Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts

Abstract

Expressed sequence tag (EST) libraries for Metarhizium anisopliae, the causative agent of green muscardine disease, were developed from the broad host-range pathogen Metarhizium anisopliae sf. anisopliae and the specific grasshopper pathogen, M. anisopliae sf. acridum. Approximately 1700 59 end sequences from each subspecies were generated from cDNA libraries representing fungi grown under conditions that maximize secretion of cuticle-degrading enzymes. Both subspecies had ESTs for virtually all pathogenicity-related genes cloned to date from M. anisopliae, but many novel genes encoding potential virulence factors were also tagged. Enzymes with potential targets in the insect host included proteases, chitinases, phospholipases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites. A diverse array of proteases composed 36 % of all M. anisopliae sf. anisopliae ESTs. Eighty percent of the ESTs that could be clustered into functional groups had significant matches (E , 10 25 ) in other ascomycete fungi. These included genes reported to have specific roles in pathogens with plant or vertebrate hosts. Many of the remaining ESTs had their best BLAST match among animal, plant and bacterial sequences. These include genes with plant and microbial counterparts that produce potent antimicrobials. The abundance of transcripts discovered for different functional groups varied between the two subspecies of M. anisopliae in a manner consistent with ecological adaptations of the two pathogens. By hastening gene discovery this project has enhanced development of improved mycoinsecticides. In addition, the M. anisopliae ESTs represent a significant contribution to the extensive database of sequences from ascomycetes that are saprophytes or plant and vertebrate pathogens. Comparative analyses of these sequences is providing important information about the biology and evolutionary history of this clade. INTRODUCTIONAt least 90 genera and more than 700 species of fungi, dispersed in virtually every major taxonomic group except the higher basidiomycetes, have been identified as insect pathogens (Roberts & Humber, 1981). In terms of diversity they rival the plant pathogens, while there are comparatively few (about 40) fungal pathogens of warmblooded animals (Rippon, 1988). Insect-pathogenic fungi such as Metarhizium anisopliae have been extensively studied as key regulatory factors in insect populations and as agents of biocontrol (Inglis et al., 2001). However, attempts to discern the physiological determinants of infection processes and produce a rational plan for strain improvement have often been thwarted by the complexity of fungal responses to host-related signals. Consequently, side effects occurring in a selected or constructed strain are hard to predict and assess, and the full range of engineering possibilities cannot be exploited, due to lack of knowledge of inter-related regulatory and metabolic processes going on in the cell.We need alternative strategies to assess genomes of M. anisoplia...

Colony sectorization of Metarhizium anisopliae is a sign of ageing

“…Gene-specific primers were designed with an anticipated product size of approximately 200 bp to guarantee high amplification efficiency. The examined genes included those for subtilisins PR1A (M73795) and PR1B (U59484), the chymotrypsin CHY1 (AJ242735), the trypsin TRY1 (AJ242736) and the esterase STE1 (AJ251924), as they are involved in fungal virulence (Freimoser et al, 2003). The primers designed from the small subunit ribosomal gene (AF218207) of M. anisopliae were used as an internal control.…”

EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. I. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia

“…This secreted phospholipase (A2) is unusual in that it is calcium dependent and contains amino acid motifs characteristic of neutral lipases and serine esterases (Tjoelker et al, 1995). These enzymes play important roles in fungal virulence towards arthropods (El-Sayed et al, 1993;Freimoser et al, 2003, Charnley & St Leger, 1991Gupta et al, 1994) and our data suggest that conidia (the dispersive spores) are pre-loaded with some of the enzymes (or with transcripts at least) that are required for cuticle degradation and establishment of the fungal infection on target hosts. The aerial conidia library contained transcripts potentially involved in spore cell wall formation including an EST similar to the sporulation specific 1,3-b-glucan synthase from Schizosaccharomyces pombe (Martin et al, 2000) and a class V chitin synthase, as well as a septin homologue (Homann et al, 1996) possibly involved in cell surface organization.…”

“…Transcript analyses performed on distinct developmental stages (germinating teliospores, diploid filamentous growth and microsclerotia development) or during pathogenic growth of the plant pathogens Ustilago maydis and Verticillium dahliae have provided bases for genome annotation and further exploration of these fungi (Neumann & Dobinson, 2003;Nugent et al, 2004;Sacadura & Saville, 2003). EST analyses of two subspecies of the entomopathogenic fungus Metarhizium anisopliae revealed distinct patterns of expression of secreted proteases and pathogenicity factors that have led to the ability to examine gene expression patterns during infection of various insect hosts (Freimoser et al, 2003(Freimoser et al, , 2005. Our analyses revealed a robust and dynamic gene expression repertoire available to the entomopathogenic fungus B. bassiana.…”

Optimization of the Chitinase Production by Different <i>Metarhizium anisopliae</i> Strains under Solid-State Fermentation with Silkworm Chrysalis as Substrate Using CCRD

“…These differences in the enzymes secretion are important as virulence factor directly related with the potential of the fungus M. anisopliae to recognize the structure of the cuticle of their host and to secrete the correct enzymatic pool. According to Freimoser et al (2003) [30], M. anisopliae can encode different chitinase isoforms and other proteins as function of the material used as substrate.…”