants were transferred into the infected hyphae of U.virens,resulting in a decrease in the transcription level of target genes,and RNAi transgenic rice plants enhanced the resistant to RFS.In this study,the pathogenic mechanism of U.virens was explored from the perspective of three different virulence factors,including cell morphology protein UvPal1,transcription factor Uv CGBP1 and effector Uv Sec117.To enrich the understanding of the pathogenic mechanism of U.virens and the interaction mechanism between U.virens and rice,create new materials and provide a new strategy for resistance to RFS through HIGS technology.