Functional Analysis Of Genes MaFKS And MaChsⅦ Related To Cell Wall Synthesis In Metarhizium Acridum

Metarhizium, one of the most important genera of entomopathogenic fungi, is an important regulatory factor in pest insect populations in nature and has been considered as a promising alternative or supplement to chemical pesticides. It can invade host directly and low likelihood of the development of insect resistance becaused of the process of Metarhizium acridum infected host is complex, thus in the increasing attention on the importance of biological prevention and control has broad application. However, some disadvantages have retarded widespread application, including short-term storage, sensitivity to environmental conditions and the resources required for mass production of conidia. The dynamic cell wall of fungi is an intermediary between the fungus and the environment and can change, depending on the life cycle stage and environmental conditions. But the study of the entomopathogenic fungal wall is still not clear. In this study, we use the Metarhizium acridum as the material whose whole genomic sequence has known. The gene encodingβ-1,3-glucan synthase and a class VII chitin synthase gene in the entomopathogenic fungus Metarhizium acridum were cloned. To investigate their function, the RNAi and gene knock-out strategies have been used.The main results are as follows:1 cloning and function analysis ofβ-1,3-glucan synthase gene MaFKS1.1 Cloning of MaFKSAccording the genomic sequence, we cloned aβ-1,3-glucan synthase gene named MaFKS (GenBank accession number AY187630). Sequence analysis demonstrated that MaFKS contains a 5820 bp open reading frame (ORF) and two introns located at the N terminus (177–238 bp) and the C terminus (5539–5600 bp). The complete ORF of MaFKS encodes a predicted protein of 1938 amino acids (aa) with an molecular mass of 221.7 kDa and a pI of 7.94. Analysis of the transmembrane domains showed that the deduced MaFKS protein has 16 transmembrane helices. And the putative MaFKS protein is most closely related to the putative FKS protein from two other entomopathogenic fungi, Beauveria bassiana and Cordyceps militaris.1.2 To study the function of MaFKS, an RNAi strategy was applied.To study the function of MaFKS, an RNAi vector was constructed (pPK2-pB-MaFKS-RNAi). Agrobacterium-mediated transformation was carried out, and obtained five RNAi mutants. Transcripts of MaFKS were detected by real-time RT-PCR. In comparison with the WT, MaFKS transcription was inhibited in RNAi transformants, with down-regulation ranging from 40% to 64.2%.1.3 MaFKS is involved in cell wall integrityTo determine the cell wall integrity of FKS-RNAi transformants, the sensitivity of two transformants and the WT to agents that disturb the cell wall or cell membrane was investigated. In comparison with WT, FKS-RNAi transformants had much less aerial mycelium on PDA plates with or without CR, CFW or SDS after 4 days of incubation. To further clarify the effect of agents that disturb the cell wall or cell membrane, we observed mycelial growth in 1/4 SDAY liquid medium supplemented with CR, CFW or SDS. Closer inspection revealed some regions of the mycelium with frequent aberrant conformations, such as blowing out of cell wall elements at hyphal tips and internal compartments in the presence of CR. All the results demonstrate that depleted MaFKS expression affected mycelium growth and increased sensitivity to the agents used, which indicates that MaFKS plays an important role in cell wall integrity in M. acridum.1.4 Depleted MaFKS expression increases the sensitivity to hyperosmotic pressure in M. acridumSubstrate hyphae of the transformants on PDA plates containing KCl were much fewer than for the WT. Moreover, the colony surface for transformants was smooth and tawny, while that for the WT was typically wrinkled and green. On PDA plates containing sorbitol or mannitol, the transformants had sparse aerial hyphae compared with WT.1.5 MaFKS is involved in conidiation in M. acridumConidial yield was measured on PDA and PDA plates containing CR, CFW, SDS, KCl, sorbitol or mannitol after 12 days of incubation. The conidial yield/mm2 was significantly higher for WT than for FKS-RNAi transformants on PDA plates containing CR, CFW, SDS, KCl, sorbitol or mannitol.1.6 FKS-RNAi transformant are defective inβ-1,3-glucan synthesisTheβ-1,3-glucan content of hyphal wall of the transformant was only 51.32% of the WT content.To further investigate changes inβ-1,3-glucan distribution, mycelia were stained with theβ-1,3-glucan-specific fluorochrome aniline blue. Fluorescence microscopy revealed that the septal regions were greatly decreased in transformant compared to WT hyphae.2 cloning and function analysis of class VII chitin synthase gene MaChsVII 2.1 According the genomic sequence, full-length MaChsVII gene was cloned and sequenced. The putative coding sequence which contained a 5427 bp ORF, and encodes a protein of 1784 aa with an molecular mass of 199.2 kDa and a pI of 5.57. The predicted MaChsVII protein has 6 transmembrane helices and a MMD which is short than the class V chitin synthase and without P-loop, switch I and switchⅡ.2.2 To study the function of MaChsVII, a gene knock-out strategy was applied.To study the function of MaChsVII, an gene knock-out vector was constructed (pPK2-pB-MaChsⅦL/R). Agrobacterium-mediated transformation was carried out, and obtained five mutants.2.3 MaChsVII is involved in cell wall integrityWe observed the colonies of the mutants on the PDA with agents that disturb the cell wall or cell membrane. The result demonstrated that the colonies of mutants were smaller than that of WT, and the mutants had much less aerial mycelium.2.4 MaChsVII is involved in tolerance to hyperosmotic pressureColonies of the mutants on the PDA with the KCl and mannitol plates were smaller than that of WT, and the growth of△M aChsⅦmutants on the on the PDA with the KCl plates was hysteretic. The result reveals that MaChsⅦinfluences the hyperosmotic pressure tolerance of M. acridum.2.5 MaChsVII disruption reduced the virulence of the fungiBioassay result showed that the LD50 of MaChsVII-disruption strains to locusts was nearly 1.2 day later than that of the wild-type after inoculation. The result indicated that MaChsVII disruption could impair the infection efficiency of M. acridum in locusts.