Thorsten Nürnberger

Innate immunity, an ancient form of defense against microbial infection, is well described for animals and is also suggested to be important for plants. Discrimination from self is achieved through receptors that recognize pathogen‐associated molecular patterns (PAMPs) not found in the host. PAMPs are evolutionarily conserved structures which are functionally important and, thus, not subject to frequent mutation. Here we report that the previously described peptide elicitor of defense responses in parsley, Pep‐13, constitutes a surface‐exposed fragment within a novel calcium‐dependent cell wall transglutaminase (TGase) from Phytophthora sojae . TGase transcripts and TGase activity are detectable in all Phytophthora species analyzed, among which are some of the most destructive plant pathogens. Mutational analysis within Pep‐13 identified the same amino acids indispensable for both TGase and defense‐eliciting activity. Pep‐13, conserved among Phytophthora TGases, activates defense in parsley and potato, suggesting its function as a genus‐specific recognition determinant for the activation of plant defense in host and non‐host plants. In summary, plants may recognize PAMPs with characteristics resembling those known to trigger innate immune responses in animals.

Summary: Activation 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.

Harpin from the bean halo-blight pathogen Pseudomonas syringae pv phaseolicola (harpin_Psph) elicits the hypersensitive response and the accumulation of pathogenesis-related gene transcripts in the nonhost plant tobacco. Here, we report the characterization of a nonproteinaceous binding site for harpin_Psph in tobacco plasma membranes, which is assumed to mediate the activation of plant defense responses in a receptor-like manner. Binding of ¹²⁵I-harpin_Psph to tobacco microsomal membranes (dissociation constant = 425 nM) and protoplasts (dissociation constant = 380 nM) was specific, reversible, and saturable. A close correlation was found between the abilities of harpin_Psph fragments to elicit the transcript accumulation of the pathogenesis-related tobacco gene HIN1 and to compete for binding of ¹²⁵I-harpin_Psph to its binding site. Another elicitor of the hypersensitive response and HIN1 induction in tobacco, the Phytophthora megasperma–derived β-elicitin β-megaspermin, failed to bind to the putative harpin_Psph receptor. In contrast to activation by β-megaspermin, harpin_Psph-induced activation of the 48-kD salicylic acid–responsive mitogen-activated protein kinase (MAPK) and HIN1 transcript accumulation were independent of extracellular calcium. Moreover, use of the MAPK kinase inhibitor U0126 revealed that MAPK activity was essential for pathogenesis-related gene expression in harpin_Psph-treated tobacco cells. Thus, a receptor-mediated MAPK-dependent signaling pathway may mediate the activation of plant defense responses induced by harpin_Psph.

The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. Some hrp gene products constitute elements of the type III secretion system, by which effector proteins are exported and delivered into plant cells. Here, we show that the hrpZ gene product from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (HrpZ_Psph), is secreted in an hrp-dependent manner in P. syringae pv. phaseolicola and exported by the type III secretion system in the mammalian pathogen Yersinia enterocolitica. HrpZ_Psph was found to associate stably with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2 nM of purified recombinant HrpZ_Psph to the cis compartment of planar lipid bilayers provoked an ion current with a large unitary conductivity of 207 pS. HrpZ_Psph-related proteins from P. syringae pv. tomato or syringae triggered ion currents similar to those stimulated by HrpZ_Psph. The HrpZ_Psph-mediated ion-conducting pore was permeable for cations but did not mediate fluxes of Cl⁻. Such pore-forming activity may allow nutrient release and/or delivery of virulence factors during bacterial colonization of host plants.

Transient influx of Ca²⁺ constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae–derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca²⁺]_cyt), which peaked at ∼1 μM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca²⁺]_cyt signature was achieved by elicitor concentrations sufficient to stimulate Ca²⁺ influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca²⁺]_cyt but not the rapidly induced [Ca²⁺]_cyt transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca²⁺ influx across the plasma membrane or of Ca²⁺ release from internal stores suggests that the elicitor-induced sustained increase of [Ca²⁺]_cyt predominantly results from the influx of extracellular Ca²⁺. Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca²⁺]_cyt, and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca²⁺]_cyt is causally involved in signaling the activation of pathogen defense in parsley.