Escherichia coli and affinity purified as fusion proteins with the epitope tag FLAG. Recombinant EPI1 specifically inhibited subtilisin A among major serine proteases, and inhibited and interacted with the pathogenesis-related P69B subtilisin-like serine protease of tomato. Interestingly, EPIC1 and EPIC2 were degraded by P69B but EPI1 protected both proteins from degradation. Co-immunoprecipitation experiments revealed that EPIC2 interacts with a novel extracellular cysteine protease of tomato. Overall, our results suggest that complex cascades of inhibition of host proteases occur in the plant apoplast during infection. Both Kazal-like and cystatin-like inhibitors are widespread in the oomycetes, but are absent in other microbial plant pathogens. Inhibition of host proteases by P. infestans protease inhibitors is proposed to be a novel mechanism of pathogen suppression of plant defenses.
Functional characterisation of a cellulose synthase from P. infestans through proteomics and RNA-interference.
Laura J. Grenville-Briggs,1 Catherine R. Bruce,1 Anna O. Avrova,2 Stephen C. Whisson,2 Alison Williams,1 Paul R. J. Birch2 and Pieter van West1.
1Aberdeen Oomycete Group, College of Life Sciences and Medicine, University of Aberdeen, Foresterhill, Aberdeen, AB24 2ZD, UK, 2Plant-Pathogen Interactions Programme, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK
A thorough understanding of the molecular events taking place during early interactions between P. infestans and host and non-host plants is crucial for developing new control measures. P. infestans produces a variety of cell types prior to penetration of the host plant and during the early stages of infection. It is possible to obtain mycelia, sporangia, zoospores, cysts, germinating cysts, appressoria, and infection vesicle-like cells in the absence of the host plant, making this system amenable to laboratory experiments. The appressorial stage of the interaction is the first point in which direct contact between the pathogen and the plant occurs via the formation of highly specialised infection structures, including the appressorium, penetration peg, and the infection vesicle. Moreover, it is during this phase that plant defence responses are initiated. We have isolated proteins that are abundant in appressoria using proteomics. Five proteins have been identified as enzymes involved in the biosynthesis of amino acids such as methionine, tryptophan, arginine, and branched chain amino acids. Two proteins involved in methionine and threonine synthesis have also been identified through a parallel SSH approach. Real-Time PCR has been used to validate these results and to investigate the expression of these genes, and potato homologues, throughout the infection cycle in potato. It would appear that P. infestans is able to rapidly respond to the nutritional status of the host plant, elevating the expression of these genes during the necrotrophic stage of infection and in response to a reduction in the expression of homologous host genes. A cellulose synthase has also been identified that is up-regulated in germinating cysts with appressoria. Gene silencing studies employing RNA interference pertubes the morphology and structure of appressoria. Here we discuss our latest results.
Relationship between secondary structure of elicitins, proteinaceous elicitors of Phytophthora sp. and defense reaction induced in tobacco cells.
V. Mikes, T. Kašparovský, J. Lochman, J. Damborsky, M. Ponchet, J.-P. Blein
Department of Biochemistry, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic
Elicitins are secreted specifically by the fungi Oomycete genera Pythium and Phytophthora. Biological function of elicitins is currently unknown. The response is induced by the interaction of elicitins with a putative receptor located on the cytoplasmic membrane. Transfer of the signal through the receptor triggers phosphorylation-dephosphorylation cascades in the tobacco resulting in alkalization of the extracellular medium, efflux of potassium and chloride ions, influx of calcium, production of the active species from oxygen, changes in composition of the cell wall and the induction of acquired systemic resistance. Binding of sterols to the cavity of cryptogein (elicitin from P. cryptogea) seems to be essential for consecutive association of the cyrptogein with a receptor and induction of a biological response in a plant. We prepared a series of mutants of cryptogein, one of the most potent elicitors of this group, with altered capacity for binding sterols (L19-R, I63-F, L15-W, L36-F, M35-F, M35-W, M59-W, M59-F). We compared the physicochemical parameters of sterol-cryptogein binding, their ability to induce the synthesis of active oxygen species, ion fluxes, their necrotic activity on tobacco suspension cells and the ability to induce the expression of pathogenesis related (PR) proteins in plants. The results showed that some of the early events are proportional to the affinity of cryptogein to bind sterols whereas the others (necrotic effect and the induction of PR protein synthesis) seemed to be dependent on the overall cryptogein structure.