The application of proteomics and recombinant antibody technology for crop improvement

Fungal phytopathogens represent a significant threat to crop production and a more thorough understanding of their ability to infect susceptible plants may offer novel information with applications in disease management. Although Sclerotinia sclerotiorum (Lib.) de Bary has been studied extensively, there are still aspects related to the ability of this phytopathogen to cause disease in susceptible plants that remain unclear. A proteome-level investigation of S. sclerotiorum mycelium and secretome was initiated and led to the identification of an arabinofuranosidase whose role in mediating virulence had not been studied previously in this fungus. The generation of a gene-disrupted mutant S. sclerotiorum that was unable to express the gene encoding the identified protein and subsequent inoculation of canola tissue revealed that at all time points tested, the degree of necrosis was significantly greater on the plant tissue inoculated with wild type mycelia. Furthermore, the effect of temperature and growth medium on gene expression was investigated in addition to a time course analysis of gene expression in pectin medium. We also used recombinant antibody technology to determine if a viable antibody-based diagnostic assay to detect S. sclerotiorum on plants could be developed and if transgenic plants expressing antibodies and antibody-antifungal protein fusions would be more tolerant to S. sclerotiorum infection. A monomeric and dimeric single chain, variable fragment (scFv) antibody with affinity for S. sclerotiorum was generated using phage display technology and the purified scFv was observed to bind S. sclerotiorum with some cross reactivity with Botrytis cinerea. The dimeric scFv displayed improved binding to the fungus and could detect the presence of mycelium in inoculated canola petals. To our knowledge, this is the first report of an scFv-dimer with affinity for S. sclerotiorum that has the potential for use in the development of a new diagnostic test. The constitutively expressed, cytosolic antifungal proteins and scFv did not appear to offer any significant tolerance to S. sclerotiorum infection based on leaf inoculations of Brassica napus canola, although additional work is required to conclusively determine the viability of the various constructs for conferring increased tolerance to fungal infection in transgenic plants, especially in agriculturally-important crops such as canola.