Cloning And Function Analysis Of Pathogenic Gene GcVIR1 From Colletotrichum Gloeosporioides

Glomerella leaf spot of apple(GLSA)caused by Colletotrichum gloeosporioides is a major foliar disease of apple in recent years,which have a serious threat to apple industry.At present,most research on this disease was mainly focused on etiology?infection cycle?epidemic regularity and control methods,whereas little on pathogenic mechanism.This study carry out research on molecular pathogenic mechanism to provide some new clues in control this disease.In this study,we obtained seven traits variation mutants.The spores production test showed that M22 and M178 produced a great deal of branch spore(nearly 10 fold of wild-type),M17 and M44 lost the ability to produce spore,M163?M183 and M285 produced nearly 1 percent that of wild-type.The assay of pathogenicity indicated that the virulence to leaf of seven mutants had varying degrees decline,M44 and M285 lost the ability of pathogenicity.Southern hybridization results had showed T-DNA was single copy in mutants.Flanking sequence of T-DNA insertion locus was obtained by hiTail-PCR.And the genes(GcRRN3,GcRXT2,GcGD1,GcCON3,GcOPT2,GcMRP1,GcVIR1)influenced by T-NDA inserting were knowed by blast the flanking sequence from genome of Glomerella cingulata 23.The RT-PCR results revealed that the transcripts of these seven genes were not detected in the T-DNA insertional mutants,and all these genes transcripts were present in the WT isolate.The full length genome sequence of GcVIR1 is 1686 bp,which encoding 562 amino acids,and not containing inxon.The complementary expression vector of Gc VIR1 was constructed and transferred into the mutant M285 to construct the strains complementary of mutant M285.The function were characterized by phenotype test and found R-GcVIR1 have the same phenotype with the wild-type.To cofirm the subcellular localization of the GcVIR1,the GcVIR1-RFP fusion expression mutant was construted.After observation fusion expression,GcVIR1-RFP fusion expression protein was located in cytoplasm.In the study of temporal expression of GcVIR1 gene,it expressed during mycelia growth,conidiation,conidium germination,and appressorium formation and high expressed during conidia.Colletotrichum gloeosporioides secrete pectinase degrading cell walls to infect host and colonization.In this study,the GcVIR1 mutant was no virulence,so we investigated the expression stability of 5 pectinase-related genes in qRT-PCR experiments.Compared with WT,the M285 mutant showed a increase of PG1 transcripts(5.89 fold),PG2 transcripts(4.58 fold)?In Colletotrichum gloeosporioides,the infection process regulated by appressorium play an important role in the pathogenesis of disease.The CMK1?CPK1?MEK1?MAF1 of MAPK(Mitogen-activated protein kinase)Cascade signaling pathways have been proven to regulate the appressorium formation.Because of the GcVIR1 mutant lost the ability to formate appressorium,we investigated the expression level of MAPK genes in M285 by qRT-PCR experiments.The result showed that these four genes was not influenced by GcVIR1,which implied the function of GcVIR1 might be downstream of these four genes.