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Isolation and characterisation of two chitinase and one novel glucanase genes for engineering plant defence against fungal pathogens

Severgnini, Susana M.E. (2006) Isolation and characterisation of two chitinase and one novel glucanase genes for engineering plant defence against fungal pathogens. PhD thesis, Murdoch University.

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Hydrolytic enzymes such as chitinases and glucanases are implicated in plant defense responses against fungal pathogens. These enzymes are responsible for the breakdown of chitin and glucan, two major components of the fungal cell walls. Genes encoding these enzymes have been used to genetically engineer plants to enhance their protection against fungal pathogens.

Western Australia has over 4000 endemic plant species and a largely unknown fungal biota. Given that fungi possessing chitinases and glucanases with novel activities have been isolated in other parts of the world, we propose that fungi from Western Australian soils may possess novel biochemical/enzymatic activities.

The aims of this research project were to isolate chitinolytic and glucanolytic fungi from soil and to clone the genes encoding for chitinase and glucanase enzymes. To achieve these aims, fungi with activity against chitin and glucan were isolated, the activity quantified by colorimetric and inhibition assays and gene fragments with homology to known chitinase and glucanase genes were isolated and their sequences determined.

Soil fungi were isolated from five locations in and around the Perth Metropolitan area of Western Australia with the use of a medium containing Rose Bengal that eliminates all actinomycetes and most bacteria and reduces the growth of fast growing mold colonies.

Forty-one isolates were obtained by this method. Twenty four chitinolytic and glucanolytic fungal isolates were identified by growing them on chitin-containing media to select for those species that utilised chitin/glucan as a carbon source. These were assayed for production of exo- and endochitinolytic and glucanolytic enzymes.

Enzyme activity was compared between crude and dialysed supernatants. Exochitinase activity was determined in the supernatants of 4-day old fungal cultures by the release of p-nitrophenol from p-nitrophenyl-N-acetyl-beta-D glucosaminide. The supernatants were measured for endochitinase activity determined by the reduction of turbidity of suspensions of colloidal chitin. Glucanase activity was determined by release of reducing sugar (glucose) from laminarin. Supernatants from eleven of the twenty four isolates showed significant levels of enzyme activity.

Eleven isolates were assayed for activity against purified cell walls of phytopathogenic fungi. Activity was determined by measuring reducing sugars in the fungal supernatants against cell wall preparations of six economically important plant pathogens.

Chitinolytic activity was detected in seven isolates against cell wall preparations of Botrytis cinerea and Rhizoctonia solani, in four isolates against Fusarium solani and Sclerotinia sclerotium; in five isolates against Ascochyta faba and in six isolates against Leptosphaeria maculans. Similarly glucanolytic activity was detected in eight isolates against B. cinerea, in seven against R. solani, in two against F. solani, in three against S. sclerotium and A. faba and in one against L. maculans.

The supernatants derived from the isolates were used in a bioassay to determine growth inhibition against live B. cinerea spores by measuring turbidity reduction. Growth inhibition was measured against a control (B. cinerea, grown in medium with no added supernatant). Boiled supernatant did not inhibit the growth of B. cinerea spores but there was 100% inhibition by the crude supernatant from ten of the twenty four isolates. Similarly, supernatants were used to assess growth inhibition against live mycelia cultures of F. solani and S. sclerotium. Growth inhibition of F solani ranged from 9- 59%, boiled and crude supernatants respectively whilst growth inhibition of S. sclerotium ranged from 46-75%, boiled and crude supernatants respectively.

Two partial chitinase genes from the soil filamentous ungus Trichoderma asperellum,(ChiA and ChiB) and a novel glucanase gene from the filamentous fungus Aspergillus (Glu1) were cloned. ChiA, was 639 bp long, encoding 191 amino acids with identity to other chitinase genes. Two highly conserved regions, characteristic of glycosyl hydrolases from family 18, were present.

ChiB, was 887 bp long and encoded a 293 amino acid sequence that was closely related to an endochitinase gene from the filamentous fungus Trichoderma asperellum. The two highly conserved regions corresponding to the substrate binding and active sites that characterise the glycosyl hydrolases from family 18, also found in ChiA, were found in this gene.

Glu1 was 2844 bp long and encoded a 948 amino acid sequence that shared high identity with a beta-1, 3-glucanase from the filamentous fungus Aspergillus oryzae. The sequence contained conserved regions found in glycosyl hydrolases from family 17 that encode for substrate binding, N-terminal sequences and putative asparagine linked glycosylation sites.

The partial putative sequence ChiA is probably a pseudogene because it has two inframe stop codons. However, once the entire sequence of ChiB is known, both ChiB and the novel glucanase gene Glu1 could be useful contenders for engineering resistance in crop plants.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Supervisor(s): O'Brien, Philip
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