Functional Analysis Of Serine-threonine Protein Kinase Genes MaSnf1and MaHog1in Metarhizium Acridum

Metarhizium is one of the most important genera of entomopathogenic fungi, it hasa great potential as biocontrol agents due to its environmental safety and low likelihoodof the development of insect resistance. Moreover, the wide occurrence and its broadspectrum of action make this fungus has been become a very important modelorganisms to study the infection process in arthropods. However, some shortcomingshave retarded their widespread application, such as sensitivity to various adversites andlow insecticidal speed. Better understanding of the mechanisms of fungal pathogenesiswould be helpful for improvement of the biocontrol fungus. Entomopathogenic funguspenetrate host needs protease, chitinase and lipase proteins hydrolysis enzymedegradation function, it also needs acidic trehalose enzymes to degradation of insecttrehalose making glucose for growth that required for fungi carbon sources in the bloodcavity of insects. Early studies showed that the expression of these hydrolysis enzymegene will be inhibited by glucose. The serine-threonine protein kinase Snf1is veryimportant in glucose repression pathways and plays a main rule in sporulation andvirulence in some pathogenic fungi. During infection, pathogenic fungi have toencounter various stresses from environment, such as heat shock and UV radiation, andfrom the host insect, such as high osmotic stress of the insect, oxidative stress duringinfection, and behavioral changes, such as behavioral fever in the desert locust.Mitogen-activated protein kinase Hog1-type MAPKs play a critical role in sensingchanges in the environment and regulating the expression of genes involved in variousdevelopment and differentiation processes as a reaction to diverse extracellular stimuli.In this study, the genes of MaSnf1and MaHog1have been identified from theentomopathogenic fungus Metarhizium acridum. The function of MaSnf1and MaHog1were characterized using gene knock-out and complementation strategy.The main results are as follows:1. Cloning and function analysis of MaSnf11.1Molecular characteristics of MaSnf1Comparison of the genomic and cDNA sequences showed that this gene, MaSnf1（JQ728546） revealed an ORF of2199bp interrupted by3introns of84,64, and117bpthat located at positions420,1340and2054of the coding region, respectively. The ORF encodes a polypeptide of732amino acids with a calculated molecular mass of82.5kDa and a pI of6.69. Sequence analysis revealed that the predicted MaSnf1contained all conserved characteristic subdomains as other the stress-activated proteinkinases, including the activation loop （A-loop） at positions216to244, a site for serineand threonine phosphorylation required for kinase activation. Based on thebioinformatic analysis of the sequence of MaSnf1, we conclude that M. acridum MaSnf1gene encodes a protein belonging to the member of the SNF1/AMPK family.1.2Targeted disruption of MaSnf1and complementationTo study the biological function of MaSnf1in M. acridum, a disruption vectorpK2-PB-MaSnf1L/R and a complementation vector pK2-Sur-MaSnf1were constructed.Agrobacterium-mediated transformation was carried out to gain MaSnf1disruptionmutants and complementary transformants and confirmed by PCR and Southernblotting.1.3MaSnf1disruption affects conidiation and conidial germinationFungal colony of each strain was photographed on PDA media. Images showedthat ΔMaSnf1had a significant reduction in growth compared to WT and CP. Theconidial yield of the ΔMaSnf1was⁵0%lower than that of WT and CP. Accordingly,the half germination time （GT50） of the ΔMaSnf1was delayed than that of the WT andCP.1.4The MaSnf1-deletion mutant has an increased sensitivity to UV-B and heatshockThe stress tolerance levels of conidial germination to UV-B and heat shock wereanalyzed to clarify the function of MaSnf1. Every strain was immediately exposed toirradiances and45℃heat shock for0h,2h,4h and6h, then incubated in darkness at28C for20h. The results revealed that ΔMaSnf1had a significant reduction comparedwith WT, while the CP was similar to that of the WT.1.5MaSnf1is involved in utilization of carbon sourcesTo investigate the role of MaSnf1in the utilization of carbon sources, variousstrains were cultured in liquid MM supplemented with different sole carbon sources,including glucose, sucrose, arabinose, fructose, xylose, galactose, trehalose and maltose.When glucose was used as the sole carbon source, no significant difference was foundin the growth among the various strains. However, in MM supplied with trehalose andxylose, the growth of the ΔMaSnf1was more significantly decreased than that of the WT, while the growth of the CP was similar to that of the WT.1.6MaSnf1is required for not only the penetration but also the postpenetrationdevelopment of M. acridumThe ΔMaSnf1exhibited a reduced level of pathogenicity, but did not lose itspathogenicity completely when tested using topical inoculation and intrahaemocoelinjection bioassays on locusts as the insect host. These results indicate that MaSnf1determines not only the penetration but also the postpenetration development of M.acridum.1.7Deletion of MaSnf1has a declined fungal growth in the hemolymph of the hostinsect in vivo and in vitroIn order to investigate the effects of MaSnf1on hyphal body