| Rice blast caused by Magnaporthe oryzae is the most serious fungal disease on rice,which causes 10-30% yield loss in the global rice production every year and seriously threatens the global food production security.At present,disease-resistant varieties and chemical pesticides are mainly used to control the disease in production.However,it’s easy for them to lose the application value in a short time due to the genetic complexity of M.oryzae and resistance to pesticides,and the development of new fungicides needs to target the key proteins of the pathogen.Therefore,it has important theoretical and practical significance for the exploration of potential fungicide targets and the development of new fungicide to identify key proteins involved in the pathogenic process of M.oryzae and reveals their regulatory mechanism at the molecular level.Mitochondria are one of the most important organelles in organisms,and play an important role in many physiological and biochemical processes such as energy generation,ion storage,apoptosis,anabolism and catabolism.Meanwhile,mitochondria are highly dynamic organelles,which morphology is precisely controlled by the dynamic balance mechanism of fission and fusion,thus ensuring the normal quantity and quality of mitochondria.At present,several dynamins have been identified in pathogenic fungus regulating mitochondrial fission/fusion and the virulence of pathogens,but few studies on their specific mechanisms have been reported.Previous studies in our laboratory have found that the zinc finger transcription factor MoMsn2 in M.oryzae participates in the biological processes of growth and development and pathogenicity by directly regulating the expression of a series of target genes.Further analysis revealed that MoMsn2 may regulate the biological processes related to mitochondria,peroxisome and other organelles to control the growth and development and pathogenicity of M.oryzae,but the specific regulatory mechanism remains unclear.Therefore,the molecular mechanism of MoMsn2 in regulating mitochondrial fission/fusion balance and the infectious hyphal growth of M.oryzae was investigated in this study.The specific research contents are as follows:(1)Transcription factor MoMsn2 regulates the mitochondrial morphology and pathogenicity of M.oryzaeTo determine whether MoMsn2 is involved in regulating biological processes related to mitochondria,we first examined the mitochondrial morphology of wild-type Guy11,theΔMomsn2 mutant and the complemented transformant MoMSN2-C by Mitotracker Red staining.Both tubular(70%)and vesicular/punctate(10%)mitochondria were observed in hyphae of Guy11 and MoMSN2-C.However,in the hyphae of ΔMomsn2 mutant,only 10%of mitochondria were tubular,and more than 60% of mitochondria are punctate or short linear.Further fluorescence observation was performed on Guy11,ΔMomsn2 and MoMSN2-C strains expressing mitochondrial marker protein Ilv2-GFP,in the meanwhile,the transmission electron microscope was used to observe the mitochondrial morphology of these three strains,and all the results showed an increase of punctate mitochondria inΔMomsn2 mutant.Further treatment of ΔMomsn2 mutant with mitochondrial fission inhibitor Mdivi-1 could restore the mitochondrial morphology of the mutant.Studies have shown that abnormal mitochondrial morphology could result in mitochondrial damage and dysfunction.Therefore,the mitochondrial membrane potential and mitophagy levels ofΔMomsn2 mutant were evaluated with or without Mdivi-1 treatment.The results showed that the mitochondrial membrane potential and mitophagy levels of ΔMomsn2 mutant were significantly lower than those of the wild-type,and could be partially restored under the treatment of Mdivi-1.The pathogenicity of ΔMomsn2 mutant was further analyzed with or without Mdivi-1 treatment,the slower infectious hyphal growth and smaller disease spots were observed compared with the wild type,and could be partially restored under the treatment of Mdivi-1.These results suggest that transcription factor MoMsn2 is involved in regulating the mitochondrial fission/fusion balance,mediating the mitochondrial membrane potential and mitophagy levels and controlling the infectious hyphal growth of M.oryzae.(2)MoMsn2 targeting genes MoUBP12 and MoUBP14 regulate the mitochondrial morphology and pathogenicity of M.oryzaeMitochondrial fission and fusion are regulated by a variety of post-translational modifications including ubiquitination.To further explore the molecular mechanism of transcription factor MoMsn2 regulating mitochondrial morphology and pathogenicity of M.oryzae,two genes involved in protein ubiquitination,MoUBP12 and MoUBP14,which encode ubiquitin specific peptidase and ubiquitin carboxyl-terminal hydrolase respectively,were identified by analyzing Ch IP-Seq and transcriptomic data obtained in previous study of our laboratory.Therefore,we hypothesized that MoMsn2 may directly regulate the expression of MoUBP12 and MoUBP14 to regulate mitochondrial fission and fusion balance.First,the relationships between MoMsn2 and MoUBP12,MoUBP14 were analyzed by electrophoretic