Cloning Of Adenylate Cyclase Gene And Its Functional Analysis In Entomopathogenic Fungus, Metarhizium Acridum

Fungal biocontrol agents, which are widespread and environmentally safe, havegreat potential in integrated pest management. However, the application ofentomopathogenic fungi such as Metarhizium acridum in the field has been held backowing to their poor efficacy. During the infection process of entomopathogenic fungi,germ tubes are produced after the fungal conidia attach to the insect cuticle, and thendifferentiate into swollen infection structures called appressoria. The appressoriaproduce penetration pegs, which penetrate the host cuticle via a combination ofmechanical pressure and cuticle degrading enzymes, then pierce the surface of the hostinto the blood cavity. They produce a large number of hyphae through budding, therebyexhausting the nutrition of the insect host. The efficacy of entomopathogenic fungi inpest control is mainly affected by various adverse environmental factors, such as heatshock and UV-B radiation, and by responses of the host insect, such as oxidative stress,osmotic stress and fever. In this study, an adenylate cyclase gene (MaAC) was clonedfrom the locust-specific entomopathogenic fungus Metarhizium acridum, which ishomologous to various fungal adenylate cyclase genes. RNA silencing was adapted toanalyze the role of MaAC in virulence and tolerance to adverse factors fromenvironment and host insect. The main results are as follows:A6,492bp of cDNA encoding adenylate cyclase (MaAC) has been isolated andsequenced (GenBank accession JQ358775). Alignment with the DNA sequence showedthat MaAC gene contained a open reading frame (ORF) and was interrupted by twointrons located at the N terminus (940bp to1276bp) and the C terminus (6,204bp to6,264bp). The complete ORF of MaAC encoded a predicted protein of2164aminoacids (aa) with a molecular mass of541.2kDa. An analysis using SignalP suggested thatthe N-terminal sequence of MaAC had no signal peptide.We conducted an RNA interference strategy to study the function of MaAC.Phosphinothricin-resistant transformants of M. acridum were generated bytransformation with the vector pK2-Pb-MaAC-RNAi. To investigate the efficiency ofRNA interference, the wild type and RNAi mutants of MaAC were analyzed byquantitative RT-PCR. Compared to the wild type, the MaAC transcription in the RNAimutants was downregulated by56.5%,67.3%and77.0%, respectively. These resultsdemonstrated that the transcription of MaAC was efficiently downregulated. The phenotypes of the MaAC RNAi mutants in vitro were analyzed on PDA andCzapek-dox medium. A variety of morphological abnormalities was observed in theMaAC RNAi mutants. On PDA, the growth of the MaAC RNAi mutants were inhibited,mycelium formation was delayed, and the colonies of RNAi mutants were smaller incontrast to the wild type. On Czapek-dox medium, the conidiation of the MaAC RNAimutants were also delayed, and the colonies of RNAi mutants were lighter incomparison to the wild type. The AC-RNAi-3mutant had the most significantdifference in contrast to the wild type, and was used as MaAC RNAi mutant in thefollowing experiments.The vegetative growth in vitro was further quantified by assaying the living cells inPD liquid culture by CellTiter96AQueous One Solution Assay. In contrast to the wildtype, the RNAi mutant grew conspicuously slowly before48h (p <0.01). These resultsindicated that MaAC affects growth in vitro.As shown in this study, the fungal growth of the MaAC RNAi mutant of M.acridum was significantly slower in vitro than that of the wild type. In order to assesswhether the growth defect of the RNAi mutant was due to reduced levels of cAMP, wequantified and compared the steady-state levels of cAMP in PD liquid culture. ThecAMP level was significantly reduced in the AC-RNAi-3mutant compared to the wildtype and the cAMP concentration of the MaAC RNAi mutant (259.4fMol/mg) wasabout twofold less than that of the wild type (486.8fMol/mg) after being cultured for30h (p <0.01). This demonstrated that MaAC was involved in cAMP production duringvegetative growth of M. acridum.Differences in virulence and invasive growth inside insects were also comparedbetween the wild type and RNAi mutant. Five days post inoculation on the pronotum,locusts infected by the wild type began to die, while those infected by the RNAi mutantdied one day later. Wen the insects were inoculated by injection of conidia intoabdominal segments, the locusts began to die four days after injection of the wild type,and the insects treated with the conidia of RNAi mutant died one day later. Accordingly,the lethal time value for50%mortality (LT50) by topical inoculation and injection of theRNAi mutant was significantly higher than that of the wild type (p <0.05), indicatingthat MaAC is required for virulence in M. acridum. However, the ΔLT50values (thevirulence difference) between the AC-RNAi mutant and wild type in two experimentswere similar (p>0.05), suggesting that the effect of MaAC on virulence was mainlyafter the fungus entered the host. To further confirm the effect of MaAC on virulence, the fungal growth in vivo wasobserved by photomicroscope and quantified by real time PCR. M. acridum mutantgrew significantly quicker than the wild type, which was further confirmed by aquantitative assay. Here, the fungal DNA of the wild type was conspicuously higher (~4times) than that of the RNAi mutant.In order to further clear the reason that MaAC affect the virulence and growth invivo, the osmosensitivity and H2O2tolerance of conidia were analyzed. Firstly,1/4SDAY was choose as a based medium, on which these strains grew with no difference10d post inoculation. However, RNAi mutants were more sensitive to osmotic stress,the colonies of the RNAi mutants were sparse in contrast to the dense ones of the wildtype on1/4SDAY+KCl (1M). The effect of externally applied H2O2on the wild typeand RNAi mutants was also tested. The most striking differences between the responseof the wild type and RNAi mutants was observed in1/4SDAY containing6mM H2O2,where the colonies of the RNAi mutants were sparser than the wild type colonies. Theseresults indicated that MaAC is involved in the tolerances of M. acridum to oxidative andosmotic stress.Furthermore, the heat and UV tolerance of conidia was analyzed to clarify thefunction of MaAC. After wet-heat exposure at45oC, the germination rate of conidiadeclined with increasing exposure time and the conidia germination rates of thewild-type strain and mutant appeared to be significantly reduced for each succeeding0.5hour interval. However, the response to tolerance was obviously different for thewild-type strain and RNAi mutant. The conidia germination rate of the wild-type strainwas higher than that of the mutant, especially, there was significant difference at2,2.5h(p <0.01). Similar results were observed in UV-B tolerance test. Exposure to UV-B for1-3h, significant difference was found in germination rate of conidia between thewild-type and RNAi mutant (p <0.01). The result indicated that RNAi mutant was moresensitive to UV-B treatment than the wild type. These data showed that the MaACaffects the tolerance of M. acridum on heat and UV.In conclusion, MaAC affects virulence, primarily by fungal growth inside theinsect, and is required for tolerance to oxidative stress, osmotic stress, heat shock andUV-B radiation. MaAC affects the fungal virulence via vegetative growth and toleranceagainst oxidative stress, osmotic stress and locust fever.