Functional Studies On Effector Protein CsSp1 From Bipolaris Sorokiniana

Wheat root rot is a worldwide disease of wheat.The main pathogen of wheat root rot is Bipolaris sorokiniana in Huanghuai wheat area.Effectors play an important role in plant-pathogen interactions.In this study,the function of effector CsSp1 was studied,which provided a direction for the further study on the pathogenic mechanism of the wheat root rot fungus,and a train of thoughts for the control of wheat root rot.In transcriptome data of wheat root infected by B.sorokiniana,we identified a gene that was highly expressed at the infection stage and named CsSP1.CsSpl predicts a 323 bp genome with two exons separated by 53 bp introns,and a 270 bp cDNA that encodes a secreted protein of 89 aa and a signal peptide of 17 aa at the N termina1.First of all,we verified that CsSp1 was up-regulated expression in the infection process by qRT-PCR,and the expression level was about 50 times higher than that in the hypha after 12h of infection.A knockout box containing HYG(hygmycin phosphate transferase gene)was constructed by using the method of split marker,and antihygmycin inverters were obtained by peg-mediated protoplast transformation and homologous recombination in vivo.The deletion mutant of CsSP1 gene was obtained by PCR preliminary screening and resequencing verification.The CsSP1 gene and the upstream 1.8Kb promoter region were jointly constructed into a pYIP carrier transferred into ΔCssp1 mutant to obtain a complementary transgene.The results of biological characters showed that CsSp1 was different from wild-type and complementary strains in terms of colony morphology,colony growth rate and spore production.However,conidia were used to inoculate wheat and barley leaves,the results showed that the disease spots formed on the leaves were significantly reduced in ΔCssp1.It indicating that CsSp1 was involved in the pathogenicity of B.sorokiniana.By using conidia to inoculate the inner epidermis of onion,ΔCssp1 could produce shape-changd appressoriums saturated infective nails,but the growth rate of penetrating cell hypha was significantly reduced,and the expansion rate of hypha end was decreased,which proved that CsSpl was nvolved in the colonization of pathogen in plant.The secretion of the signal peptide was verified by yeast YTK12,indicating that CsSpl is a secretory protein.CsSP1-GFP was mainly located in the nucleus and had cytoplasmic background in N.benthamiana,while the expression of CsSp1 didn’t trigger N.benthamiana cell death.The fluorescence signals of CsSP1-GFP and CsSP1-NS-GFP in the nucleus of inner epidermis of onion.CsSp1 and GFP were expressed in N.benthamiana,and then inoculated with Phytophthora capsici.Compared with GFP,P.capsici in CsSpl produced smaller lesions.So CsSpl could trigger the immunity of plants to resist the infection of pathogens.In the transcriptome data of B.sorokiniana infected wheat,the expression of phenylalanine ammonia-lyase(PAL)related genes was up-regulated during the infection stage,and PAL mediated the SA synthesis pathway.TaPAL,TaPR-1 and TaPR-2 was significantly reduced in wheat leaves inoculated ΔCssp1 compared with WT,expression significantly reduced.CsSpl can trigger immunity in plants to resist pathogenic infection.In order to further verify the function of CsSp1,we screened wheat yeast two-hybrid cDNA library and obtained the proteins that may interact with CsSp1.As a conclusion,the effector protein CsSpl regulates the morphological development and growth of B.sorokiniana,plays an important role in pathogen infection and colonization of plants,and also may participate in the SA pathway as an elicitor.