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Trichoderma harzianum and T. atroviride are filamentous fungi commonly found in soil. Both display biocontrol capabilities against a range of phytopathogenic fungi including Rhizoctonia solani and Botrytis cinerea which are known pests of hundreds of commercially important crops including tomatoes, potatoes, beans, cucumber, strawberries, cotton and grapes. These Trichoderma species secrete a combination of enzymes degrading cell walls and antibiotics to overgrow and kill fungal phytopathogens. They are seen as an environmentally friendly alternative to chemical fungicides currengly used on crops. A proteomic approach was taken to separate and identify proteins from a strain of T. harzianum with well established biocontrol properties. Several methods were developed in this thesis to display the whole proteome content and several subcellular proteome fractions from T. harzianum. Proteins were separated by two-dimensional electrophoresis and identified by mass spectrometric methods. The resulting proteomic maps represent the first extensive array of cellular and sub-cellular proteomes for T. harzianum. Cellular protein patterns of T. atroviride (T. harzianum P1) grown on media containing either glucose or R. solani cell walls were compared by differential gel electrophoresis to identify a suite of new proteins involved in the biological control response. Twenty four T. atroviride protein spots up-regulated in the presence of the R. solani cell walls were identified by mass spectrometry and N-terminal sequencing. Proteins identified from this study included previously implicated enzymes degrading cell walls and three novel proteases, vacuolar serine protease, vacuolar protease A and trypsin-like protease. The genes encoding two of these proteases, vacuolar protease A and vacuolar serine protease have been cloned by degenerate primer PCR and genomic walking PCR and sequenced. The gene sequences and protein sequences derived from these genes have been partially characterised. |
