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Reactive oxygen species are believed to perform multiple roles during plant defense and possibly as cellular signaling molecules. In animals, nitric oxide (NO) is an important redox-active signaling molecule. Here we show that infection of resistant, but not susceptible, tobacco with tobacco mosaic virus resulted in enhanced NO synthase (NOS) activity. Furthermore, administration of NO donors or recombinant mammalian NOS to tobacco plants or tobacco suspension cells triggered expression of the defense-related genes encoding pathogenesis-related 1 protein and phenylalanine ammonia lyase (PAL). These genes were also induced by cyclic GMP (cGMP) and cyclic ADP-ribose, two molecules that can serve as second messengers for NO signaling in mammals. Consistent with cGMP levels. Furthermore, NO-induced activation of PAL was blocked by 6-anilino-5,8-quinolinedione and 1H-(1,2,4)-oxadizole[4,3-alpha]quinoxalin-1-one, two inhibitors of guanylate cyclase. Although 6-anilino-5,8-quinolinedione fully blocked PAL activation, inhibition by 1H-(1,2,4)-oxadiozole[4,3-alpha]quinoxalin-1-one was not entirely complete, suggesting the existence of cGMP-independent, as well as cGMP-dependent, NO signaling. We conclude that several critical players of animal NO signaling are also operative in plants

The Arabidopsis genes EDS1 and NDR1 were shown previously by mutational analysis to encode essential components of race-specific disease resistance. Here, we examined the relative requirements for EDS1 and NDR1 by a broad spectrum of Resistance (R) genes present in three Arabidopsis accessions (Columbia, Landsberg-erecta, and Wassilewskija). We show that there is a strong requirement for EDS1 by a subset of R loci (RPP2, RPP4, RPP5, RPP21, and RPS4), conferring resistance to the biotrophic oomycete Peronospora parasitica, and to Pseudomonas bacteria expressing the avirulence gene avrRps4. The requirement for NDR1 by these EDS1-dependent R loci is either weak or not measurable. Conversely, three NDR1-dependent R loci, RPS2, RPM1, and RPS5, operate independently of EDS1. Another RPP locus, RPP8, exhibits no strong exclusive requirement for EDS1 or NDR1 in isolate-specific resistance so P. parasitica, although resistance is compromised weakly by eds1. Similarly, resistance conditioned by two EDS1-dependent RPP genes, RPP4 and RPP5, is impaired partially by rdr1, implicating a degree of pathway cross-talk. Our results provide compelling evidence for the preferential utilization of either signaling component by particular R genes and thus define at least two disease resistance pathways. The dna also suggest that strong dependence on EDS1 or NDR1 is governed by R protein structural type rather than pathogen class

A 5-kD plant defensin was purified from Arabidopsis leaves challenged with the fungus Alternaria brassicicola and shown to possess antifungal properties in vitro. The corresponding plant defensin gene was induced after treatment of leaves with methyl jasmonate or ethylene but not with salicylic acid or 2,6-dichloroisonicotinic acid. When challenged with A. brassicicola, the levels of the plant defensin protein and mRNA rose both in inoculated leaves and in nontreated leaves of inoculated plants (systemic leaves). These events coincided with an increase in the endogenous jasmonic acid content of both types of leaves. Systemic pathogen-induced expression of the plant defensin gene was unaffected in Arabidopsis transformants (nahG) or mutants (npr1 and cpr1) affected in the salicylic acid response but was strongly reduced in the Arabidopsis mutants ein2 and coi1 that are blocked in their response to ethylene and methyl jasmonate, respectively. Our results indicate that systemic pathogen-induced expression of the plant defensin gene in Arabidopsis is independent of salicylic acid but requires components of the ethylene and jasmonic acid response.

