| Rice blast caused by Magnaporthe oryzae is one of the most serious fungal diseases in rice production.At present,rice blast is mainly controlled by breeding and using resistant varieties,supplemented by chemical pesticide control.However,due to the changeability of the pathogen and the emergence of drug resistance and so on,the resistant varieties and chemical agents can not be used for a long time.Therefore,the effective way to control rice blast is to find out the potential target of fungicide and to develop new fungicides with.long-term,high efficiency and low toxicity.Besides,the premise to determine the target of fungicide is to understand the biological function of key genes involved in the growth and pathogenic process of the pathogenic bacteria.Thus,depth analysis of the pathogenic molecular mechanism of M.oryzae can provide theoretical basis and experimental basis for the mining of new targets of fungicide and the formulation of new disease control strategies.Transcription regulation is one of the main mechanisms that affect the expression of specific genes in the process of cell development and differentiation,and this process is mainly controlled by transcription factors.Previous research in our laboratory found a transcription factor MoMsn2,which can directly or indirectly regulate the transcription of a series of genes to participate in the control of multiple biological processes of M.oryzae,such as vegetative growth,asexual reproduction,stress response,cell wall integrity,pathogenicity and fatty acid metabolism.In order to further analyze the regulatory mechanism of MoMsn2,we selected two genes from the 332 potential target genes identified in the previous study and carried out depth functional analysis,including β-1,3 exoglucanase encoding gene MoMTG7 which may be related to cell wall integrity and peroxisome acetyl CoA synthetase encoding gene MoPCS60 which may be related to fatty acid metabolism.The main results are as follows:First,MoMTG7 is involved in the regulation of the formation of functional appressorium,stress response and pathogenicity of M.oryzae.Electrophoretic mobility shift assay and quantitative PCR analysis revealed that the promoter region of MoMTG7 combines with MoMsn2;and the expression of MoMTG7 was significantly down-regulated in △Momsn2 mutant,which showed that MoMTG7 is a target gene regulated by MoMsn2.We obtained the knockout mutant △Momtg7 by homologous recombination and carried out a series of analysis on its biological phenotype.It was found that the growth of the mutant on the complete medium slowed down and its pathogenicity decreased significantly.And further analysis showed that the formation of appressorium was delayed,the bud tube became longer,the turgor decreased,and the spread of infection hypha slowed down.Furthermore,stress response analysis showed that the sensitivity of △Alomtg7 to Congo red,a cell wall stress agent,increased significantly,while the sensitivity to salt stress,osmotic stress and oxidative stress decreased significantly.These results indicated that MoMTG7 is involved in the regulation of vegetative growth,functional appressorium formation,stress response and pathogenicity of M.oryzae.Second,MoPCS60 is involved in the regulation of fatty acid metabolism and pathogenicity of M.oryzae.Electrophoretic mobility shift assay and quantitative PCR analysis revealed that the promoter region of MoPCS60 combines with MoMsn2,and under the induction of sodium oleate,the expression level of MoPCS60 in AMomsn2 mutant was significantly lower than that in wild type,and these results suggested that MoPCS60 may be a target gene regulated by MoMsn2 during oleic acid metabolism.We obtained the knockout mutant △Mopcs60 by homologous recombination and carried out a series of analysis on its biological phenotype.It was found that the vegetative growth of AMopcs60 slowed down,the formation of appressorium was delayed,and the invasion rate and pathogenicity were significantly reduced.Further analysis showed that MoPcs60 was involved in the metabolism of fatty acids.The growth of △Mopcs60 on sodium oleate medium slowed down,and at the same time,the number of peroxisomes in △Mopcs60 decreased and the number of lipid droplets increased significantly.These results indicated that MoPcs60 plays an important role in regulating fatty acid metabolism and pathogenicity of M.oryzae.In summary,MoMTG7 and MoPCS60 are target genes of MoMsn2.MoMsn2 controls the growth and pathogenicity of M.oryzae by regulating the expression level of these genes.The results of this study further analyzed and improved the molecular regulatory mechanism of MoMsn2 on cell wall integrity and fatty acid metabolism of M.oryzae,and it also provided potential targets for the development of new targets of fungicide. |
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