Functional Analyses Of The Gti1/Pac2 Family Proteins MaGti1 And MaPac2 In Metarhizium Acridum

The entomopathogenic fungi,as an important resource of biocontrol agents,are of great potential in pest control due to their low possibility of producing insect resistance,environmental friendliness,and considerable diffusion capacity.However,some disadvantages,such as sensitivity to adverse conditions,poor efficiency,and high production costs,have limited their large-scale applications.Therefore,enhancing basic research intensity in entomopathogenic fungi should be beneficial to promote the production and application of mycopesticides.The Gti1/Pac2 family proteins are important transcriptional factors in fungi,and play crucial roles in regulating the growth and pathogenicity of fungi.In this study,two of the Gti1/Pac2 family protein genes,MaGti1 and MaPac2,were cloned in the model entomopathogenic fungus Metarhizium acridum.And the roles of MaGti1 and MaPac2in the growth,stress resistance,infection and pathogenicity of M.acridum were determined using the knockout and complemention strategies.The main results are as follows:1.Bioinformatics analysis of the Gti1/Pac2 family proteins in M.acridum.Two genes encoding the Gti1/Pac2 family proteins,MaGti1 and MaPac2,were found in the complete genome sequence of M.acridum.Their total length of c DNAs are severally 1482 bp and 1269 bp,encoding proteins of 493 and 422 amino acids.Both proteins contain the typical Gti1/Pac2 family domain（Pfam09729）.2.Disruption of MaGti1 or MaPac2 affects the growth of M.acridum.To determine the functions of MaGti1 and MaPac2,the knockout and complemention strains of these two genes were successfully constructed,respectively.The conidial germination rates and conidial yield of each strain on 1/4 SDAY medium were determined.The results showed that deletion of MaGti1 or MaPac2 both promoted the conidial germination.Additionally,MaGti1^ΔDOCK（functional domain deletion）,MaGti1^S75A（CDK phosphorylation site serine was replaced by an alanine residue）and MaGti1^T69A（PKA phosphorylation site threnine was replaced by an alanine residue）also promoted the conidial germination.The conidial yield ofΔMaPac2 was decreased significantly compared to that of WT,but there was no significant difference betweenΔMaGti1 and WT.3.Disruption of MaGti1 or MaPac2 affects the stress tolerance of M.acridum.The conidia of each strain were treated with heat-shock or UV-B,and inoculated on 1/4 SDAY medium supplemented with kinds of chemical reagents.Then the conidial gemination or the colony growth were examined to evaluate the contributions of MaGti1 and MaPac2 to fungal stress tolerance.The results showed that the IT₅₀s of theΔMaGti1,MaGti1^ΔDOCK,MaGti1^S75A,MaGti1^T69A andΔMaPac2 strains were significantly increased relative to that of WT after the heat-shock treatment.After the UV-B treatment,the IT₅₀s ofΔMaGti1,MaGti1^ΔDOCK and MaGti1^S75A were significantly decreased and the IT₅₀ ofΔMaPac2 was significantly increased compared to that of WT.These results demonstrated that both MaGti1 and MaPac2 were involved in thermo-and UV-tolerances of M.acridum,and the intact Gti1/Pac2 family domain of MaGti1 is crucial for fungal stress tolerances.There was no significant difference in colony morphology betweenΔMaGti1 and WT on 1/4 SDAY medium supplemented with CR,CFW and H₂O₂.However,there was obvious decrease in colony size betweenΔMaPac2 and WT,indicating that the MaPac2-disruption affected the tolerance of M.acridum to CR,CFW and H₂O₂,while the MaGti1-disruption had no effect.4.Disruption of MaGti1 or MaPac2 affect the virulence of M.acridum.The effects of MaGti1 or MaPac2 deletion on the pathogenicity of M.acridum were severally examind by the topical inoculation and intrahaemocoel injection.It was found thatΔMaGti1 andΔMaPac2 was both significantly lower than that of WT in virulence in the topical inoculation.Then,the appressorium formation of each strain on the locust hind wings was analyzed.As a result,the appressorium formation rate ofΔMaGti1 was significantly lower than that of WT.Similarly,the appressorium formation rate ofΔMaPac2 was significantly reduced in the early stage.At 28 h,however,there was no difference in the appressorium formation rate between theΔMaPac2 and WT.In the intrahaemocoel injection assay,the virulence ofΔMaGti1 andΔMaPac2 was also both significantly lower than that of WT.These results demonstrated that both MaGti1-disruption and MaPac2-disruption affected the virulence of M.acridum.5.MaGti1 regulates the conidiation pattern shift of M.acridum.After incubating each strain on the microcycle conidiation medium SYA,it was found that the conidiation pattern of WT andΔMaPac2 was microcycle conidiation,while those ofΔMaGti1,MaGti1^ΔDOCK,and MaGti1^S75A were normal conidiation.And the transcriptome sequencing analysis was performed to further reveal the mechanism of MaGti1 regulating the conidiation pattern shift of M.acridum.The results showed that 228 differencially expressed genes（DEGs）were isolated.Of the 228 DEGs,including 86（37.7%）known proteins,142（62.3%）putative proteins,91（39.91%）up-regulated genes and 137（60.09%）down-regulated genes.These DEGs are mainly related to conidiation,mycelium growth,cell membrane,transport and virulence,suggesting that MaGti1 regulates the conidiation pattern shift by regulating the expression of genes related to conidiation and mycelium growth.