Functional characterization of extracellular protease inhibitors of Phytophthora spp and their targets tomato proteases

The interplay between proteases and protease inhibitors during plant-pathogen interaction represents a common strategy for defense and counterdefense. The plant pathogens Phytophthora infestans and Phytophthora mirabilis secrete effectors such as protease inhibitors that facilitate host colonization through a defense-counterdefense mechanism. The P. infestans serine protease inhibitors EPI1 and EPI10 physically bind and inhibit the tomato serine protease P69B. On the other hand, the P. infestans cysteine protease inhibitor EPIC2B targets PIP1, a papain-like protease that has close similarity to another tomato cysteine protease Rcr3, which is required for the fungal resistance and Avr2 hypersensitivity in Cf-2 tomato. The objective of this research is to characterize these protease inhibitors and their association with specific targets in the host. We studied the structure and activities of these protease inhibitors and their target proteases using recombinant proteins expressed in Escherichia coli and Nicotiana benthamiana. PIP1-His was pulled down using coimmunoprecipitation with anti-FLAG resin from N. benthamiana apoplast by recombinant protein FLAG-EPIC2B, suggesting physical interaction of EPIC2B and PIP1. Similarly, tomato protease Rcr3pim was shown to be a common target for both the Cladosporium fulvum effector Avr2 and P. infestans effector EPIC2B using pull down assays and DCG-04 activity profiling. However, unlike Avr2, EPIC2B is a reversible inhibitor of Rcr3pim and does not trigger hypersensitivity on Cf-2/Rcr3pim tomato. We also found that the rcr3-3 mutant of tomato that carries a premature stop codon in the Rcr3 gene exhibits enhanced susceptibility to P. infestans, suggesting a role for Rcr3 pim in basal defense. These findings are consistent with the predictions of the guard model and suggest that effectors from unrelated pathogens can target the same tomato defense protease Rcr3pim. It appears that relative to C. fulvum, P. infestans evolved a cunning effector that carries virulence activity without triggering plant innate immunity. Like EPIC1, PmEPIC1, an EPIC1 homolog from P. mirabilis, was also found to bind Rcr3pim but not PIP1. Unlike EPIC2B, neither EPIC1 nor PmEPIC1 binds or inhibits PIP1. This work was possible by the ability to express and purify proteins in the apoplast of N. benthamiana . Three tomato proteases with C-terminal 6XHistidine tag were successfully expressed in N. benthamiana apoplast. Our findings suggest that C-terminal His-tagging of proteins in N. benthamiana apoplast is efficient enough to enable purification of functional proteins. Further studies will focus on the three dimensional structural of the protease-protease inhibitor complexes, identifying the interactors of EPIC1 and PmEPIC1 in their respective host cells, and further characterizing the biochemical activities of these protease inhibitors.