5.3.3
CLONING OF ANTIFUNGAL ANTIBODY GENES

V MANATUNGA1, H SANATI2 and PA O'BRIEN1

1School of Biological Sciences and Biotechnology, and WA State Agricultural Biotechnology Centre, Murdoch University, Murdoch, WA 6150, Australia; 2National Research Centre for Genetic Engineering and Biotechnology, PO Box 14155-6343, Tehran, Iran

Background and objectives
Rhizoctonia solani is a common soil fungus which infects the roots of many plant species. Some isolates of the fungus cause a disease known as bare patch disease. This has become a major problem in cereals, legume and pasture crops in Australia. In necrotrophic fungi such as R. solani virulence is correlated with the production of pectin-degrading enzymes which lead to maceration of the host tissues. Resistance to necrotrophic pathogens can be increased by transforming plants with genes encoding inhibitors of pectic enzymes [1].

Genes for antibodies active against fungal enzymes can potentially be used to engineer resistance in the same way as pectinase inhibitors. Antibody genes can now be cloned and expressed in a variety of organisms including plants. To test this we are investigating the placement of genes for antipectinase antibodies into plants as a means of generating resistance to R. solani.

Materials and methods
Fungal enzyme activities were assayed using standard methods. Antibody genes were cloned from mouse spleen mRNA using a Pharmacia RPAS Kit as described by the manufacturer. Conditions for fungal growth and enzyme production were as described (Zamani, Biotechnology Programme, Murdoch University, unpublished).

Results and conclusions
Fungal extracellular proteins concentrated from the R. solani-inoculated culture medium were used as antigen to immunize six mice (BALB/c). Antiserum from all six mice reacted specifically with fungal antigen, whereas antiserum from non-immunized mice did not.

Amplification of first-strand spleen cDNA produced correct size bands of immunoglobulin variable heavy (VH) and variable light (VL) chain genes in two separate reactions. Those VH and VL genes were joined together with a linker molecule to make a single chain variable fragment (ScFv), and a major band of the appropriate size for an assembled ScFv gene (750 bp) was cloned into the pCANTAB 5E phagemid vector. Phage displaying antipectinase ScFv antibodies were isolated by biopanning against immobilized pectinases. 192 colonies were isolated by this procedure. All of these gave positive results in ELISA tests against fungal enzymes. These clones are being used for production of soluble antibodies in E. coli.

References
1. Powell ALT, D'Hallewin G, Ball BD et al., 1994. In 4th International Congress for Plant Molecular Biology, International Society for Plant Molecular Biology, Amsterdam.