Functional Analysis Of Mid1 Gene In Metarhizium Acridum

Entomopathogenic fungi are a kind of important bio-control microorganisms. Compared with other pesticides, they have advantages of strong selectivity,no environmental pollution and easy to transform. However, entomopathogenic fungi also have obvious shortcomings such as poor efficiency and seriously dependence on environment which strongly restrict the development of fungal pesticides.Therefore, clarifying fungi pathogenesis and improving virulence would lay foundation for the development of the biocontrol fungi.Previous reports showed that the pathogenicity of many fungi are regulated by Ca²⁺.Mid1, as a part of putative stretch-activated calcium channel, is not only the way where external Ca²⁺ gets into the cytoplasm, but an important factor to affect fungus physiological function by changing the concentration of Ca²⁺. However, the functions of Mid1 in entomopathogenic fungi are not clear. In this study, Mid1 gene was functionally analyzed by deleting it from the entomopathogenic fungus Metarhizium acridum.The main results are as follows:?1? Bioinformatic analysis of the Mid1The Mid1 cDNA?XP007808776.1? had an ORF of 1818 bp, which encodes a predicted protein of 603 amino acids residues with a calculated molecular weight of 87.43 kDa and an isoelectric point of 6.27. The Mid1 contained a C-terminal signal peptide, a hydrophobic transmembrane domains and two highly conserved Cysteine domains C1, C2 with other fungal Mid1. Phylogenetic analysis of Mid1 indicated that Mid1 protein was closely related to Mid1 from the entomopathogenic fungus M. anisopliae and had 95% identity.?2? Mid1 deletion and complementationTo construct Mid1 deletion, Mid1 was replaced by the Bar as a selectable marker gene through homologous recombination. Deletion and recovery of Mid1 was confirmed by PCR and Southern blot.?3? Expression pattern of Mid1The transcription level of Mid1 in the mycelium, conidia?3d, 6d, 9d and 12d?, appressorium and hyphal body were analyzed. Quantitative real-time polymerase chain reaction?qRT-PCR? revealed strong expression of Mid1 during appressorium formation period.?4? Virulence is attenuated in the ?Mid1 mutantBioassay against locust indicated that pathogenicity of ?Mid1 was attennuated compared with WT and CP in topical inoculation. The LT50 values were significantly delayed in the topical inoculation assays, but no difference was found in LT50 between WT and ?Mid1 in injection assays. These results suggested that Mid1 may possibly affect the process of penetrating into insect cuticle.?5? Mid1 affected the growth of hyphal bodies in insect haemocoelIn order to study the affect of Mid1 to the growth of M. acridum in insect haemocoel, the number of hyphal bodies was analyzed in different time. The results indicated that ?Mid1 hyphal body appeared in haemolymph later than that of WT and CP. Quantitative analysis showed that hyphal bodies in haemolyph significantly decreased in ?Mid1 compared to WT. However, there were no different in the number of hyphal bodies in injection assay.?6? Disruption of MaMid1 effected appressorium formation.Germination and appressorium formation on locust wings were analyzed. The results showed that the spore germination rate of ?Mid1 has no significant difference compared to the wild-type strain and CP. However, appressorium formation rate of ?Mid1 was significantly lower than the wild-type strain and CP.?7? Mid1 effected the expression of genes related to penetration processBioassay results showed that Mid1 affected the process of penetrating the insect cuticle. Expression of genes Pr1, Pr2, CHI, CHII and lipase, which have been reported to involve in penetration process, were analyzed by RT-PCR. Results showed that Pr1, CHI and lipase were all downregulated, indicating that Mid1 was responsible for the expression of gene related to penetration proecess.?8? Mid1 was involoved in cell wall integrityColony diameter of ?Mid1 was significant smaller than WT and CP, when the fungi were grown on the 1/4SDAY added with CFW and CR. However, ?Mid1 had no significant difference in tolerance to UV irradiation and heat stress compared with WT and CP.?9? ?Mid1 mutant was more sensitive to metal ionsThe growth of fungi was tested on 1/4 SDAY added with different metal ions such as Ca²⁺, Fe²⁺, Mg²⁺, Mn²⁺ and the calcium chelator EGTA. The growth of all strains were inhibited but the ?Mid1 was more sensitive to these ions,indicating that ?Mid1 was sensitive to different metal ions.?10? Mid1 regulated trafficking of calciumA specific calcium Fluorescent staining, Fluo-3AM, was used to analyze the calcium in funal cell. Results suggested that the calcium fluorescent of ?Mid1 was weaker than that of WT and CP. The transcriptional level of Mid1 under environment of different metal ions were analyzed by qRT-PCR. Results showed that Mid1 was up-regulated when Ca²⁺ was present, while under the Fe²⁺, Na⁺, K⁺, EGTA, Mid1 was down-regulated. These results indicated that Mid1 was involved in the regulation of calcium traffick.In summary, our results suggested that Mid1 was involved in growth, virulence and contributed to penetration processes through affecting appressorium formation, the number of hyphal bodies in insect haemocoel. Therefore, to clarify the function of mid1 helps further understanding the mechanisms of pathogenesis in entomopathogenic fungi.