The Function Of Hydrophobin Genes In Beauveria Bassiana

Fungal-insect interactions have profound consequences in environmental microbiology, ecosystem balance, biodiversity, and pathogen evolution. In nature, entomopathogenic fungi are important regulatory factors in the pest populations, about 60% of total infected death pest was caused by entomopathogenic fungi. Compared to other microbial pathogens, such as bacteria and virus, entomopathogenic fungus can invade and infect host through cuticle. Therefore, the pathogenesis of entomopathogenic fungus is more complicated than other microbial pathogens. The entomopathogenic fungus Beauveria bassiana, can exist as a saprophyte and plant endophyte, but has evolved the ability to infect and parasitize a broad range of insects and other arthropods. The major infectious propagules are aerial conidia, although several other single celled forms can be produced including in vitro blastospores, submerged conidia, and in vivo insect hemolymph derived cells known as in vivo hyphal bodies, each of which display distinct surface properties. Interaction between the conidial surface and the insect epicuticle is the first step in pathogenesis. Two adhesions, MAD1 and MAD2, one implicated in insect and other in plant interactions have been isolated from the related entomopathogenic fungus, Metarhizium anisopliae, however similar proteins have not yet been reported for B. bassiana.Hydrophobins are small amphipathic proteins found that function in diverse physiological roles in filamentous fungi, including virulence. Although hydrophobins have been supposed as mediator of ashesion between entomopathogenic fungi and hosts, to date, no molecular determinants of fungal-insect host interactions have been reported. In this project, by using gene knock out technology combined with SEM, AFM and immune fluorescent, the function of hydrophobins in the development and pathology of entomopathogenic fungi Beauveria Bassiana was researched.The main results achieved in the following aspects:①Selection of a new marker for the entomopathogenic fungus Beauveria bassianaA genetic transformation system for B. bassiana based upon the use of a sulfonylurea resistance cassette derived from the Magnaporthe grisea, acetolactate synthase gene (sur) was developed. The transformation frequency ranged from 100-150 transformants /μg DNA. A construct bearing the sur gene and the enhanced green fluorescent protein gene egfp as a visual marker was used to successfully transform B. bassiana. The transformants containing pSUR-eGFP cassette exhibited both sulfonylurea resistance and fluorescence activity.②Optimization of transformation efficiencyThe efficiency of transformation was optimized by adjusting the pH of selective medium. When using pSUR-eGFP as target plasmid, the efficiency of transformation was improved from about 40 transformants /μg DNA before adjustment (pH 7.5) to over 150 transformants /μg DNA after adjustment (pH 6.5). The improved transformation method is obviously higher transformation effiency than other method in B. beauveria. Furthermore, the prepared competent cell can be kept for at least three months without affecting the transformation efficiency. The following procedure of transformation is very similar to yeast transformation which will take less than 1 h.③Cloning upstream and downstream fragment of hyd1 and hyd2A 4.3 kb (hyd1) and 2.9 kb (hyd2) DNA fragment which containe promoter and entire ORF of hyd1 and hyd2 were cloned by YADE (Y shape adaptor dependent extension) method. The sequences have been submitted to NCBI and the accession number is EF452344 and EF520285, respectively.④Function analysis of hydrophobinsa. The location and expression of hydrophobins Protein localization experiments revealed that Hyd1 localized to the surface of aerial conidia and submerged conidia, but was not detected in blastospores. In addition, germinated conidia retained a Hyd1 signal at the conidial base, which did not extend to the growing germ tube and hyphae. The Hyd2 signal appeared weaker than the Hyd1, but the protein could be readily detected on the surface of conidia and similar to Hyd1 was retained on the conidial base of germinated conidia, but could not be detected in the growing germ tube. However, in contrast to Hyd1, Hyd2 was not detected in either submerged conidia or blastospores. In addition, neither protein could be detected on the surfaces of insect derived in vivo hyphal bodies isolated from infected M. sexta.b. The model of hydrophobins in the form of conidia rodletsBased on the results from SEM and AFM, our hypothesis about the function of hydrophobin in the form of conidia surface is that one protein (Hyd1) forms the boundaries or underlying structure of the rodlet fascicle or bundle, potentially acting as the nucleating partner for the other protein (Hyd2) which forms the characteristic filaments or rodlets contained within these structures. c. The function of hydrophobins on the development of B. bassianaMeasurements of cell surface hydrophobicity using a microbial adhesion to hydrocarbons (MATH) assay indicated that both hyd1 and hyd2 contribute approximately equally to cell surface hydrophobicity. Loss of both proteins resulted in a cumulative decrease in cell surface hydrophobicity.Δhyd1 conidia are easily formed clumps or aggregates in liquid, andΔhyd2 conidia displayed greatly reduced adhesion to hydrophobic substrate.The loss of hyd1 and hyd2 did not affect the conidia germination on PDA and SDB medium and the stress resistance to KCl, calcofluor white, sorbitol, SDS and Congo Red.d. The loss of hydrophobins affected the distribution of carbohydrate epitopesLectin andβ-1, 3-glucan antibody mapping using flow cytometry revealed differential rearrangement of surface carbohydrate epitopes. Compared to wild-type parent, a 2-4 fold increased exposure of these epitopes was obsverd forΔhyd1 andΔhyd1Δhyd2, whereas only a small variation in the tested lectin andβ-1, 3-glucan antibody.e. Hydrophobins influenced the virulence of B. bassianaBioassay result showed that compared to wild-type, the hydrophobin mutants decreased virulence to G. mellonella when applied topical infection while not in hemocol injection application. It indicated the influence of hydrophobins in virulence occurred in the pre-penetration. The deduced reason is that the hydrophobin mutants decreased addension and germination on cuticle.