Functional Analysis Of Bcmcta, A Putative Monocarboxylate Transpoter Gene In Botrytis Cinerea

Botrytis cinerea is an important plant pathogenic fungus,which can infect more than 200 kinds of plants,including cucumber,tomato,strawberry,grape,apple and so on.In previous study,our research team constructed Botrytis cinerea T-DNA insertion mutant library,and screened mutants which were defective in pathogenecity.T-DNA insertion sites of these mutants were analyzied by amplifying the flank segments of T-DNA with TAIL-PCR and sequencing.It was found that in one of the mutants?74?,BC1G₁5016.1,a putative monocarboxylate transporter?MCT?gene,was inserted by T-DNA.This mutant?74?showed obviously reduced virulence compared to the wild-type.To our knowledge,it is the first reported functional defective mutant of an MCT homolog gene in B.cinerea,and this gene is named as BcmctA.In this study,the function of BcmctA gene was anlyzed by gene deletion and complementation.First,an entry vector pETHG-MAD-MAU was constructed by the Gateway system,and the BcmctA deletion binary vector pCAMBIA-Bar-?mctA was generated by LR recombination reaction between pETHG-MAD-MAU and the destination vector pCAMBIA-Bar-RfA.Through Agrobacterium tumefaciens mediated transformation,hygromycin-resistant transformants were obtained from Botrytis cinerea B05.10.These transformants were analysed by RT-PCR and Southern blot analysis,and 2 BcmctA deletion mutants??15??34?were confirmed.The BcmctA gene complementation was also performed with the A.tumefaciens-mediated method as described above,and the glufosinate-resistant transformants were obtained from the BcmctA deletion mutant??34?.Finally,2 BcmctA complementation strains?C11?C25?were confirmed.The experiments showed that disruption of the Bcmct A gene decreased the growth rate on the medium with monocarboxylate?acetate or pyruvate?as the sole carbon sources,but not affected on lactate.The pyruvate contents in BcmctA deletion mutants decreased about 35 % compared with the wild strain.Besides,the conidial yield was increased about two times in BcmctA disruption mutant.The pathogenicity assay indicated that disruption of BcmctA significantly reduced the virulence of B.cinerea on cucumber and tomato leaves.It was found that gene complementation strain restore most of characteristics as wild-type.Our results demonstrated that BcmctA is related to pyruvate uptake and pathogenicity of B.cinerea on cucumber and tomato leaves.