| Chitin is the key component of the cell wall in almost all fungi to date, which binds to glucan, another key component in cell wall, to form the tough skeleton of the cell wall, thus sustaining the shape of the cell and preventing the invasive microorganism.At the same time, chitin also plays important role as the Pathogen-Associated Molecular Pattern (PAMP) in the innate immune response.And the key enzymes involving in the process of chitin synthesis named chitin synthases have already to be find related to the pathogenicity of pathogenic fungi(take the plant pathogenic fungi, M. oryzae, for example). Metarrhizium robertsii, used in this study, is one of the important insect-pathogenic fungi and being explored for biocontrol with limatations of slow killing speed and instability.However,the research of chitin synthases which may be related to the pathogenicity in M. robertsii is unpublished.Therefore, functional characterization of all chitin synthases from M. robertsii may shed light on the mechanism of insect-pathogenic fungi, which contributes to find the soild theoretical foundations for biocontrol.The progress made in the research of the chitin synthases in M. robertsii is as follows:With the chitin synthases sequence from S. cerevisiae and M. oryzae as queries, we find there may be seven chitin synthase genes in Metarrhizium robertsii.Then, through domain structure analysis (PFAM database), the seven proteins were confirmed to have tipical chitin synthase domain and conversed CON1 domain (QxxEY, EDRxL, QxRRW).Finally, we name the seven proteins as ChsⅠ(Maa-02999),ChsⅡ(Maa-02740),ChsⅢ(Maa-03168),ChsⅣ(Maa-00137),ChsⅤ( Maa-01111), ChsⅥ(Maa-01099)andChsⅦ(Maa-01112)respectively through domain analysis and phylogeny analysis.Through the expression pattern analysis, we find except ChsⅥ, the remaining chitin synthases are highly expressed in almost all the life stages of M. robertsii.In addition, the expression pattern of ChsV and ChsⅦ are similar, both of which have high expression level in the development phase and infection phase of Metarrhizium robertsii,with the highest expression level in appressorium.We then constructed the mutants of seven chitin synthases from Metarrhizium robertsii to find out the function of the chitin synthase gene family.During saprophytic growth,ΔChsⅠ, ΔChsⅡ、ΔChsⅤ and ΔChsⅦ were inhibited in complete medium. Besides, Compared to the wild type, the conidiation yield of ΔChsⅤ and ΔChsⅦ reduced significantly, both of which formed abnormal conidiophore with fewer branches. And through SEM (scanning electron microscope) observation of the spore from seven chitin synthases mutants, the hydrophobic rodlet structure on the spore surface of ΔChsⅤ and ΔChsⅦ were impaired.During the pathogenicity assay, the pathogenicity of ΔChsⅤ and ΔChsⅦ reduced significantly compared to the wild type. Further experiment like appressoria observation, ΔChsⅤ and ΔChsⅦ formed abnomal appressoria with big vacuoles occupied. Besides, through turgor pressure measurement assay, the turgor pressure of ΔChsⅤ and ΔChsⅦ reduced significantly compared to the wild type.Under heat stress, ΔChsⅡ and ΔChsⅤ are lethal, while ΔChs1 and ΔChsⅦ are inhibited significantly. Besides, through the analysis of chitin synthase gene family from nine 9 phyla with 232 fungal species,we find there are 95 fungal species with ChsⅤ and ChsⅦ from Pezizomycotina, which were produced by tandem duplication.In the 95 fungal species,there are 78 species were pathogenic fungi and the remaining 17 species are saprophytic fungi, among which 10 had thermotolerance and the other 7 were close taxonomic relatives of pathogenic fungi or the fungi with thermotolerance.Through the analysis of the represented strains to heat stress, we confirmed that some of the chitin synthases mutants from the species with ChsV and ChsⅦ tandem duplication are turly sensitive to heat stress. |
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