An Effector-Targeted Protease Contributes to Defense against Phytophthora infestans and Is under Diversifying Selection in Natural Hosts

Abstract

Since the leaf apoplast is a primary habitat for many plant pathogens, apoplastic proteins are potent, ancient targets for apoplastic effectors secreted by plant pathogens. So far, however, only a few apoplastic effector targets have been identified and characterized. Here, we discovered that the papain-like cysteine protease C14 is a new common target of EPIC1 and EPIC2B, two apoplastic, cystatin-like proteins secreted by the potato (Solanum tuberosum) late blight pathogen Phytophthora infestans. C14 is a secreted protease of tomato (Solanum lycopersicum) and potato typified by a carboxyl-terminal granulin domain. The EPIC-C14 interaction occurs at a wide pH range and is stronger than the previously described interactions of EPICs with tomato defense proteases PIP1 and RCR3. The selectivity of the EPICs is also different when compared with the AVR2 effector of the fungal tomato pathogen Cladosporium fulvum, which targets PIP1 and RCR3, and only at apoplastic pH. Importantly, silencing of C14 increased susceptibility to P. infestans, demonstrating that this protease plays a role in pathogen defense. Although C14 is under conservative selection in tomato, it is under diversifying selection in wild potato species (Solanum demissum, Solanum verrucosum, and Solanum stoliniferum) that are the natural hosts of P. infestans. These data reveal a novel effector target in the apoplast that contributes to immunity and is under diversifying selection, but only in the natural host of the pathogen.

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

“…Likewise, Arabidopsis Responsive to Desiccation21 (RD21) is a PLCP required for immunity against Botrytis cinerea Lampl et al, 2013), while its Nicotiana benthamiana ortholog C14 contributes to immunity against Phytophthora infestans (Kaschani et al, 2010;Bozkurt et al, 2011). Suppression of an aleurain-like PLCP delays floret senescence in broccoli (Brassica oleracea) and increases susceptibility to pathogens in N. benthamiana (Eason et al, 2005;Hao et al, 2006) Furthermore, Arabidopsis gVPE is required for toxin-induced PCD, while its N. benthamiana ortholog is required for virus-induced PCD (Hatsugai et al, 2004;Kuroyanagi et al, 2005).…”

“…This probe was subsequently used to monitor PLCP activities during immunity and senescence (Martínez et al, 2007a;Shabab et al, 2008), to study protease activation (Gilroy et al, 2007;Wang et al, 2008;Gu et al, 2012), and to reveal the selectivity of endogenous and pathogen-derived protease inhibitors (Rooney et al, 2005;Tian et al, 2007;Shabab et al, 2008;van Esse et al, 2008;Song et al, 2009;Kaschani et al, 2010;Lampl et al, 2010;Hörger et al, 2012;Lozano-Torres et al, 2012;van der Linde et al, 2012;Mueller et al, 2013;Dong et al, 2014). Although powerful, a disadvantage of DCG-04 profiling is that this biotinylated probe involves an indirect detection using streptavidin-horseradish peroxidase, which reduces throughput and resolution.…”

Dual disease resistance mediated by the immune receptor Cf-2 in tomato requires a common virulence target of a fungus and a nematode

“…The dual resistance specificity of Cf-2 results from perturbations in the apoplastic papain-like cysteine protease Rcr3 pim induced by two structurally different effectors. Recently it has been shown that the oomycete plant-pathogen Phytophthora infestans secretes cystatin-like effectors that also interact with Rcr3 pim (15,33,34). Although the perturbations of Rcr3 pim induced by Gr-VAP1 of G. rostochiensis and by Avr2 of C. fulvum may be structurally different, both trigger a Cf-2-mediated programmed cell death and disease resistance.…”

Broad-range Glycosidase Activity Profiling

“…ABPP has had a significant impact on plant science. After the introduction of probes for papain-like cysteine proteases (13,14), these probes revealed increased protease activities in the tomato and maize apoplasts during immune responses (15,16) and that these immune proteases are targeted by unrelated inhibitors secreted by fungi, oomycetes, and nematodes (17)(18)(19)(20)(21)(22)(23)(24). Likewise, probes for the proteasome displayed unexpected increased proteasome activity during immune responses (25) and revealed that the bacterial effector molecule syringolin A targets the nuclear proteasome (26).…”