Cloning And Functional Analysis Of The Pathogenic Related Gene Mzbot In Metarhizium Anisopliae

Metarhizium anisopliae, a common filamentous fungi, has a wide geographical distribution; many strains of M. anisopliae have been developed for the control of insect pest species in agriculture and forestry. M. anisopliae has a number of advantages, such as no harm to human and livestock, host specificity, environmentally friendly pest control, no toxic residues, no pest resistance problem. Moreover, it is a key fungal model for elucidating the basis of entomopathogenicity, especially after whole genome sequencing. So far, the researches on the pathogenic mechanisms of entomopathogenic fungi has been comprehensively launched, and cloning, screening, functional identification of the pathogenic related genes of fungi is also very active. These are also because the effect of biological control of insects by M. anisopliae requires improvement to be accepted as an alternative control method. For instance, a relatively low virulence, short shelf life, environmental dependence severely restricted the application of M. anisopliae as a biological pesticide. The researches on virulence genes and stress resistant genes of fungus would provide gene resources for the improvement of fungi strains for pests biological control.We used suppression subtractive hybridization in M. anisopliae and construct a cDNA library from expressed genes. One EST from the library, WTEA78, was highly expressed when M. anisopliae was incubated on locust wings. Based on the WTEA78 sequence, we cloned the gene from the normalized full-length cDNA library. The 1251bp cDNA termed Mzbot (GenBank accession HQ011919 ) contained a full length gene. This gene was predicted to encode a protein similar to a putative zinc-binding oxidoreductase ToxD of Neosartorya fischeri (51% identity, accession number XP₀01258138). Then we analyzed the genetic control phenotypes through RNAi and overexpression of this gene in M. anisopliae.The main results obtained were as follows:1. Gene cloning, sequence analysis and expression pattern in wild type In this report, a putative zinc-binding oxidoreductase ToxD gene, Mzbot, was isolated from Metarhizium anisopliae. We predicted functions based on the putative amino acid sequence of the Mzbot gene using bioinformatics tools on line. analyzed the expression of this gene during the infection of M. anisopliae by RT-PCR. The 1251bp cDNA termed Mzbot (GenBank accession HQ011919 ) contained a full length geneas well as upstream and downstream non-translated regions (a 1089bp ORF, a 69 bp 5'UTR and 93 bp 3'UTR were identified using ORF Finder and BlastX on line). Four exons and three introns (283bp-381bp,661bp-715bp,800bp-866bp) were found in the genomic DNA. The deduced open reading frame (ORF) of Mzbot cDNA encoded a protein of 362 amino acids, of 39.3kDa mass and a predicted pI of 5.41. No signal peptide was found at N-terminal sequence of the putative protein when analyzed using SignalP.The Mzbot protein was a acidic protein. The formula of this putative protein was C1786H2772N456O530S7, which has 47 negatively charged residues (Asp + Glu) and 39 positively charged residues (Arg + Lys). The GRAVY of this putative protein was -0.135, indicating it's a hydrophilic protein. The Instability index was 19.67, indicating it's a stable protein. This gene was predicted to encode a protein similar to a putative zinc-binding oxidoreductase ToxD of Neosartorya fischeri (51% identity, E-Value =6e-85, accession number XP₀01258138). It also has homology with known or presumed zinc-binding oxidoreductase ToxD from other fungi. Bioinformatics analysis suggested that the protein has no lead peptide which was used directing to the mitochondria and also no nuclear localization signals (NLS), and it was localized in the cytoplasm using bioinformatics tools on line.No significant transmembrane structural regions in this protein was found. This is consistent with the subcellular localization using bioinformatics tools. In the putative protein, a N-glycosylation site was found at the aa residues 163 (NKTV); cAMP- and cGMP-dependent protein kinase phosphorylation sites were found at the residues 12, 223 (RKAT); Protein kinase C phosphorylation sites were found at the residues 195, 242, 251 (SSK); Casein kinase II phosphorylation sites were found at the residues 27(TLPD), 48(TTLD), 67(TVEE), 159 (SQDD), 217(TVGE), 269(SDVE), 298(TYYE); N-myristoylation sites were found at the residues 57(GTLVGC), 91(GGNDAR), 170(GGSTAT), 255(GTYVNL), 329(GGLIGV).Functional domain analysis indicating that this putative protein had two domains as a distant homology to GroES in N-terminal domain and NAD(P)-binding domains. All of these protein had these characteristics. Oxidoreductase activity is correlated with an N-terminal GroES-like catalytic domain and C-terminal NAD(P)-binding domains. These may be involved in antioxidant protection in the life processes in organisms. In the wild type, RT-PCR analysis demonstrated continuous and strong expression of Mzbot when M. anisopliae incubated on locust wings at 28℃after 15h, but little expression when M. anisopliae grew in locust haemocoe. These indicated that the primary role of this gene may be used to adapt to locust wings microenvironment of M. anisopliae, and may be associated with resistance to stress factors.2. Gene knockdown and overexpression, and expression analysis in mutants In order to study the function of the gene, we analyzed the genetic control pheno -types through RNAi and overexpression of this gene in M. anisopliae. A dual promoter RNAi vector containing the mzbot fragment as well as a overexpression vector contain -ing the mzbot full-longth gene was constructed. Electrotransformation was employed for M. anisopliae transformation. RT-PCR analyses were employed for the regulatory effects of Mzbot gene expression in the RNAi and overexpression mutants. Analysis of transcript accumulation showed lesser production in the RNAi mutant but more accumulation in the overexpression mutant when compared with that of the wild type. Strains with the greatest extents of change were selected for further biological analysis.3. Phenotypic variation after gene knockdown and overexpression in M. anisopliae The colony morphologies of the RNAi and overexpression mutants were analyzed on the 1/4SDAY medium. On the nonamended 1/4SDAY medium, the morphology of RNAi mutant also was different from that of the wild type. The RNAi mutant colonies showed a concave center cracked off from edge while the wild type colonies were umbonate. The morphology of overexpression mutant also was not different from that of the wild type. Plasticity in fungal morphology is common. We postulate that a problem in handling oxidative stress may be related to this change in growth morphology. ROS levels regulate development and intercellular communications in other systems including fungi.To observe the phenotype variation of the mutant strains, we assayed the growth of the wild type, RNAi and overpression mutant under different conditions. Compared with the wild type, no effects were detected when the mutant grew on higher osmoticum -1/4SDAY supplemented with 1M KCl. There were also no different effects detected when the conidia grew on 1/4SDAY under different pHs. Greater inhibition was observed with the RNAi mutant and more resistance of the overexpressing mutant was observed compared with the wild type when H2O2 was present in the medium. A role of the oxidoreductase expressed from mzbot in oxidative stress is consistent with the higher resistance of the overexpressing mutant and lower resilience of the RNAi mutant to exogenous H2O2 when plate growth or spore germination was measured. Therefore, this protein may be novel in its role in protection against oxidative stress. To investigate the role of Mzbot during the infection of M. anisopliae, an insect bioassay was conducted. The median lethal time for the RNAi mutant was significantly longer than that for the wild type (P<0.01). Therefore, the gene mzbot plays a role in virulence because underexpression in the RNAi mutant reduced killing in the insect host. However, overexpression of mzbot was not advantageous suggesting that other parameters in the host pathogen interaction are rate limiting.