Study On The Functions Of MrPex14/17 From Metarhizium Robertsii

Metarhizium robertsii is an insect pathogenic fungus that is widely used in biological control of pests and diseases.In recent years,it has been reported that it is also used as a biological fertilizer.Like other entomogenous insecticides,biofungal insecticides based on Metarhizium robertsii spores have short shelf life,easy to change control effects and long-term effectiveness of insecticides,which leads to their small market share;Therefore,the pathogenic and stress-resistant mechanisms of Metarhizium robertsii and other insect pathogenic fungi need to be further studied in depth to provide a theoretical basis for the efficient field application of biological control.In this study,Metarhizium robertsii was used as the research object,and the sequence characteristics of MrPex14/17（MAA＿05331）were analyzed.The knockout strains（MT,ΔMrPex14/17）and backfill were obtained using strategies such as gene knockout and backfill mutants.Strain（Comp,ΔMrPex14/17-Comp）,and through a variety of phenotypic analysis and identification with wild-type strains（WT,Mr2575）to clarify the biological function and regulatory mechanism of Metarhizium robertsii MrPex14/17.The main analysis results are as follows:1.MrPex14/17 sequence feature analysisSearch in NCBI to obtain the full-length sequence of MrPex14/17.The results of sequence analysis revealed that there was a conserved Pex14＿N domain at the N-terminus（Accession:pfam04695）;at the C-terminus,there was a weak similarity to the yeast Sc Pex17（CAA96116.1）.In addition,the phylogenetic tree was constructed and found to be highly related to Metarhizium acridum and Ophiocordyceps sinensis.2.MrPex14/17 subcellular localizationThe EGFP-MrPex14/17 fusion protein was co-transformed with the peroxisomal molecular marker m Cherry-PTS1（Peroxisomal Targeting Signals1）into WT,and the subcellular localization of MrPex14/17 in peroxisomes was observed by laser confocal.It shows that MrPex14/17 fused to express EGFP in WT is distributed in green dots while the peroxisomal molecular marker m Cherry-PTS1 is shown in red dots and the red and green overlap,indicating that MrPex14/17 is located in Peroxisomal from Metarhizium robertsii.3.Obtaining MrPex14/17 gene knockout strains and supplementary strainsAccording to the genome location of MrPex14/17（MAA＿05331）,select flanking sequences and full-length sequences at both ends to construct its knockout vector plasmid and complement vector plasmid;and obtain the MrPex14/17 gene by Agrobacterium-mediated transformation and other methods Knockout strains and replacement strains.4.Growth analysis of MrPex14/17 deletion in Metarhizium robertsiiThe growth and development analysis of three different strains（WT,MT,Comp）revealed that:compared with WT/Comp,the MT spore germination rate increased by 26.7%at 12h;the spore production decreased by 46.5%at 14d.The results showed that MrPex14/17 deletion played a key role in the growth and development of Metarhizium robertsii.5.MrPex14/17 deletion responds to chemical stress in Metarhizium robertsiiSpore suspensions of three different strains（WT,MT,Comp）were spotted and cultured on PDA,PDA-Congo red,PDA-CFW,and PDA-H₂O₂medium respectively.The test results found that:compared with WT/Comp,the MT colony diameter decreased by 8.9%,17.2%,and 10.1%on Congo red,CFW,and H₂O₂,respectively.It showed that after the deletion of MrPex14/17,the integrity of Metarhizium robertsii cell wall decreased and the reactive oxygen response decreased.6.Pathogenicity analysis of MrPex14/17 deletion in Metarhizium robertsiiBiological determination of three different strains（with Galleria mellonella as a test insect）.In the body wall infection test:compared with WT/Comp,MT lethality decreased,and further calculation of LT₅₀found that MT LT₅₀（9.46±0.49d）was significantly longer than WT LT₅₀（5.31±0.78d）and Comp（4.44±0.33 d）;In the direct injection infection test,compared with WT/Comp,the MT virulence was not significantly reduced;further we measured the attachment cell formation rate and cuticle penetration ability of the three strains.The experiment found that:compared with WT/Comp,most of MT cannot generate appressorium structure.In the hydrophobic dish appressorium formation test and cicada wing appressorium formation test,the MT appressorium generation rate decreased by 73.2%and 82.0%;cuticle in the penetration ability test,compared with WT/Comp,MT colony diameter decreased by36.5%.These results indicate that the deletion of MrPex14/17 leads to a decrease in the rate of appressorium formation of the strain,which affects the ability of the stratum corneum to penetrate and ultimately leads to a significant decrease in virulence.In summary,the absence of MrPex14/17 resulted in a significant reduction in spore production,a decrease in reactive oxygen response,a decrease in cell wall integrity,a decrease in virulence,a decrease in the rate of attachment cells,and a decrease in the ability of the stratum corneum to penetrate;these results indicate that MrPex14/17 17 participated in the growth and development and pathogenesis of Metarhizium robertsii.