Protease inhibitors as a possible new factor in agricultural plant protectionagainst microbial and fungal attack

Abstract: Extracellular serine proteases are produced by a wide range of microbial and fungalpathogens of agricultural plants. For example, they are involved in potato blight and wiltingcaused by Phytophthora infestans and Fusarium oxysporum, respectively. To improve potatoresistance to the pathogens, we prepared plants containing a gene derived from the wax mothGalleria mellonella and expressing the silk protease inhibitor 2 (SPI2). SPI2 is secreted by thesilk glands and inhibits microbial peptidases such as subtilisin and proteinase K. The syntheticSPI2 gene (modified to match the codon usage in potato) was fused with mGFP-5 to facilitatedetection of the transgene and its protein product. A construct of the fusion gene with plantregulatory elements (promoter 35S and terminator OCS) was prepared in a plasmid and insertedas a restriction fragment EcoRI – HindIII into the binary vector pRD400. The final construct wasintroduced into Agrobacterium tumefaciens (strain GV2260 with plasmid pGV2260). Leaf discsof potato cultivar Velox, which exhibits medium resistance to Phytophthora infestans, weretransformed with A. tumefaciens. Transgenic plants were regenerated in the presence ofkanamycin antibiotics and the content of SPI2 DNA was confirmed in the plantlets by PCR.Polyclonal antibodies were raised commercially against the natural SPI2 purified from the extractof G. mellonella silk using the RP HPLC. Western blotting was used to verify antibody reactionwith a protein of expected size in the extracts of transformed potato plants. The presence of thefusion protein in resistant plants was confirmed with additional analytical methods. IndirectELISA was employed for the quantification of SPI2. The results showed up to 3 fold increase ofprotease inhibition in transformed plants compared to the control background level. Potato plantsexpressing the SPI2 gene were tested for resistance to late blight either in vitro (isolates ofPhytophthora were collected from infected potato plants in the latter half of growing season) orin field trials (actual isolates of Phytophthora). The results revealed heterogeneity amongtransgenic lines, i.e. some lines were more resistant and others were more susceptible than thenon-transgenic plants.