Function Analysis Of Monocarboxylate Transporter CgMCT1 In Colletotrichum Gloeosporioides

Glomerella leaf spot of apple is mainly caused by Colletotrichum gloeosporioide,which infects both leaves and fruits of the apple.It often caused early leaf drop and severely weakened the tree.The infected fruits showed the lesion when the fruits were nearly ripe,which resulted in fruit drop at pre-harvest.In-depth studies on the molecular mechanisms of C.gloeosporioide not only help us to understand the pathogenic nature of the pathogen but also provide important reference information for control strategies of plant disease.Data of apple genome and C.gloeosporioide genome have provided an opportunity to study the interaction of plant-Colletotrichum.In this paper,we identified a pathogenicity-related gene CgMCT1 of C.gloeosporioide by screening the T-DNA insertion mutant library.We found that the CgMCT1 is involved in the growth,development,conidiation and pathogenicity of C.gloeosporioide,by using molecular biology techniques,such as the gene knockout,fluorescence-fused expression and subcellular co-localization,combined with phenotype analysis and pathogenic tissue structure observation.The main findings were obtained as follows:In this study,a non-pathogenic mutant(N7)was screened from a C.gloeosporioide T-DNA insertional mutant library.The right flanking sequence of the T-DNA insertion site was cloned by hiTail-PCR,analyzed by sequence alignment.The gene marked by the T-DNA in N7 encoded a putative monocarboxylate transporter(MCT),we designated it as CgMCT1.The transcripts of the CgMCT1 have two alternative splicings,named MctS1 and MctS2.both MctS1 and MctS2 have the same functions.The phenotypic analyses showed that the Δcgmct1 mutant affected hyphal growth,pigmentation and conidiation in C.gloeosporioide.Both CgMCT1p-e GFP and q RT-PCR expression assays showed that CgMCT1 is expressed in hyphae,conidia,germ tubes and appressoria.The highest expression of CgMCT1 was found in conidia.The CgMCT1 protein contains a typical transmembrane domain,co-localization of CgMCT1-Tag RFP with MTS-e GFP indicates that the CgMCT1 is localized in the mitochondria.The CgMCT1 did not mediate the uptake of five monocarboxylates(lactate,pyruvate,acetate,oxalate and citrate).However,the CgMCT1 was induced to be up-regulated in MM medium cultures supplemented with exogenous lactic acid.The enhanced transcriptional activity of the CgMCT1 was a response to the presence of extracellular lactate.These results speculate that the CgMCT1 is located on the mitochondrial tegument and is involved in the transmembrane transport of lactate.The Δcgmct1 mutant was non-pathogenic to wounded or non-wounded leaves.ROS was accumulated in infected cells.Thus,the Δcgmct1 mutant showed a defect in scavenging the host ROS during fungal infection,suggesting that the deletion of CgMCT1 influenced plant defence response.Collectively,our study indicated that the CgMCT1 plays important roles in the growth,development,conidiogenesis and pathogenicity of C.gloeosporioides.Our results facilitated the understanding of functions of the monocarboxylate transporter and also provided target sites for developing neotype fungicides.