Preliminary Functionally Characterization Of A Gene Cluster Regulated By Zn₂Cys₆ Transcription Factors In Magnaporthe Oryzae

Rice blast,caused by Magrraporthe oryzae,is one of the fungal diseases which threaten yield and quality of rice,brings massive loss to rice production.M.oryzae has a typical infection process in plant pathogenic fungi and has emerged as a model for host-fungal pathogen interactions.It is of great significance to study gene functions in the fungus for fungal pathogenesis and control of the rice blast.Gene clusters can be broadly defined as the close linkage of two or more genes that participate in a metabolic or developmental pathway.Clusters of functionally related genes are a general feature of prokaryotic gene organization,such as "pathogenic islands" in plant pathogenic bacteria,but are much less investigated in eukaryotes.In recent years,the genes in some metabolic pathways were found clustered in filamentous fungi,such as the gene cluster in metabolic pathway of quinic acid in Neurospora crassa and that in the synthesis of melanin in Aspergillus nidulans.However,little is reported on the function of the gene cluster in plant pathogenic fungi.In order to further understand the gene cluster in fungi,and to reveal the biological function of gene cluster in the infection of plant fungal pathogens,we explored the function of a putative gene cluster in M.oryzae.The hypothetical proteins in M.oryzae genome,MGG₁0528,MGG₁0529,MGG₁0530,MGG₁0531 and MGG₁0532 are located within an approximately 15.8-kb region on chromosome I.MGG₁0528 and MGG₁0529 are adjacent to each other with opposite encoding directions,possess similar gene structure and both encode proteins with Zn2Cys6 domains.MGG₁0530 encodes a sugar transporter.MGG₁0531 encodes a hypothetical protein with unknown function.MGG₁0532 encoding a protein in NLP（necrosis and ethylene-including-like protein）family,which have frequently been shown to trigger cell death and activate the defense signaling reactions in dicotyledonous plants.These characters remind us these genes may be a gene cluster which mediating a metabolic pathway.To known the functions and relations of these genes,in the present work,we knocked out the MGG₁0529,MGG₁0530 and MGG₁0532 and phenotypically analysis the mutants.The replacement vectors of MGG₁0529,MGG₁0530 and MGG₁0532 with hygromycin resistance were constructed and integrated into the genome of the wild-type strain Guy11 through AtMT transformation.The transformants were screened on selective medium and then tested by PCR to select the mutants,and which were mono-conidial isolated and confirmed by Real-Time PCR finally.The expression levels of MGG₁0529 and MGG₁0532 were increased in MGG₁0529 knockout mutants.The expression level of MGG₁0532 was decreased and that of MGG₁0529 was slightly increased in MGG₁0532 knockout mutants.While the knockout of MGG₁0532 had no significant effect on the expression levels of the other genes.These results suggested that MGG₁0529 may have a regulation to MGG 10532.On the complete medium,the △MGG 10529,△MGG₁0530 and OMGG₁0532 mutants were normal in the colony morphology and growth rate compared with that of the wild-type Guy 11,indicating that the three genes were not involved in the vegetative growth and sporulation.Growth experiment on the media with different carbon sources indicated that the mutants can used the common carbon sources normally.Inoculation test on barley and wheat leaves showed the pathogenicity of mutants were not different significantly to that of the wild-type Guyl 1.No significant changes were found in conidial germination and appressorial formation of the mutants to the wild-type.These results indicated that MGG₁0529,MGG 10530 and MGG₁0532 are not directly involved in the pathogenic process of fungus.In mating experiment,the mutants formed numerous perithecia at the junctions region with opposite mating type strain 2539,suggesting that MGG 10529,MGG₁0530 and MGG 10532 is not required for sexual reproduction in M.oryzae.On the active oxygen tolerance test,we found that the growth of the mutants are reduced on the media supplemented with reactive oxygen species.On the media containing 200}Lg/mL Congo red,the growth inhabitation of OMGG₁0530 mutants were significantly increased,while that of AMGG 10529 and AMGG 10532 mutants were found no significant difference to that of the wild-type Guyll.The GFP-10528 and GFP-10529 fusion proteins were constructed and used to study the subcellular localization of the two genes.The conidia of transformants expressing GFP-10528 and GFP-10529 emitted GFP fluorescence in the cytoplasm and as well GFP accumulation in punctate areas of the cells under a laser confocal microscopy,suggesting the 2 proteins are cytoplamically distributed and also located in some organelles.Summarily,our results indicate that MGG₁0528 and MGG₁0529 are distributed in cytoplasm and some organelles.The single knock out of MGG 10529,MGG₁0530 or MGG₁0532 did not affect the vegetative growth,conidiation,utilization of common carbon sources,pathogenicity and pathogenicity-related developments.However,the three genes were all involved in the resistance of the fungus to reactive oxygen species.And meanwhile,MGG₁0530 was found affect the cell wall strength.The biological functions of the the genes and their relationships between them need to be further investigated.