Comparative Transcriptomic Analysis For Responsing To Heat Stress And D Ifferential Function Roles Of Two Metalloproteases (Mrmep1 And 2) In Metarhizium Robertsii

The entomopathogenic fungi Metarhizium is widely used in biological control of a variety of insect pests.The degree of commercialization of Metarhizium and the efficiency of field application,however,is limited by poor thermotolerance.At present,the heat tolerance of Metarhizium is limited to the study of a single gene.The molecular mechanism underlying the response to heat stress in the conidia of entomopathogenic fungi remains unclear.Therefore,in order to elucidate the molecular mechanism profoundly and further improve the biocontrol effect of Metarhizium anisopliae,in this study Metarhizium robertsii was used as the experimental material.Here,we conducted high-throughput RNA-Seq to analyze the differential gene expression between control and heat treated conidia of M.at the transcriptome level.A number of genes underlying the response to heat stress were found.Above all,transcriptome analysis revealed two zinc metalloproteinase genes,Mrmepl(EFY97549)and Mrmep2(EFY97706)was significantly up-regulated in M.robertsii after heat shock.These results suggested that two metalloproteases genes may be involved in the heat stress response in conidia.Herein,we characterized two MEPs,Mrmepl and Mrmep2,in M.robertsii using gene deletion.The detailed results were as follows:1.Using high-throughput RNA-seq sequencing technology,the differential expression profiles of the conidia of Metarhizium sinensis before and after heat treatment were compared and analyzed,and 3,510 differentially expressed genes were identified.Of these,a total of 2,722 genes were significantly up-regulated under conditions of heat stress while 788 genes were significantly down-regulated.2.Then the GO and KEGG Pathway enrichment analysis were carried as well as the functional analysis of the 3,510 DEGs in the response to heat stress.Among these differentially expressed genes,many were related to metabolic processes,biological regulation,cellular processes and response to stimuli.The majority of genes involved in endocytic pathways,proteosome pathways and regulation of autophagy were up-regulated,while most genes involved in the ribosome pathway were down-regulated.As expected,significant changes in expression levels of genes encoding heat shock proteins and proteins involved in trehalose accumulation were observed in conditions of heat stress.These results suggested that the two genes may be involved in the heat stress response in conidia.3.The two MEPs(Mrmepl and Mrmep2)sequences were amplified using rapid amplification of cDNA ends(RACE).The predicted molecular weight and theoretical isoelectric point(pI)of the deduced Mrmep1and Mrmep2 proteins were 68.66kDa and 27.89 kDa,and 6.17 and 5.66,respectively.Both Mrmep1and Mrmep2 were found to contain predicted signal peptide sequences,suggesting that they are extracellular proteins.Mrmepland Mrmep2 are Zinc MEPs by a phylogenetic tree of MEPs from Metarhizium spp..4.To validate the role of the two MEPs during pathogenesis,we developed M.robertsii null mutants lacking the Mrmepl or Mrmep2 genes as well as a double mutant.The results showed that the growth rates of ?Mrmepl and ?Mrmep2 mutants decreased by 16.2%and 16.5%,respectively,relatived to the wild-type(WT)strain.Both mutants were less sensitive to cell wall-perturbing agents,sodium dodecyl sulfate and Congo red,than the WT strain,whereas the fungi did not show any obvious changes in fungal sensitivity to ultraviolet B irradiation or heat stress.The conidial yield of ?Mrmep1,?Mrmep2,and?Mrmep1?Mrmep2 mutants decreased by 56.0%,23%,and 53%,respectively.Insect bioassay revealed that median lethal time values against Galleria mellonella increased by 25.5%(?Mrmep1),19%(?Mrmep2),and 28.8%(?Mrmep1?Mrmep2)compared with the WT strain with a concentration of 1×107 conidia mL-1,suggesting attenuated fungal virulence in the ?Mrmep1,?Mrmep2,and ?Mrmep1?Mrmep2 strains.During fungal infection,transcription levels of Mrmepl was 1.6-fold higher than Mrmep2 at 36 h post inoculation.Additionally,expression levels of gallerimycin gene were 1.2-fold,2.18-fold,and 2.5-fold higher in insects infected with the ?Mrmep1,?Mrmep2,or?Mrmep1?Mrmep2 mutant than those infected with the WT strain,respectively.These results revealed that Mrmepl and Mrmep2 had different functions.In summary,this paper had preliminarily revealed the molecular mechanisms of the heat stress response of conidia of M.robertsii and the different functions of two metalloproteases(Mrmep1and Mrmep2)in the growth,sporulation,cell wall integrity and virulence formation of M.robertsii.These results provide important genetic resources and theoretical basis for further creating transgenic strains with high virulence and environmental tolerance using metalloproteases.