A Study On Roles And Mechanisms Of PCG6 In Asexual Development And Pathogenesis Of Magnaporthe Oryzae

Rice blast is a widely distributed and devastating fungal disease on rice throughout the world and is a model system that is widely used for studying fungus-plant interactions.Deciphering the pathogenesis of the rice blast fungus will contribute to designing novel strategies for controlling the rice blast and many other fungal diseases.Though many studies have been carried out to investigate the molecular mechanism on pathogenesis in Magnaporthe oryzae from diverse aspects,studies are lacked on the roles of post-transcriptional gene control in pathogenesis.Previously,an insertion mutant LF13 of M.oryzae was isolated from Agrobacterium tumefaciens-mediated transformation（ATMT）mutant library.The mutant LF13 formed smaller colony on oatmeal tomato agr（OTA）plate compared with the wild-type P131.Genetic analysis indicated that growth defects of the mutant LF13 was co-segregated with the hygromycin resistant marker.Southern blot analysis showed that a single copy of T-DNA was integrated into the genome of the mutant LF13.The flanking sequences of T-DNA in the mutant LF13 was isolated by thermal asymmetric interlaced PCR（TAIL-PCR）approaches and the integration site of the T-DNA was located between 1,004-bp and 478-bp upstream of the translation start site of a putative gene MGG₀1781.6（GenBank access number is KC492050,and here it was named as PCG6）with loss of one 525-bp fragment.This gene encoded a deduced protein with 964-aa in length and contains five KH domains by SMART and InterPro analysis.The Pcg6 orthologs were then searched through BLAST in GenBank.Results showed that Pcg6 was a putative RNA-binding protein and Pcg6 orthologs were widely existed and highly conserved among filamentous fungi.To determine the biological roles of PCG6 in asexual development and pathogenesis of M.oryzae,the deletion mutants of PCG6 were generated by the homologous recombination approach.In this study,PCG6-deletion mutant was used to investigate the role of Pcg6 in the disease cycle of M.oryzae.The null mutant of PCG6 exhibited severely attenuated virulence on barley and rice seedlings compared to the wild-type strain.Microscopic observation revealed that Pcg6 had pleiotropic effects on fungal morphogenesis,including reduction in mycelial growth,conidiation,appressorium development,penetration and infectious growth in host cells.To determine sub-cellular localization of Pcg6,a PCG6-eGFP fusion construct pLPCG6 under control of its native promoter was generated and transformed into the pcg6 null mutant k3.More than ten transformants including k3C were isolated and showed the wild-type phenotypes on vegetative growth,conidiation,and plant infection.The green fluorescence signals were observed to uniformly distribute among cytoplasm of vegetative hyphae,conidia,germ tubes,appressoria,and infectious hyphae of k3C.Thus,Pcg6 was cytoplasm localized and constitutively expressed.The result of pBLAST showed that Pcg6 exhibited 32%identity with a functionally characterized protein Khd4 from Ustilago maydis.It was reported that Khd4 recognized the AUACCC cis-element.The author speculated that whether Pcg6 exhibited similar binding capacity with Khd4 and tested this possibility by the EMSA assay.And the results showed that Pcg6 binded the RNA motif AUACC.To reveal the target genes recognized by Pcg6,the author firstly compared the transcriptomes of the pcg6 null mutant k3 and the wild-type P131 by the RNA-seq technology,and then selected the genes having AUACC motifs which were up-or down-regulated over two-fold.Finally,2225 up-regulated and 1129 down-regulated genes were found to be the potential targets.The RNA-seq results were then testified by qRT-PCR and the qRT-PCR results were consistent with the RNA-seq data.The potential target genes were classified based on the KEGG pathway and majority of these genes were participated into metabolism process,including amino acid metabolism,carbohydrate metabolism and lipid metabolism.Among the genes directly regulated by Pcg6,one sub-group genes involving in the glutamate metabolism were focused on.Most of the genes involving in the glutamate metabolism contained the Pcg6-binding motif AUACC in their ORF,and the expression levels of these genes,especially the key genes MGG₀7187,MGG₁4279 and MGG₀8074 were significantly changed,suggesting they were directly regulated by Pcg6.The contents of glutamate and glutamine in different strains were measured by HPLC approach.In the pcg6 null mutant k3,the content of glutamine was significantly elevated and glutamate was declined.To investigate the relationship between amino acids level and fungal development,MGG₀7187（MoGLTl,glutamate synthase）was knocked out and MGG₁4279（MoGLN,glutamine synthetase）and MGG₀8074（glutamate dehydrogenase）were overexpressed separately.Results showed that the ΔMogltl null mutants were greatly reduced in virulence towards host plants,due to low efficiency in appressorium formation,appressorial penetration,and infection hyphae development.Moreover,the ΔMogltl null mutant accumulated much less glutamate and could not grow on minimal media without glutamic acid.Exogenous addition of glutamic acid could restore the defects of the ΔMogltl null mutants in colony growth on minimal media,conidiation,and plant infection.In addition,the overexpression of MGG₁4279 and MGG₀8074 both caused the decrease of glutamate and led to the reduction in mycelial growth,conidiation and virulence.This study indicated that glutamate is required for fungal development and virulence in M.oryzae.Furthermore,genes containing AUACC motifs involved in pathogenicity like MPG1,PTH11 and PTH12 were down-regulated in k3.Exogenous addition of cAMP or IBMX restored the appressorial formation and penetration defects of k3,which indicated that Pcg6 could control the expression level of these pathogenesis related genes involved in cAMP signal pathway.In this study,a multi-KH-domain protein Pcg6 in M.oryzae was identified and played important roles in colony growth,conidiation and pathogenesis of.Moreover,Pcg6 up-regulated the genes involved in glutamate metabolism and down-regulated the genes participating in cAMP signal pathway.Furthermore,the overexpression of genes involved in glutamate synthesis caused reduction in hyphal growth,conidiation and virulence to hosts.This study provides a new perspective for understanding the molecular mechanism of pathogenicity of rice blast fungus.