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 tomato ACC oxidase gene family is comprised of three members designated AC01, AC02 and AC03. These are highly homologous throughout the protein coding regions but do show a degree of sequence divergence within the 3' untranslated regions. These regions have been cloned and used as gene-specific probes to analyse the differential expression of the tomato ACC oxidase gene family in various tissues at different stages of development. Results indicate that all three genes are transcriptionally active and display a high degree of inducibility in a number of organs at various stages of the life cycle. Both AC01 and Ac03 transcripts accumulate during the senescence of leaves, fruit and flowers. In addition, it appears that AC01 is wound-inducible in leaves. All three ACC oxidase genes are expressed during flower development, with each showing a temporally distinct pattern of accumulation. In addition, the ACC oxidase transcripts are also spatially regulated throughout flower development; AC01 is predominantly expressed in the petals and the stigma and style, AC02 expression is mainly restricted to tissues associated with the anther cone whereas AC03 transcripts accumulate in all of the floral organs examined apart from the sepals. ACC oxidase enzyme assays and Western blot analysis indicate that both enzyme activity and ACC oxidase protein increase with transcript abundance in several tissues. The physiological role of the differential expression of the ACC oxidase gene family, in relation to the regulation of ethylene synthesis, during these various developmental processes is discussed.

INTRODUCTIONCytoplasmic free Ca 2 ϩ ([Ca 2 ϩ ] cyt ) serves as a second messenger in plant processes as diverse as root nodule formation, phytochrome phototransduction, stomatal closure, geotropism, circadian rhythm, pollen tube growth, and stress adaptation (Rudd and Franklin-Tong, 1999). Stimulusspecific and spatially and temporally defined Ca 2 ϩ signatures of characteristic magnitude, frequency, and duration are assumed to maintain signal specificity of transduction cascades (Thuleau et al., 1998;Trewavas, 1999). Subsequently, the binding of [Ca 2 ϩ ] cyt to calmodulin, Ca 2 ϩ -dependent protein kinases, Ca 2 ϩ -dependent protein phosphatases, Ca 2 ϩ -gated ion channels, or Ca 2 ϩ -activated phospholipases facilitates downstream signal transduction directed toward activation of a signal-specific cellular response (Blumwald et al., 1998).Expression of the Aequorea aequorea apoaequorin gene in the cytoplasm of plant cells provides a means for accurate, noninvasive quantification of changes in [Ca 2 ϩ ] cyt (Knight et al., 1991). When reconstituted with coelenterazine, holoaequorin acts as a bioluminescent indicator of [Ca 2 ϩ ] cyt . Since the pioneering work of Knight et al. (1991), aequorin technology has been widely applied in plants to report changes in [Ca 2 ϩ ] cyt in response to abiotic stimuli, such as touch, wind, cold, heat, and drought (Knight et al., 1991(Knight et al., , 1992(Knight et al., , 1997Haley et al., 1995;Gong et al., 1998;Plieth et al., 1999); blue light ; circadian rhythm (Johnson et al., 1995); ozone (Clayton et al., 1999); anoxia (Sedbrook et al., 1996); oxidative stress (Price et al., 1994); and hypoosmotic shock (Chandra and Low, 1997;Takahashi et al., 1997;Cessna et al., 1998).Numerous recent studies have provided evidence that Ca 2 ϩ plays a pivotal role in activating the plant's surveillance system against attempted microbial invasion (Yang et al., 1997;Scheel, 1998). Activation of plant defense is believed to be receptor mediated through recognition of pathogenderived elicitors (Yang et al., 1997;Scheel, 1998;Nürnberger, 1999). In contrast to elicitors from phytopathogenic bacteria, elicitors of fungal or oomycete origin appear to be recognized by high-affinity receptors residing in the plasma membrane of plant cells. Although several such plasma membrane binding sites have been characterized kinetically and structurally, our knowledge of the molecular mode of fungal pathogen perception in plants remains fragmentary: only one elicitor receptor has been isolated thus far, a soybean 70-kD plasma membrane protein that binds Phytophthora sojae-derived ␤ -glucans (Umemoto et al., 1997).Receptor-ligand interaction initiates an intracellular signal transduction cascade that mediates activation of the defense against the pathogen. Cellular components shown to be modulated by elicitor treatment include GTP binding proteins (Bischoff et al., 1999); plasma membrane ion channels 1 Current address: LION Bioscience AG, Im Neuenheimer Feld 515-517, D-69120 Heidelberg, Germany. 2 To whom corre...

Transient influx of Ca(2+) 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 ([Ca(2+)](cyt)), which peaked at approximately 1 microM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca(2+)](cyt) signature was achieved by elicitor concentrations sufficient to stimulate Ca(2+) influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca(2+)](cyt) but not the rapidly induced [Ca(2+)](cyt) transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca(2+) influx across the plasma membrane or of Ca(2+) release from internal stores suggests that the elicitor-induced sustained increase of [Ca(2+)](cyt) predominantly results from the influx of extracellular Ca(2+). Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca(2+)](cyt), and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca(2+)](cyt) is causally involved in signaling the activation of pathogen defense in parsley.

The enzyme ACC oxidase, catalysing the last step in the biosynthesis of the plant hormone ethylene, is encoded by a small multigene family in tomato, comprising three members, LEACO1, LEACO2 and LEACO3. LEACO1 is the major gene expressed during ripening, leaf senescence, and wounding (Barry et al., 1996). To investigate the transcriptional regulation of ACC oxidase gene expression, chimeric fusions between the beta-glucuronidase reporter gene and 97 bp of 5' UTR plus 124, 396 and 1825 bp, respectively, of 5' untranscribed LEACO1 sequence were constructed and introduced into Lycopersicon esculentum (Mill cv. Ailsa Craig) and Nicotiana plumbaginifolia. Analysis of transgenic tomatoes indicated that the region containing nucleotides -124 to +97 of the LEACO1 gene is sufficient to confer a marked increase in GUS activity during fruit ripening, albeit at very low levels. Fusion of 396 and 1825 bp of LEACO1 upstream sequence resulted in strong and specific induction of GUS expression in situations known to be accompanied by enhanced ethylene production. Reporter gene expression was similar to that of the endogenous LEACO1 gene, with major increases especially during fruit ripening, senescence and abscission of leaves and, to a lesser extent, of flowers. Analysis of transgenic N. plumbaginifolia plants confirmed the pattern of LEACO1 promoter activity detected in tomato leaves and flowers. Reporter gene expression was also induced following wounding, treatment with ethylene, and pathogen infection. Histochemical analysis illustrated localized GUS activity in the pericarp of ripening fruit, abscission zones of senescent petioles and unfertilized flowers, and at wound sites. These results demonstrate that ACC oxidase is regulated at the transcriptional level in a wide range of cell types at different developmental stages and in response to several external stimuli.