Enhanced biocontrol options for the Australian sugar industry: a proteomic approach

The filamentous fungus Metarhizium anisopliae is a naturally occurring biological control agent of many insects including the greyback canegrub (Dermolepida albohirtum), a sugarcane pest in Australia. While there have been some gene-based approaches into identifying determinants for biological control and developing improved strains, this study provides a new comparative proteomics approach into identifying key proteins produced by M. anisopliae during infection of greyback canegrubs. Pathogenicity-related proteins have been identified by both liquid and solid culture approaches using proteomic technologies. Proteome maps of healthy canegrubs, canegrubs infected with Metarhizium and fungus only were produced and analysed. Comparative proteome analysis of proteins produced in solid culture provided a view into cellular reactions triggered in the canegrub in response to Metarhizium infection. Some of the proteins identified included cytoskeleton proteins, proteases, peptidases, metalloproteins and proteins involved in signal transduction. Liquid culture approach was used to display secreted proteins of Metarhizium growing on the whole greyback canegrubs and their isolated cuticles. Proteins identified included 64-kDa serine carboxypeptidase, 1,3 β-exoglucanase, dynamin GTPase, THZ kinase, calcineurin like phosphoesterase and phosphatidylinositol kinase. These proteins have not been previously identified from the culture supernatant of M. anisopliae during infection. To our knowledge, this is the first proteomic map established to study the extracellular proteins secreted by M. ansiopliae (FI-1045), a strain currently used for biological control of greyback canegrubs. Metarhizium anisopliae strain FI-1045 was further subjected to UV mutagenesis to select mutants that can tolerate better environmental conditions such as varying temperature and pH ranges. M. anisopliae mutant strain (NM10) was isolated and bioassays against greyback canegrubs proved that the mutant strain was more virulent than the parental strain. Two-dimensional electrophoresis was employed to display secreted proteins of the M. anisopliae mutant strain (NM10) growing on the whole greyback canegrubs and their isolated cuticles, in order to identify various proteins involved in infection of canegrubs. Eighty six secreted proteins were identified in this approach, amongst them six proteins that have not been previously identified from the culture supernatant of M. anisopliae during infection. These included the 56-kDa aspartyl aminopeptidase, 29-kDa secreted aspartyl protease, cyclin-dependent protein kinase, thymidylate kinase, septin and adenylate kinase. Finally, mutant strain NM10, generated by UV-mutagenesis was stably transformed and found to be highly resistant to benomyl, a commonly used fungicide in agriculture. Benomyl resistant transformants were found to tolerate 40 times higher concentration of benomyl than then amount that inhibits the parental strain. Laboratory bioassays proved that four transformants resistant to benomyl retained virulence characteristics against greyback canegrubs. The proteomic methods established, developed and applied in this thesis proved their strength and suitability in the visualisation, detection and identification of proteins produced by the fungus during infection of greyback canegrubs. Genetic manipulation techniques such as mutagenesis and transformation methods described in this thesis demonstrated successful steps in improving the Metarhizium strain while retaining pathogenicity against the greyback canegrubs. Combining the proteomics data obtained in this work with other "omics‟ data such as genomics, transcriptomics, metabolomics and bioinformatics, will lead to a more complete understanding of the biology of canegrub infection by Metarhizium at the molecular level.