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

Abstract

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.

Transcriptional Variability Associated With CRISPR-Mediated Gene Replacements at the Phytophthora sojae Avr1b-1 Locus

“…The “all-in-one” CRISPR/Cas9 system ( Fang et al, 2017 ) has been successfully applied in several different Phytophthora species except for P. infestans ( van den Hoogen and Govers, 2018 ; Chen et al, 2019 ; Wang W. et al, 2019 ; Ochola et al, 2020 ; Zhang et al, 2020 ). In most cases, the efficiency of homozygous replacement events was low (<10%).…”

“…As a result, knock-out and complementation of target genes have become basic requirements for functional genomics research in P. sojae . The tool has subsequently been deployed in other Phytophthora species including P. capsici ( Chen et al, 2019 ; Wang W. et al, 2019 ), P. palmivora ( Gumtow et al, 2018 ), P. parasitica ( Zhang et al, 2020 ), and P. litchii ( Situ et al, 2020 ), as well as in the animal pathogenic oomycete Aphanomyces invadans ( Majeed et al, 2018 ).…”

Optimizing the PBS1 Decoy System to Confer Resistance to Potyvirus Infection in Arabidopsis and Soybean

“…The PBS1/RPS5 decoy system should be capable of conveying immunity to any biotrophic pathogen that requires a protease as part of its infection or replication machinery, providing that the protease accumulates inside the host cell and recognizes a cleavage sequence of seven or fewer amino acids. This has the potential to be broadly applicable, as bacterial, viral, and fungal pathogens as well as oomycetes and nematodes, are known to secrete proteases as part of their infection cycle (Alfano and Collmer 2004;Gardner et al 2018;Lim et al 2011;Orbach et al 2000;Raffaele et al 2010;Zhang et al 2020). We expect that at least of some of these are translocated into host cells.…”

Phytophthora capsici CBM1‐containing protein CBP3 is an apoplastic effector with plant immunity‐inducing activity

“…Agrobacterium-mediated transient expression and TRV-based gene silencing were performed as described previously (Nie et al, 2019;Zhang et al, 2020). The levels of BAK1 and SOBIR1 in TRV-treated N. benthamiana plants were determined by RT-qPCR using NbAct as the reference gene.…”