Functional Analysis Of The Heat Shock Protein Genes (MHF6 And MHF15) And Deubiquitinating Enzyme Gene (MoUBP14) In Magnaporthe Oryzae

Rice blast caused by Magnaporthe oryzae（anamorph Pyricularia oryzae,teleomorph Magnaporthe oryzae）is one of the most serious diseases that threaten the rice production in the world.The annual average loss is up to 10%-30% of the total yield,which is a serious threat to the world food safety.Due to its socioeconomic importance and the operability of M.oryzae in molecular genetics,the M.oryzae has gradually become a model pathogenic fungus in studying pathogenic mechanism.Further research on the pathogenicity mechanism of M.oryzae is not only conducive to the development of new fungicides,but also serving an important guiding for controling of other diseases.In this study,a non-pathogenic T-DNA insertional mutant was obtained by Agrobacterium tumefaciens-mediated T-DNA tagging transformation and the T-DNA tagged gene MHF15,which belongs to heat shock protein 40（HSP40）family genes,was identified.Furthermore,biological functions of another HSP40 family gene MHF6 and deubiquitinating enzyme family gene MoUBP14 were functionally characterizedin M.oryzae.The results obtained are listed as follows:1.The heat shock protein genes（MHF6 and MHF15）are essential for conidiation and pathogenicity in M.oryzae.To identify new pathogenicity-related genes of M.oryzae,a T-DNA insertional mutagenesis library with 2400 transformants was constructed.The results of pathogenicity assays indicate that the transformat T1210 completely lost the ability of causing disease on its host.The T-DNA-tagged gene MHF15（MGG₀6486）putatively coding a HSP40 protein was successfully identified by Tail-PCR and bioinformatics analysis.To our knowledge,the biological function of MHF15 has not been reported previously in M.oryzae.Phenotypic analysis showed that the colony growth of the mutant T1210 was significantly reduced and conidiation and the pathogenicity of the mutant were completely lost.However,we were unable to obtain the deletion mutant of MHF15 after checking more than 2000 transformants from many independent transformation experiments（>30 times）,indicating that MHF15 is indispensible for the survival of M.oryzae.In order to verify that the defects of the T1210 mutant were caused by the insertion of T-DNA,gene complementation experiments were carried out.The experiment results showed that the introduction of a complete MHF15 into the T1210 mutant could restore its defective phenotypes.Also,introduction of a complete MHF15 into the corresponding yeast mutant YNL₀64C could partially complement its defects.According to previous reports and bioinformatics analysis,a total of 25 HSP40 family genes have been identified in the genome of M.oryzae,which were named as MHF1-25（Magnaporthe oryzae heat shock protein forty）respectively.We knocked out several genes（MHF1,MHF3,MHF6,MHF10,MHF11 and MHF25）of the M.oryzae HSP40 family.Phenotypic analysis revealed that the（35）mhf6 mutant was completely nonpathogenic to susceptible hosts.The colony growth rate of the mutant had no significant difference compared with the wild type,but conidiation and pathogenicity were completely blocked.Moreover,reintroduction of the whole MHF6 gene into the（35）mhf6 mutant could restore all phenotypic defects.In addition,we found that both T1210 and（35）mhf6 mutant were more sensitive to temperature.These data suggest that MHF15 and MHF6 are essential for conidiation and pathogenicity in M.oryzae.2.The deubiquitinating enzyme gene MoUBP14 is essential for vegetative growth,conidiation and pathogenicity by the rice blast fungus.MGG₀8270 encodes a putative ubiquitin carboxyl-terminal hydrolase 14,which belongs to the deubiquitinating enzyme family.Therefore,we named it MoUBP14（Magnaporthe oryzae ubiquitin-specific protease）.Biological function of MoUBP14 was characterized by targeted gene deletion and gene complementation strategy.The results indicated that the colony growth rate of the（35）Moubp14 mutant was significantly decreased compared with the wild type strain.The mutant was unable to produce conidia and nonpathogenic.The sensitivity of the（35）Moubp14 mutant to saltions was increased.Taken together,we conclude that MoUBP14 is essential for vegetative growth,conidiation and pathogenicity by the rice blast fungus.Furthermore,six genes putatively encoding deubiquitinating enzymes were deleted,respectively.Deletion of MoUBP12（MGG₀5666）resulted in a defect in conidiation,while deletion of each other genes did not cuase any detectable phenotypic alterations.