Plant disease-resistance (R) proteins are thought to function as receptors for ligands produced directly or indirectly by pathogen avirulence (Avr) proteins. The biochemical functions of most Avr proteins are unknown, and the mechanisms by which they activate R proteins have not been determined. In Arabidopsis, resistance to Pseudomonas syringae strains expressing AvrPphB requires RPS5, a member of the class of R proteins that have a predicted nucleotide-binding site and leucine-rich repeats, and PBS1, a protein kinase. AvrPphB was found to proteolytically cleave PBS1, and this cleavage was required for RPS5-mediated resistance, which indicates that AvrPphB is detected indirectly via its enzymatic activity.

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...

SummaryWe have identified an Arabidopsis mutant that displays enhanced disease resistance (edr2) to the biotrophic powdery mildew pathogen Erysiphe cichoracearum. Inhibition of fungal growth on edr2 mutant leaves occurred at a late stage of the infection process and coincided with formation of necrotic lesions approximately 5 days after inoculation. Double-mutant analysis revealed that edr2-mediated resistance is suppressed by mutations that inhibit salicylic acid (SA)-induced defense signaling, including npr1, pad4 and sid2, demonstrating that edr2-mediated disease resistance is dependent on SA. However, edr2 showed normal responses to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000. EDR2 appears to be constitutively transcribed in all tissues and organs and encodes a novel protein, consisting of a putative pleckstrin homology (PH) domain and a steroidogenic acute regulatory protein-related lipid-transfer (START) domain, and contains an N-terminal mitochondrial targeting sequence. The PH and START domains are implicated in lipid binding, suggesting that EDR2 may provide a link between lipid signaling and activation of programmed cell death mediated by mitochondria.

SUMMARYIn plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins.