Innate immunity constitutes the first line of defense against attempted microbial invasion, and it is a well-described phenomenon in vertebrates and insects. Recent pioneering work has revealed striking similarities between the molecular organization of animal and plant systems for nonself recognition and anti-microbial defense. Like animals, plants have acquired the ability to recognize invariant pathogen-associated molecular patterns (PAMPs) that are characteristic of microbial organisms but which are not found in potential host plants. Such structures, also termed general elicitors of plant defense, are often indispensable for the microbial lifestyle and, upon receptor-mediated perception, inevitably betray the invader to the plant's surveillance system. Remarkable similarities have been uncovered in the molecular mode of PAMP perception in animals and plants, including the discovery of plant receptors resembling mammalian Toll-like receptors or cytoplasmic nucleotide-binding oligomerization domain leucine-rich repeat proteins. Moreover, molecular building blocks of PAMP-induced signaling cascades leading to the transcriptional activation of immune response genes are shared among the two kingdoms. In particular, nitric oxide as well as mitogen-activated protein kinase cascades have been implicated in triggering innate immune responses, part of which is the production of antimicrobial compounds. In addition to PAMP-mediated pathogen defense, disease resistance programs are often initiated upon plant-cultivar-specific recognition of microbial race-specific virulence factors, a recognition specificity that is not known from animals.

We show that oomycete-derived Nep1 (for necrosis and ethylene-inducing peptide1)-like proteins (NLPs) trigger a comprehensive immune response in Arabidopsis thaliana, comprising posttranslational activation of mitogen-activated protein kinase activity, deposition of callose, production of nitric oxide, reactive oxygen intermediates, ethylene, and the phytoalexin camalexin, as well as cell death. Transcript profiling experiments revealed that NLPs trigger extensive reprogramming of the Arabidopsis transcriptome closely resembling that evoked by bacteria-derived flagellin. NLP-induced cell death is an active, light-dependent process requiring HSP90 but not caspase activity, salicylic acid, jasmonic acid, ethylene, or functional SGT1a/SGT1b. Studies on animal, yeast, moss, and plant cells revealed that sensitivity to NLPs is not a general characteristic of phospholipid bilayer systems but appears to be restricted to dicot plants. NLP-induced cell death does not require an intact plant cell wall, and ectopic expression of NLP in dicot plants resulted in cell death only when the protein was delivered to the apoplast. Our findings strongly suggest that NLP-induced necrosis requires interaction with a target site that is unique to the extracytoplasmic side of dicot plant plasma membranes. We propose that NLPs play dual roles in plant pathogen interactions as toxin-like virulence factors and as triggers of plant innate immune responses.

SummaryActivation of non-cultivar-specific plant defense against attempted microbial infection is mediated through the recognition of pathogen-derived elicitors. Previously, we have identified a peptide fragment (Pep-13) within a 42-kDa cell wall transglutaminase from various Phytophthora species that triggers a multifacetted defense response in parsley cells. Many of these oomycete species have now been shown to possess another cell wall protein (24 kDa), that evoked the same pattern of responses in parsley as Pep-13. Unlike Pep-13, necrosis-inducing Phytophthora protein 1 (NPP1) purified from P. parasitica also induced hypersensitive cell death-like lesions in parsley. NPP1 structural homologs were found in oomycetes, fungi, and bacteria, but not in plants. Structure-activity relationship studies revealed the intact protein as well as two cysteine residues to be essential for elicitor activity. NPP1-mediated activation of pathogen defense in parsley does not employ the Pep-13 receptor. However, early induced cellular responses implicated in elicitor signal transmission (increased levels of cytoplasmic calcium, production of reactive oxygen species, MAP kinase activation) were stimulated by either elicitor, suggesting the existence of converging signaling pathways in parsley. Infiltration of NPP1 into leaves of Arabidopsis thaliana Col-0 plants resulted in transcript accumulation of pathogenesis-related (PR) genes, production of ROS and ethylene, callose apposition, and HR-like cell death. NPP1-mediated induction of the PR1 gene is salicylic acid-dependent, and, unlike the P. syringae pv. tomato DC3000(avrRpm1)-induced PR1 gene expression, requires both functional NDR1 and PAD4. In summary, Arabidopsis plants infiltrated with NPP1 constitute an experimental system that is amenable to forward genetic approaches aiming at the dissection of signaling pathways implicated in the activation of non-cultivar-specific plant defense.

Many plant pathogens secrete toxins that enhance microbial virulence by killing host cells. Usually, these toxins are produced by particular microbial taxa, such as bacteria or fungi. In contrast, many bacterial, fungal and oomycete species produce necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) that trigger leaf necrosis and immunity-associated responses in various plants. We have determined the crystal structure of an NLP from the phytopathogenic oomycete Pythium aphanidermatum to 1.35Å resolution. The protein fold exhibits structural similarities to cytolytic toxins produced by marine organisms (actinoporins). Computational modeling of the 3-dimensional structure of NLPs from another oomycete, Phytophthora parasitica, and from the phytopathogenic bacterium, Pectobacterium carotovorum, revealed a high extent of fold conservation. Expression of the 2 oomycete NLPs in an nlp-deficient P. carotovorum strain restored bacterial virulence, suggesting that NLPs of prokaryotic and eukaryotic origins are orthologous proteins. NLP mutant protein analyses revealed that identical structural properties were required to cause plasma membrane permeabilization and cytolysis in plant cells, as well as to restore bacterial virulence. In sum, NLPs are conserved virulence factors whose taxonomic distribution is exceptional for microbial phytotoxins, and that contribute to host infection by plasma membrane destruction and cytolysis. We further show that NLP-mediated phytotoxicity and plant defense gene expression share identical fold requirements, suggesting that toxin-mediated interference with host integrity triggers plant immunity-associated responses. Phytotoxin-induced cellular damage-associated activation of plant defenses is reminiscent of microbial toxin-induced inflammasome activation in vertebrates and may thus constitute another conserved element in animal and plant innate immunity.crystal structure ͉ immunity ͉ pathogen ͉ plant immunity ͉ phytotoxin