Indirect activation of a plant nucleotide binding site–leucine-rich repeat protein by a bacterial protease

Abstract

Nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins mediate pathogen recognition in both mammals and plants. The molecular mechanisms by which pathogen molecules activate NBS-LRR proteins are poorly understood. Here we show that RPS5, a NBS-LRR protein from Arabidopsis, is activated by AvrPphB, a bacterial protease, via an indirect mechanism. When transiently expressed in Nicotiana benthamiana leaves, full-length RPS5 protein triggered programmed cell death, but only when coexpressed with AvrPphB and a second Arabidopsis protein, PBS1, which is a specific substrate of AvrPphB. Using coimmunoprecipitation analysis, we found that PBS1 is in a complex with the N-terminal coiled coil (CC) domain of RPS5 before exposure to AvrPphB. Deletion of the RPS5 LRR domain caused RPS5 to constitutively activate programmed cell death, even in the absence of AvrPphB and PBS1, and this activation depended on both the CC and NBS domains. The LRR and CC domains both coimmunoprecipitate with the NBS domain but not with each other. Thus, the LRR domain appears to function in part to inhibit RPS5 signaling, and cleavage of PBS1 by AvrPphB appears to release RPS5 from this inhibition. An amino acid substitution in the NBS site of RPS5 that is known to inhibit ATP binding in other NBS-LRR proteins blocked activation of RPS5, whereas a substitution thought to inhibit ATP hydrolysis constitutively activated RPS5. Combined, these data suggest that ATP versus ADP binding functions as a molecular switch that is flipped by cleavage of PBS1.B oth plants and animals employ nucleotide binding siteleucine-rich repeat (NBS-LRR) proteins to mediate detection of pathogen molecules (1). There appear to be at least two distinct mechanisms by which NBS-LRR proteins detect pathogens: either by binding pathogen-derived molecules directly or by sensing the modification of host proteins by pathogen-derived molecules (2). It is presently unclear how either mechanism causes activation of signaling by NBS-LRR proteins. We have been investigating these processes by using the plant NBS-LRR protein RPS5, which mediates detection of the protease AvrPphB from the bacterial pathogen Pseudomonas syringae (3-6).In plants, NBS-LRR proteins were first identified as the products of classically defined disease-resistance genes (R genes) (7-9), which are genes that confer resistance to infection by specific pathogen strains. R gene-mediated resistance is typically manifested by activation of a programmed cell death response referred to as the hypersensitive response (HR) that is localized to the site of pathogen ingress (10). In the last decade, R genes have been cloned from a large range of plant species, with the majority being found to encode NBS-LRR proteins (11). Plant NBS-LRR proteins can be subdivided into two broad categories defined by the presence of a Toll-interleukin receptor (TIR) domain or a non-TIR domain, most often a coiled-coil (CC) domain, at the amino terminus. The function of the CC and TIR domains in pathogen perception and signaling is un...

Structural Determinants at the Interface of the ARC2 and Leucine-Rich Repeat Domains Control the Activation of the Plant Immune Receptors Rx1 and Gpa2

“…From these data, a picture emerges in which the LRR domain is not only involved in pathogen recognition, but also plays a role in maintaining an autoinhibited resting state in the absence of pathogens via its interactions with the other domains (Bendahmane et al, 2002;Hwang and Williamson, 2003;Ade et al, 2007;Qi et al, 2012). A similar role as regulatory domain has been found for the sensor domains of other NLRs, such as the mammalian Apaf1 (Hu et al, 1998).…”

“…Previous studies have shown that the CC/TIR, NB-ARC, and LRR domains in plant immune receptors interact and cooperate with each other in an interdependent manner Leister et al, 2005;Ade et al, 2007;Rairdan et al, 2008). From these data, a picture emerges in which the LRR domain is not only involved in pathogen recognition, but also plays a role in maintaining an autoinhibited resting state in the absence of pathogens via its interactions with the other domains (Bendahmane et al, 2002;Hwang and Williamson, 2003;Ade et al, 2007;Qi et al, 2012).…”

“…This mechanism seems to be conserved between proteins from organisms as distant as bacteria, metazoans, and plants (Rairdan and Moffett, 2007;Danot et al, 2009;Takken and Tameling, 2009). The conformational change coincides with the exchange of bound ADP for ATP in the NB-ARC, probably stabilizing the active conformation (Tameling et al, 2006;Ade et al, 2007). Hydrolysis of the bound ATP is hypothesized to return the domains to their inactive state.…”

CAST AWAY, a Membrane-Associated Receptor-Like Kinase, Inhibits Organ Abscission in Arabidopsis

“…PBS1 is cleaved by an effector of the pathogen Pseudomonas syringae, activating the nucleotidebinding site (NB)-LRR protein, RPS5, which triggers programmed cell death (Warren et al, 1999;Swiderski and Innes, 2001;Shao et al, 2003;Ade et al, 2007). A recent study has revealed that, like PBS1, BIK1 and several PBS1-like (PBL) RLCKs are also substrates of the bacterial AvrPphB protease effector .…”

“…Although receptor-like cytoplasmic kinases (RLCKs) account for at least 125 of the 610 annotated RLKs in Arabidopsis (Arabidopsis thaliana), much remains to be learned about their functions within cell signaling complexes (Shiu and Bleecker, 2001;Goring and Walker, 2004;Shiu et al, 2004;Jurca et al, 2008). Several of the 46 RLCKs assigned to the class VII subfamily have been found to function in pathogen response and developmental signaling pathways (Swiderski and Innes, 2001;Shao et al, 2003;Murase et al, 2004;Muto et al, 2004;Veronese et al, 2006;Ade et al, 2007;Lu et al, 2010;Zhang et al, 2010;Liu et al, 2011).…”