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.

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize.

HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector.

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria (P. syringae) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.

Thaumatin represents a unique class of the sweet-tasting plant proteins. Transgenic cucumber (Cucumis sativus L.) plants with stable integrated constructs consisting of the cauliflower mosaic virus 35S promoter and thaumatin II cDNA were produced. Transformed cucumber plants were obtained using Agrobacterium tumefaciens, with one, two or five integration sites in diploid cucumber and with inheritance confirmed by a 3:1 Mendelian ratio and normal morphologies and viable seeds. Inter-and intra-transformant variabilities in the expression of the thaumatin II gene were observed. The variability was independent of integrated copy number of the T-DNA. Variation in thaumatin II protein accumulation levels in the ripe fruits and the lack of correlation between protein and mRNA levels were observed, suggesting that thaumatin may be controlled at the levels of both transcription and translation. Transgenic fruits accumulating thaumatin II protein exhibited sweet phenotype and positive correlation between thaumatin accumulation levels and sweet taste intensity was noticed. Thaumatin II belongs to the pathogenesis-related (PR) proteins family. Some of the T2 progeny plants expressing thaumatin II protein did not exhibit tolerance for pathogenic fungus Pseudoperonospora cubensis. These results, together with previously reported results, suggest no relationship between transgenic protein levels and the increased tolerance phenotype.