Transgenic tobacco deficient in the H2O2-removing enzyme catalase (Cat1AS) was used as an inducible and noninvasive system to study the role of H2O2 as an activator of pathogenesis-related (PR) proteins in plants. Excess H2O2 in Cat1AS plants was generated by simply increasing light intensities. Sustained exposure of Cat1AS plants to excess H2O2 provoked tissue damage, stimulated salicylic acid and ethylene production, and induced the expression of acidic and basic PR proteins with a timing and magnitude similar to the hypersensitive response against pathogens. Salicylic acid production was biphasic, and the first peak of salicylic acid as well as the peak of ethylene occurred within the first hours of high light, which is long before the development of tissue necrosis. Under these conditions, accumulation of acidic PR proteins was also seen in upper leaves that were not exposed to high light, indicating systemic induction of expression. Short exposure of Cat1AS plants to excess H2O2 did not cause damage, induced local expression of acidic and basic PR proteins, and enhanced pathogen tolerance. However, the timing and magnitude of PR protein induction was in this case more similar to that in upper uninfected leaves than to that in hypersensitive-response leaves of pathogen-infected plants. Together, these data demonstrate that sublethal levels of H2O2 activate expression of acidic and basic PR proteins and lead to enhanced pathogen tolerance. However, rapid and strong activation of PR protein expression, as seen during the hypersensitive response, occurs only when excess H2O2 is accompanied by leaf necrosis

Synergistic viral diseases of higher plants are caused by the interaction of two independent viruses in the same host and are characterized by dramatic increases in symptoms and in accumulation of one of the coinfecting viruses. In potato virus X (PVX)/potyviral synergism, increased pathogenicity and accumulation of PVX are mediated by the expression of potyviral 5' proximal sequences encoding P1, the helper component proteinase (HC-Pro), and a fraction of P3. Here, we report that the same potyviral sequence (termed P1/HC-Pro) enhances the pathogenicity and accumulation of two other heterologous viruses: cucumber mosaic virus and tobacco mosaic virus. In the case of PVX-potyviral synergism, we show that the expression of the HC-Pro gene product, but not the RNA sequence itself, is sufficient to induce the increase in PVX pathogenicity and that both P1 and P3 coding sequences are dispensable for this aspect of the synergistic interaction. In protoplasts, expression of the potyviral P1/HC-Pro region prolongs the accumulation of PVX (-) strand RNA and transactivates expression of a reporter gene from a PVX subgenomic promoter. Unlike the synergistic enhancement of PVX pathogenicity, which requires only expression of HC-Pro, the enhancement of PVX (-) strand RNA accumulation in protoplasts is significantly greater when the entire P1/HC-Pro sequence is expressed. These results indicate that the potyviral P1/HC-Pro region affects a step in disease development that is common to a broad range of virus infections and suggest a mechanism involving transactivation of viral replication

The phenylpropanoid-derived natural product salicylic acid (SA) plays a key role in disease resistance. However, SA administered in the absence of a pathogen is a paradoxically weak inductive signal, often requiring concentrations of 0.5 to 5 mM to induce acquired resistance or related defense mechanisms or to precondition signal systems. In contrast, endogenous SA accumulates to concentrations of 70 micromolar at the site of attempted infection. Here, we show that although 10 to 100 micromolar SA had negligible effects when administered to soybean cell suspensions in the absence of a pathogen, physiological concentrations of SA markedly enhanced the induction of defense gene transcripts, H2O2 accumulation, and hypersensitive cell death by an avirulent strain of Pseudomonas syringae pv glycinea, with optimal effects being at approximately 50 micromolar. SA also synergistically enhanced H2O2 accumulation in response to the protein phosphatase type 2A inhibitor cantharidin in the absence of a pathogen. The synergistic effect of SA was potent, rapid, and insensitive to the protein synthesis inhibitor cycloheximide, and we conclude that SA stimulates an agonist-dependent gain control operating at an early step in the signal pathway for induction of the hypersensitive response. This fine control mechanism differs from previously described time-dependent, inductive coarse control mechanisms for SA action in the absence of a pathogen. Induction of H2O2 accumulation and hypersensitive cell death by avirulent P. s. glycinea was blocked by the phenylpropanoid synthesis inhibitor alpha-aminooxy-beta-phenylpropionic acid, and these responses could be rescued by exogenous SA. Because the agonist-dependent gain control operates at physiological levels of SA, we propose that rapid fine control signal amplification makes an important contribution to SA function in the induction of disease resistance mechanisms