mobility shift assay and quantitative real-time PCR.The results showed that MoMsn2 could specifically bind to the promoter regions of MoUBP12 and MoUBP14,and both of them were significantly down-regulated in the ΔMomsn2 mutant.Gene knockout mutants of MoUBP12 and MoUBP14 were obtained by protoplast transformation,which pathogenicity decreased significantly.Changes in mitochondrial morphology were observed in hyphae of both ΔMoubp12 and ΔMoubp14 mutants marked with Mitotracker Red,in which tubular mitochondria increased in hyphae of ΔMoubp12mutant and punctate mitochondria increased in hyphae of ΔMoubp14 mutant,and the mitochondrial membrane potential of both mutants were significantly lower than that of wild type.The mitochondrial morphology,mitochondrial membrane potential and infectious hyphal growth of ΔMoubp12 and ΔMoubp14 could be partially restored by treatment with mitochondrial fission inducer glycerol and inhibitor Mdivi-1.In order to further clarify the biological functions of MoUbp12 and MoUbp14 in regulating the mitochondrial morphology and infectious hyphal growth,the ubiquitination levels ofΔMoubp12 and ΔMoubp14 mutants were analyzed by Western blot.The results showed that the overall ubiquitination levels of ΔMoubp12 and ΔMoubp14 mutants were significantly increased compared with the wild type and the complemented strain.In order to explore the upstream and downstream relationship between MoUbp12 and MoUbp14,the double mutant of MoUBP12 and MoUBP14 and the strains expressing MoUbp12-GFP and MoUbp14-GFP in the double mutant were obtained by protoplast transformation,the ubiquitination level of MoUbp2-GFP and MoUbp14-GFP was detected by Western blot.The results showed that the ubiquitination level of MoUbp14-GFP in the double mutant was significantly higher than that in ΔMoubp14/MoUbp14 strain,suggesting that MoUbp12 regulates the deubiquitination process of MoUbp14.Further Mitotracker Red staining and observation showed that the deletion of MoUBP12 restored the mitochondrial morphology of the ΔMoubp14 mutant,suggesting that MoUbp12 may play a role of deubiquitination upstream of MoUbp14.These results suggest that MoUBP12 and MoUBP14,as the target genes of MoMsn2,are involved in regulating the ubiquitination degradation process of M.oryzae,and control the infectious hyphal growth of M.oryzae by mediating the mitochondrial division/fusion balance.(3)MoUbp14 regulates the mitochondrial morphology and pathogenicity of M.oryzae by degrading MoUbi4These studies have proved that MoUBP14 played an important role in regulating the mitochondrial morphology and pathogenicity of M.oryzae as the target gene of MoMsn2.To further reveal the molecular regulatory mechanism of MoUbp14,yeast two-hybrid(Y2H)system was used to screen the binding protein of MoUbp14,and the polyubiquitin MoUbi4(MGG＿01282)was identified interacted with MoUbp14.The gene knockout mutant of MoUBI4 was obtained by further protoplast transformation,which pathogenicity was significantly reduced and mitochondria were highly integrated,and both of them could be partially restored by treatment with mitochondrial fission inducer glycerol,suggesting that MoUbi4 regulated the pathogenicity of M.oryzae through the mitochondrial morphology.To explore the biological significance of interaction between MoUbp14 and MoUbi4,strains of Guy11 and ΔMoUbp14 expressing MoUbi4-GFP were obtained and the protein expression level of MoUbi4-GFP was detected.The results showed that the protein expression level of MoUbi4-GFP in ΔMoUbp14 was significantly higher than that in Guy11,suggesting that MoUbp14 could degrad the polyubiquitin MoUbi4.Further treatment of ΔMoUbp14 mutant with 26 S proteasome inhibitor MG132 showed partial recovery of the mitochondrial morphology and infectious hyphal growth of M.oryzae.In the meanwhile,the potential binding proteins of MoUbi4 were identified by Co-IP,and it’s found that MoUbi4 may connect to the mitofusins such as MoFzo1 and MoMgm1.These results suggested that polyubiquitin MoUbi4 is involved in regulating the mitochondrial morphology and pathogenicity of M.oryzae,while MoUbp14 may promote mitochondrial fusion to ensure the infectious hyphal growth of M.oryzae by degrading MoUbi4.In conclusion,this study preliminarily revealed the molecular mechanism of transcription factor MoMsn2 maintaining the mitochondrial morphology to control the infectious hyphal growth of M.oryzae by regulating the expression of the target genes MoUBP12 and MoUBP14.And the ubiquitin carboxyl-terminal hydrolase MoUbp14 is involved in the regulation of this important biological process by degrading MoUbi4.The mitochondrial fission/fusion balance mediated by MoMsn2 is necessary to maintain the normal function of mitochondria and ensure the normal infection of M.oryzae in host plants.These findings further clarify the importance of the dynamic balance of mitochondrial fission/fusion for pathogens infecting hosts,and provide lessons and references for the study on biological functions and regulation of pathogen organelles. |
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