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.

Fig. 1.

Reconstitution of the RPS5- mediated HR in N. benthamiana. (A) Coexpression of RPS5, PBS1, and AvrPphB induces an HR-like response in N. benthamiana. Leaves were photographed 20 h after induction of transgene expression. The arrowhead indicates the HR. R, RPS5; P, PBS1; A, AvrPphB; C, AvrPphB (C98S); EV, empty pTA7002 vector control. (B) Assessment of the RPS5 domains required for the HR. The indicated RPS5 domains were coexpressed in the presence of PBS1 and AvrPphB or alone in N. benthamiana. Leaves were photographed 20 h after induction of transgene expression.

Fig. 2.

RPS5 and PBS1 coimmunoprecipitate. (A) Coimmunoprecipitation of RPS5 and PBS1 requires the RPS5 CC domain and occurs independent of AvrPphB. FL, full-length RPS5; EV, empty pTA7002 vector control. (B) Coimmunoprecipitation of RPS5 and PBS1 requires PBS1 kinase activity. (C) AvrPphB coimmunoprecipitates with RPS5 only in the presence of PBS1. The indicated combinations of constructs were transiently expressed in N. benthamiana. Proteins were immunoprecipitated with anti-cMyc, and immunoblots were performed with the antibodies indicated on the right. G252R and K115N indicate amino acid substitutions in PBS1 that eliminate kinase activity of PBS1 in vivo. C98S, the protease-inactive form of AvrPphB.

Fig. 3.

RPS5 domains show homo- and heterotypic interactions. The indicated combinations of constructs were transiently expressed in N. benthamiana. Proteins were immunoprecipitated with anti-HA antibodies, and immunoblots were performed with the indicated antibodies. The two lanes on the far right of each blot were run on a separate gel from the other lanes.

Fig. 4.

Mutations in the ATP binding site affect RPS5 signaling. (A) A K189N substitution in the P-loop blocks the RPS5-mediated HR. Wild-type RPS5 or the K189N mutant was coexpressed with PBS1 and AvrPphB in N. benthamiana leaves. (B) The K189N mutation also blocks the HR induced by the autoactive RPS5 CC–NBS construct. (C) A D266E substitution in the Walker B motif autoactivates RPS5. (B and C) The indicated constructs were expressed by themselves. All photos were taken 24 h after induction.

Fig. 5.

Model for RPS5 activation. Free RPS5 is unable to activate defense responses because of the negative regulatory action of the LRR domain. In the uninfected cell, most, if not all RPS5 is bound to PBS1 and ADP (D) and primed for a response to pathogen attack. PBS1 is cleaved through the cysteine protease action of AvrPphB after injection by P. syringae. PBS1 cleavage is detected by RPS5, resulting in a conformational change that enables exchange of ATP (T) for ADP. The ATP-bound form of RPS5 then engages downstream signaling molecules, activating the defense response.