Functional Analysis Of The Protein O-mannosyltransferases(PMTs) In Metarhizium Acridum

Entomopathogenic fungi have great potential for development as biological control agents. Thus, elucidating the fungal pathogenesis and the detailed knowledge of resistance to stress would be helpful for improvement of the biocontrol fungus. In phytopathogenic fungi and human pathogenic fungi, O-mannosylation plays essential roles in development, stress tolerance and virulence. However, the functions of PMTs in M. acridum are largely unknown. In this paper, the PMTs of M. acridum have been identified and characterized. The main results are as follows:1. Knockout or knockdown of Ma Pmt1/2/4Bioinformatics analysis indicated that c DNA genomic sequences revealed three PMT homologues, contained Pmt1, Pmt2 and Pmt4. Alignments of the three M. acridum Pmt proteins with other species Pmtps demonstrated high conservation, containing ten transmembrane regions, one large luminal loop and a luminal C-terminus and the expression products of Ser/Thr rich residues. The vector of homologous recombination of Ma Pmt1, Ma Pmt4 gene and the RNA interference vector of Ma Pmt2 gene were constructed. The knockout or knockdown strains and the recovery strains were verified by Southern bloting or q RT-PCR.2. Knockout or knockdown of Ma Pmt1/2/4 affected fungal growth and stress toleranceIn the PDA solid medium, the GT50 of ΔMa Pmt4 and Pmt2-KDs had significant decrease compared with WT. However, deletion of Ma Pmt1 had no significant differences in conidial germination and conidial yield. However, the conidial yields of Pmt2-KDs reduced by 50% than that of WT. Ma Pmt1, Ma Pmt2, Ma Pmt4 of M. acridum increased its sensitivity on UV-B and heat shock treatment.3. Knockout or knockdown of Ma Pmt1/2/4 were severely attenuated for virulenceThe results of bioassays demonstrated that Ma Pmt1/2/4 were required for fungal virulence. Disruption of Ma Pmt1 reduced fungal virulence due to the decrease of turgor pressure of appressoria; Disruption of Ma Pmt4 reduced fungal virulence due to the decrease of appressorium formation, turgor pressure of appressoria and growth in host hemolymph; Knockdown of Ma Pmt2 reduced fungal virulence due to the decrease of appressorium formation and turgor pressure of appressoria.4. Knockout or knockdown of Ma Pmt1/2/4 affected its cell wall composition and structureAs revealed by transmission electron microscopy(TEM), the Ma Pmt1/2/4 mutants lost partial outer layer of conidial walls, which became thinner or obscure between cell periphery and cytoplasm, as revealed by more accumulation of electron dense material(stain) in cytoplasm. Fluorescent staining with antibodies or fluorescent dye, we investigated the changes of cell wall composition and structure. We found the cell wall of Ma Pmt1/2/4 mutants showed differences in its surface composition and structure. Content analysis found that Knockout or knockdown of Ma Pmt1/2/4 reduced the mannoproteins and chitin contents.In summary, these results will help to furhter elucidate the mechanisms of fungal pathogenesis. The knowledge will provide theoretical foundation for developing stable, high-efficiency mycoinsecticide.