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Cloning recombinant genes for antibodies inhibitory to fungal pectinases

Manatunga, Vidyani (2001) Cloning recombinant genes for antibodies inhibitory to fungal pectinases. PhD thesis, Murdoch University.

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Plant diseases caused by necrotrophic fungal pathogens cause considerable damage to crop cultivars worldwide. Traditional approaches to control fungal diseases are confined to breeding plant resistance or the application of fungicides. The use of fungicides represents a major cost to crop production and poses a number of environmental concerns. For many necrotrophic fungi there are no known sources of natural resistance that can be used in plant breeding.

Necrotrophic fungi produce an array of enzymes, which degrade the plant cell wall. Cell wall-degrading enzymes, particularly pectic enzymes, are a major cause of pathogenesis by necrotrophic fungal pathogens. Engineering plants for resistance to necrotrophic fungi is therefore a viable option for control of fungal diseases and may be achieved by transforming plants with genes that can inhibit the fungal pectic enzymes. Antibodies are known to possess enzyme inhibitory properties. Genes for such antibodies could be cloned and might be a good source of genes for engineering resistance. The aim of this study was to develop a novel approach to engineer resistance to fungal pathogens by cloning recombinant genes for antibodies against fungal pectinase enzymes.

Investigations were performed using Rhizoctonia solani as a model necrotrophic fungal pathogen. Anti-fungal antibody production in mice was induced by inoculation of mice with R. solani extracellular enzyme preparation. Antibody production was observed using an enzyme linked immunosorbent assay (ELISA) and western blot assay. Immunoglobulin G (IgG) was purified from pooled mice sera by affinity column chromatography and used in enzyme inhibition assays. Purified IgG from the antigen immunised mice inhibited the activity of polygalacturonase (PG), cellulase, and chitin synthase from R. solani. No inhibition of PG and cellulase was observed with control IgG from the control-antigen (culture media protein) immunised mice. Mice with high affinity antisera were identified by ELISA and used for cloning the antibody genes.

Reverse-transcriptase polymerase chain reaction (RT-PCR) was used to amplify the VL and VH genes from the spleen messenger RNA (mRNA) of mice. A PCR assembly process was used to link these amplified VL and VH genes randomly together for expression as single chain variable fragment (ScFv) of antibody genes. The assembled genes were cloned into the pCANTAB 5E vector and transformed into E. coli TGI. A library of recombinant phage displaying the cloned antibody bound to the M13 gill protein was produced and panned against the fungal antigen. One hundred and ninety two antigen binding clones were isolated by affinity chromatography against R. solani antigen. Recombinant phage were transformed into E. coli HB2151 for soluble ScFv antibody production. The identified (positive) clones were screened for soluble antibody secretion. Antibody accumulation was examined in the culture supernatant, periplasmic extract, and whole cell extract. Most of the antibodies were found in the supernatant. Optimum conditions for antibody production involved a 48h incubation period at 16°C. Two clones that showed stable antibody production were selected for testing the effect of soluble antibody on enzyme activity. Two clones were selected for the stable antibody production during repeat culturing. ScFv gene structures of those two clones (B1 and B4) were determined by DNA sequencing.

The effect of these B1 and B4 soluble antibodies on various enzymes was investigated: pectic enzymes of exo-polygalacturonase (exo-PG); endo-polygalacturonase (endo-PG); pectin lyase (PNL); cell maceration enzymes of Rhizoctonia solani, Fusariwn solani, Botrytis cinerea, Sclerotinia sclerotiorum, Ascochyta fabei, Colletotrichum gloeosporoides, Leptosphaeria maculans, and Phytophthora cinnamomi; and a commercially available pectinase from Aspergillus niger. With the exception of A. fabei, and C. gloeosporides, all the fungal exo-PG enzymes were inhibited by both B1 and B4. The antibodies (both B1 and B4) inhibited the endo-PG enzyme activity of F. solani, B. cinerea, S. sclerotiorum, and A. fabei, but not C. gloeosporides or the commercial enzyme from A. niger. B1 and B4 also inhibited the cell maceration enzymes of F. solani, B. cinerea, S. sclerotiorum, A. fabei, and C. gloeosporides. Only the PNL enzyme activity of B. cinerea, and S. sclerotiorum was inhibited. However, the inhibitory effect of these antibodies was different for each enzyme. When the antibodies bound strongly to the enzyme there was strong inhibition of the enzyme, and when there was weak binding partial or uninhibition was observed. Western blot analysis of the fungal enzymes with B1 and B4 antibodies confirmed these results.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Biological Sciences and Biotechnology
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): O'Brien, Philip
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