Characterization Of Botrytis Cinerea Pathogenicity Related Exocyst Complex Component Sec5

Botrytis cinerea is an important pathogenic fungus that can cause severe plant grey mold,meanwhile it is an important model organism to study interaction between plant and pathogen.Analysis of gene functions with genetic approach combining cytological and biochemical methods will speed up our understanding about the fungal biology of B.cinerea and its molecular pathogenic mechanism.In this study,a mutant ?Bcsec5 with reduced pathogenicity was obtained via the Agrobacterium tumefaciens-mediated transformation(ATMT).The integration of the hph gene was characterized by PCR analysis with specific primers.Southern blotting showed that the mutant contained single copy of T-DNA.DNA sequence flanked the integration sites of the T-DNA was amplified using high-efficiency thermal asymmetric interlaced PCR(TAIL-PCR).The flanking DNA fragments were sequenced and analyzed successfully.Analysis revealed that the transfer-DNA was integrated into a gene coding a hypothetical protein.GenBank BLAST showed it is a homologous gene of exocyst complex component Sec5 in the secretory system.Monokaryotic transformants were purified through single spore isolation and characterized by PCR.The disruption of gene was confirmed by mRNA expression analysis using RT-PCR.Comparison between the mutant and wild strain indicated the different phenotype as the insertion of T-DNA.The phenotypes of mutant showed slow growth rate,lower conidia germination rate,changed mycelial morphology,higher chitin content and decreased pathogenicity on tomato leaves.Culture on PDA plates with CFW,SDS,sorbitol,NaCl,KC1 or MgCl2,the mutant show no obvious difference comparing to WT.In addition,tansmission electron microscopy of mutant ABcsec5 showed that a proportion of mutant?Bcsec5 cells had vesicles with electron-dense substance.This may be related to deficiency in polarised exocytosis.These results suggest that sec5 involved in the regulation of growth and development and pathogenicity to host plant in Botrytis cinerea.The full-length gene sequence of SEC5 including promoter,transcribed region and terminater sequence was cloned and a binary vector was constructed.This vector had been transformed into the A.tumefaciens for genetic complementation of the mutant and function confirmation.And the transcribed region of SEC5 was cloned into vector ImpGWB505 which fuses green fluorescent protein(GFP)to the C-terminal end.This vector has been transformed into the A.tumefaciens for protein localization.