| Metarhizium roberstii is an important insect pathogenic fungus,which is widely used in the control of agricultural and forestry pests.However,the application of Metarhizium roberstii has obvious shortcomings as a new biological pesticide,such as the poor and slowly infection effect,the unstable control effect in the field environment,which seriously hinders the large-scale application of M.roberstii in agricultural production.Therefore,searching the molecular mechanism involved in the growth and pathogenesis of M.roberstii and improving the toxicity and the ability to resist adversity from the genetic engineering is a key way to effectively control agricultural and forestry pest by using M.roberstii.G protein signal regulator(RGS)is a GTPase activating protein,which promotes the decomposition of GTP with Galpha by enhancing the activity of GTPase on Galpha subunit,and then negatively regulates G protein signal transduction.In this paper,we studied the different function of different G protein signaling regulators in the growth,development,stress resistance and pathogenesis of M.roberstii systematically.The results are as follows:1.G protein signal regulator RGS is a negative regulator in G protein signal pathway.7 MrRGS proteins homologous to G protein signal regulator MoRGS of Magnaporthe oryzae were identified from the whole gene sequence of M.roberstii.The amino acid homology between MrRGS 1-7 protein of M.roberstii and MoRgsl-8(except MoRgs5)protein of M.oryzae was very high,reaching 59%,43%,78%,52%,45%,45%and 46%,respectively.The seven pairs of proteins all contained the same functional domains and transmembrane structures at specific locations.2.The different Mrrgs transcription levels in different growth and development stages of M.roberstii differed greatly.Further biological function analysis showed that these different MrRGS had different functions in growth and development.For example,the deletion of Mrrgsl,Mrrgs2 and Mrrgs6 had a great influence on the growth rate of M.roberstii,and the deletion of these three genes also affected the sporulation ability of the strain.The deletion of four genes,namely,Mrrgs3,Mrrgs4,Mrrgs5 and Mrrgs7,had little effect on the growth rate and sporulation.The expression of genes involved in regulating cell growth or sporogenesis in M.roberstii was further detected.The results showed that the expression of the key genes related to sporogenesis,wet A and StuA was significantly decreased in mutant ?Mrrgsl,while the expression of other sporogenesis-related genes such as flbA,flbB,flbC,flbD,brlA and vosA was also significantly decreased.The expression of spore-related key genes,AbaA and wetA also decreased significantly in mutant ?Mrrgs2,and the expression of flbA and brlA genes also decreased significantly.The expression off flbB,flbD and brlA decreased significantly in ?Mrrgs6 mutant.It is suggested that MrRGS is an important factor regulating the growth and development of M.roberstii,and participates in the physiological processes such as vegetative growth and conidiaproduction.3.Different G-protein signaling regulators have different ability to regulate the pathogenicity of M.roberstii.Bioassays showed that deletion of Mrrgs3,Mrrgs4 and Mrrg 7 had the greatest effect on virulence,while deletion of Mrrgs2 had little effect on virulence.The virulence of ?Mrrgs4 and ?Mrrgs4 decreased by 17.7%and 28.4%respectively in the injection test,while the virulence of M.roberstii with deletion of Mrrgs1,Mrrgs3,Mrrgs4,Mrrgs5 and Mrrgs7 decreased by 29.9%,33.5%,53.4%,29.3%and 32.4%respectively in the immersion test.In order to explore the mechanism of its influence on virulence,we studied the hydrophobicity of mycelium,the formation of appressorium and the content of cAMP in mycelium systematically.The results are as follows:(1)The hydrophobicity of wild type and mutant strains was determined on the colony.It was found that the hydrophobicity of ?Mrrgs3 and ?Mrrgs5 decreased significantly.Then,the expression of two hydrophobic related genes in ?Mrrgs Mutants was detected.The results showed that the expression of MHP1 and MPG1 genes in 5 mutants decreased compared with wild strains except ?Mrrgs2 and ?Mrrgs6.The results showed that the mutations of Mrrgs3 and Mrrgs5 genes directly affected the hydrophobicity of M.roberstii.(2)Apart from ?Mrrgs2 and AMrrgs6,the appressorium formation rate of mutants on hydrophobic dishes and cicada wings decreased significantly in all five mutants,among which ?Mrrgsl,?Mrrgs3,?Mrrgs5 and ?Mrrgs7 decreased most significantly.(3)The level of intracellular cAMP was determined by high performance liquid chromatography(HPLC).Compared with wild type,the content of cAMP in six ?Mrrgs decreased in different degrees,among which the content of cAMP in four mutants ?Mrrgsl,dMrrgs3,?Mrrgs4 and ?Mrrgs5 decreased most significantly.These results indicated that RGS protein was involved in regulating the intracellular cAMP level of M.roberstii.4.The different Mrrgs genes in M.roberstii also have different functions for chemical agents resistance,heat,and UV stress.Chemical reagent stress experiments showed that the effects of different Mrrgs genes on cell wall integrity,antioxidant and osmotic resistance of M.roberstii were significantly different.For example,the growth inhibition rates of ?Mrrgs2,?Mrrgs3,?Mrrgs5,?Mrrgs6 and ?Mrrgs7 on the medium containing hydrogen peroxide were 26.3%,20.2%,8.3%,10.7%and 29.6%lower than those of wild-type strains,respectively.The growth inhibition rates of ?Mrrgs2,?Mrrgs3 and?Mrrgs6 on NaCl-containing media were 6.8%,6.4%and 8.8%lower than those of wild-type strains,respectively.The inhibition rate of NaCl on growth of ?Mrrgs7 was 18.5%higher than that of wild-type M.robertii.The results of spore germination experiments under heat and ultraviolet stress showed that the relative germination rate of ?Mrrgs7 decreased significantly by 27.3%after being treated at 42? for 1 h.The relative germination rate of ?Mrrgs5 decreased significantly when irradiated for 12 seconds under 60 J ultraviolet lamp,reaching 37.0%.In conclusion,different G protein signal regulators have different functions in the growth and pathogenesis of M.roberstii.Among them,Mrrgsl,Mrrgs2 and Mrrgs6 genes play a positive role in regulating the growth rate and sporulation ability of M.roberstii.Further analysis at the transcriptional level revealed that these three G protein signal regulators ultimately affect sporulation ability by regulating the expression of sporulation-related genes.In addition,in the study of pathogenicity,we found that the genes of Mrrgsl,Mrrgs3,Mrrgs4,Mrrgs5 and Mrrgs7 affect the formation of appressorium by regulating the hydrophobicity of M.roberstii,thus affecting its virulence. |
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