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

Navrátil O., Kodrík D., Kludkiewicz B., Vinokurov K.S., Sehnal F., Horáčková V.

Keywords: potato, transgenosis, microbial and fungal pathogens, silk protease inhibitor, genetically modified organisms, ELISA, Western blotting
Abstract: Extracellular serine proteases are produced by a wide range of microbial and fungal pathogens of agricultural plants. For example, they are involved in potato blight and wilting caused by Phytophthora infestans and Fusarium oxysporum, respectively. To improve potato resistance to the pathogens, we prepared plants containing a gene derived from the wax moth Galleria mellonella and expressing the silk protease inhibitor 2 (SPI2). SPI2 is secreted by the silk glands and inhibits microbial peptidases such as subtilisin and proteinase K. The synthetic SPI2 gene (modified to match the codon usage in potato) was fused with mGFP-5 to facilitate detection of the transgene and its protein product. A construct of the fusion gene with plant regulatory elements (promoter 35S and terminator OCS) was prepared in a plasmid and inserted as a restriction fragment EcoRI – HindIII into the binary vector pRD400. The final construct was introduced into Agrobacterium tumefaciens (strain GV2260 with plasmid pGV2260). Leaf discs of potato cultivar Velox, which exhibits medium resistance to Phytophthora infestans, were transformed with A. tumefaciens. Transgenic plants were regenerated in the presence of kanamycin 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 extract of G. mellonella silk using the RP HPLC. Western blotting was used to verify antibody reaction with a protein of expected size in the extracts of transformed potato plants. The presence of the fusion protein in resistant plants was confirmed with additional analytical methods. Indirect ELISA was employed for the quantification of SPI2. The results showed up to 3 fold increase of protease inhibition in transformed plants compared to the control background level. Potato plants expressing the SPI2 gene were tested for resistance to late blight either in vitro (isolates of Phytophthora were collected from infected potato plants in the latter half of growing season) or in field trials (actual isolates of Phytophthora). The results revealed heterogeneity among transgenic lines, i.e. some lines were more resistant and others were more susceptible than the non-transgenic plants.
Fulltext: contact IEB authors
IEB authors: Oldřich Navrátil