Functional Analysis Of Chitin Synthase Family In Metarhizium Acridum

With the development of modern molecular biology techniques, the mechanisms of fugal pathogenesis in insects have been further revealed. Metarhizium acridum has become a model entomopathogenic fungus to investigate the insect fungal pathogenesis. Chitin is an important component of the fungal cell wall and is synthesized by chitin synthase family. Filamentous fungi generally have seven CHS genes. At present, the roles of chitin synthase family in the entomopathogenic fungi are largely unknown. In this view, we identified and characterized the chitin synthase gene family of Metarhizium acridum. The manily results are as follows:①Metarhizium chitin synthase family have been grouped into seven classes, named Ma Chs I, Ma Chs II, Ma Chs III, Ma Chs IV, Ma Chs V, Ma Chs VI, Ma Chs VII.②The expression levels of CHS genes in conidium, appressorium and hyphal boby were assayed by q RT-PCR. In comparison with other CHS genes, Ma Chs III display the highest relative expression level in all samples except hyphal body of 5 days. In hyphal body of 5 days, Ma Chs VII has the highest expression level. Ma Chs VI has the lowest expression level among the CHS genes in all samples. Compared with in conidium and hyphal body, Ma Chs II exhibited higher expression level during appressorium formation. Other CHS genes had higher expression level in conidia than those of in appressorium and hyphal boby.③Phenotypic analysis showed that ΔMa Chs III, ΔMa Chs V grew weaker than wild type in CR, CFW and H2O2, ΔMa Chs VII grew weaker than wild type in CR, CFW, H2O2, SDS, SOR, Na Cl and 1/4SDAY control. The tolerances of various strains to heat shock and UV-B were analyzed. Disruption of Ma Chs II, Ma Chs III, Ma Chs IV, Ma Chs V and Ma Chs VI affect fungal sensitivity to heat shock, delection of Ma Chs II, Ma Chs IV, Ma Chs VI affect fungal sensitivity to UV-B. No difference was observed between wild type and ΔMa Chs I.④The conidial yield and geimination of various strains were assayed. Compared with the wild-type strain, the conidial yields of Ma Chs disruption mutants except ΔMa Chs I were decreased. ΔMa Chs VII displayed the largest reduction in conidial yield. With respect to conidial germination, the 50% germination time of ΔMa Chs I and ΔMa Chs IV were delayed, while the 50% germination time of ΔMa Chs II, ΔMa Chs III and ΔMa Chs V were accelerated. No difference was observed between ΔMa Chs VI and WT.⑤Bioassays were conducted by topical inoculation and hemocoel injection. Compared with the wild-type strain, ΔMa Chs III exhibited reduced virulence by topical inoculation and hemocoel injection. Interestingly, disruption of Ma Chs V increased fungal virulence by hemocoel injection. ΔMa Chs VII lost the virulence by hemocoel injection.⑥The cell wall structures of various strains were analyzed by transmission electron microscope. Conidium cell walls of mutants were thicker than the wild-type.