Two Phytophthora parasitica cysteine protease genes, PpCys44 and PpCys45, trigger cell death in various Nicotiana spp. and act as virulence factors.

Proteases secreted by pathogens have been shown to be important virulence factors modifying plant immunity, and cysteine proteases have been demonstrated to participate in different pathosystems. However, the virulence functions of the cysteine proteases secreted by Phytophthora parasitica are poorly understood. Using a publicly available genome database, we identified 80 cysteine proteases in P. parasitica, 21 of which were shown to be secreted. Most of the secreted cysteine proteases are conserved among different P. parasitica strains and are induced during infection. The secreted cysteine protease proteins PpCys44/45 (proteases with identical protein sequences) and PpCys69 triggered cell death on the leaves of different Nicotiana spp. A truncated mutant of PpCys44/45 lacking a signal peptide failed to trigger cell death, suggesting that PpCys44/45 functions in the apoplastic space. Analysis of three catalytic site mutants showed that the enzyme activity of PpCys44/45 is required for its ability to trigger cell death. A virus-induced gene silencing assay showed that PpCys44/45 does not induce cell death on NPK1 (Nicotiana Protein Kinase 1)-silenced Nicotiana benthamiana plants, indicating that the cell death phenotype triggered by PpCys44/45 is dependent on NPK1. PpCys44- and PpCys45-deficient double mutants showed decreased virulence, suggesting that PpCys44 and PpCys45 positively promote pathogen virulence during infection. PpCys44 and PpCys45 are important virulence factors of P. parasitica and trigger NPK1-dependent cell death in various Nicotiana spp.