differentiation andgrowth, the growth phenotype of various strains in insect hemolymph in vivo and invitro were characterized. We observed that numerous hyphal bodies in the hemolymphof locusts after topical inoculated with WT or CP on day6, but hyphal bodies werehardly found in locusts infected with ΔMaSnf1. Up to day8, ΔMaSnf1produced a largenumber of hyphal bodies in locust hemolymph. Similar phenomena were observed afterinjection. Fungal growth cultured in the haemolymph with or without hemocytes of thelocust in vitro was also observed. Microscopy inspection showed that the hyphal bodiesof the ΔMaSnf1were significantly lower than that of WT and CP.1.8MaSnf1disruption affects conidial germination and appressorium formation onlocust hind wingsSince MaSnf1affects the virulence, we measured the conidial germination andappressorium formation of WT, ΔMaSnf1and CP on locust hind wings. The resultsshowed that although germ tubes and appressoria of ΔMaSnf1were morphologicallysimilar to those of WT and CP, the frequency of conidial germination and appressoriumformation ability was severely impaired in the ΔMaSnf1.1.9MaSnf1affects the expression of some hydrolase genes in M. acridumThe expression of some hydrolase genes in M. acridum were quantitatived byqRT-PCR technology. We found that the expression of chitinase and protease inΔMaSnf1are lower than WT at the stage of the appressorium formation. In the bloodcavity of insects, the expression of acidic trehalose enzymes in ΔMaSnf1was decreasedsignificantly compared with WT. Thus, MaSnf1affects the expression of somehydrolase genes in M. acridum. 2. Cloning and function analysis of MaHog12.1Coning and sequence analysis of MaHog1According to the M. acridum whole genome information, we cloned one gene,named MaHog1. Analysis of the cDNA sequence （JQ691634） demonstrated thatMaHog1has an open reading frame of1077bp encoding a predicted protein of358amino acids with an estimated molecular mass of41.1kDa and pI of5.65. Eight intronsof76,65,67,53,56,68,61, and53bp were found in MaHog1genomic DNA, locatedat positions57,181,376,487,640,1053,1200, and1445of the coding region,respectively. On the basis of bioinformatic analysis of the MaHog1sequence, weconclude that MaHog1encodes a protein belonging to the member of theHog1/Sty1/p38MAPK family.2.2Targeted disruption of MaHog1and complementationTo study the biological function of MaHog1in M. acridum, a disruption vectorpK2-PB-MaHog1L/R and a complementation vector pK2-Sur-MaHog1wereconstructed. Agrobacterium-mediated transformation was carried out to gain MaHog1disruption mutants and complementary transformants and confirmed by PCR andSouthern blotting.2.3MaHog1is required for tolerance to high osmolarity, oxidative stress and hightemperature, and sensitivity to cell wall disturbing agentsTo study the role of MaHog1in multistress responses, spot assays were performedto investigate the stress sensitivity of various strains. On standard PDA medium, noobvious differences in growth rate were observed between the wild type andtransformants. Under hyperosmotic stress conditions （PDA containing0.5M NaCl or1.5M sorbitol）, the growth of ΔMaHog1was dramatically reduced compared to that of WTand CP. Furthermore, MaHog1disruption increased sensitivity to high temperature （35℃） and oxidative stress（6mM H₂O₂）. In addition, ΔMaHog1exhibited remarkableresistance to the cell wall disturbing agents CR and CFW.2.4MaHog1is required for not only the penetration but also the postpenetrationdevelopment of M. acridumTo investigate the effect of MaHog1disruption on virulence, both topicalinoculation and intrahaemocoel injection bioassays were conducted. The ΔMaHog1exhibited a reduced level of pathogenicity, but did not lose its pathogenicity completely.These results indicate that MaHog1determines not only the penetration but also the postpenetration development of M. acridum.2.5MaHog1affects fungal growth in the hemolymph of the host insect in vivo andin vitroThe effects of MaHog1on hyphal body differentiation and growth in insecthemolymph in vivo and hemolymph cultured in vitro were investigated. On day5afterinjection, numerous hyphal bodies were observed in the hemolymph of locusts injectedwith WT or CP, but hardly any hyphal bodies were found in locusts infected withΔMaHog1. Up to day8after injection, ΔMaHog1produced a large number of hyphalbodies in locust hemolymph. Similar phenomena were observed for topical inoculation:growth was much quicker for the WT and CP groups than for the ΔMaHog1. For culturein locust hemolymph in vitro, the number of hyphal bodies at24h after inoculation wassignificantly lower for ΔMaHog1compared to WT and CP.2.6MaHog1is required for conidial germination and appressorium formation onthe locust hind wing, and is not required for conidial surface hydrophobicity.Since MaHog1affects the virulence, we measured the conidial germination andappressorium formation of WT, ΔMaHog1and CP on locust hind wings. The resultsshowed that although germ tubes and appressoria of ΔMaHog1were morphologicallysimilar to those of WT and CP, the frequency of conidial germination and appressoriumformation ability was severely impaired in the ΔMaHog1. The conidial surfacehydrophobicity of the various strains was also examined. The results indicate thatMaHog1disruption did not affect cell surface hydrophobicity in M. acridum.