The active oxygen species hydrogen peroxide (H2O2) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides. In uninoculated lettuce leaves, H2O2 was typically present within the secondary thickened walls of xylem vessels. Inoculation with wild-type cells of Pseudomonas syringae pv phaseolicola caused a rapid hypersensitive reaction (HR) during which highly localized accumulation of H2O2 was found in plant cell walls adjacent to attached bacteria. Quantitative analysis indicated a prolonged burst of H2O2 occurring between 5 to 8 hr after inoculation in cells undergoing the HR during this example of non-host resistance. Cell wall alterations and papilla deposition, which occurred in response to both the wild-type strain and a nonpathogenic hrpD mutant, were not associated with intense staining for H2O2, unless the responding cell was undergoing the HR. Catalase treatment to decompose H2O2 almost entirely eliminated staining, but 3-amino-1,2,4-triazole (catalase inhibitor) did not affect the pattern of distribution of H2O2 detected. H2O2 production was reduced more by the inhibition of plant peroxidases (with potassium cyanide and sodium azide) than by inhibition of neutrophil-like NADPH oxidase (with diphenylene iodonium chloride). Results suggest that CeCl3 reacts with excess H2O2 that is not rapidly metabolized during cross-linking reactions occurring in cell walls; such an excess of H2O2 in the early stages of the plant-bacterium interaction was only produced during the HR. The highly localized accumulation of H2O2 is consistent with its direct role as an antimicrobial agent and as the cause of localized membrane damage at sites of bacterial attachment

Plant resistance (R) genes have evolved specific recognition capabilities in defense against pathogens. The evolution of R gene function and maintenance of R gene diversity within a plant species are therefore of great interest. In the Arabidopsis accession Wassilewskija, the RPP1 region on chromosome 3 contains four genetically linked recognition specificities, conditioning resistance to different isolates of the biotrophic oomycete Peronospora parasitica (downy mildew). We show that three of four tightly linked genes in this region, designated RPP1-WsA, RPP1-WsB, and RPP1-WsC, encode functional products of the NBS-LRR (nucleotide binding site-leucine-rich repeat) R protein class. They possess a TIR Toll, interleukin-1, resistance domain that is characteristic of certain other NBS-LRR-type R proteins, but in addition, they have unique hydrophilic or hydrophobic N termini. Together, the three RPP1 genes account for the spectrum of resistance previously assigned to the RPP1 region and thus comprise a complex R locus. The distinct but partially overlapping resistance capabilities conferred by these genes are best explained by the hypothesis that each recognizes a different pathogen avirulence determinant. We present evidence suggesting that the RPP genes at this locus are subject to the same selective forces that have been demonstrated for structurally different LRR-type R genes

Response surface methodology-central composite rotatable design (RSM-CCRD) was applied to explore the optimum media formulation for maximising canthaxanthin (CTX) biosynthesis by Dietzia natronolimnaea HS-1. The effects of three variables of enzymatically hydrolysed molasses (EHM) (16.6-33.4 g/l), sodium (Na)-citrate (21.64-28.36mM), and yeast extract (6.32-9.68 g/l) concentrations on the production of CTX, total carotenoid (TCT), and biomass dry weight (BDW) were appraised. The results showed that the quadratic effects of EHM, yeast extract, and Na-citrate contents in terms of second-order polynomial regression equations (R2 = 0.968-0.986), respectively, had the most significant effects on the produced TCT and CTX. The predicted maximum accumulation of BDW (8.88 g/l), TCT (7.24 mg/l), and CTX (6.40 mg/l) under the optimum concentrations of the media variables (26.16 g/l EHM, 8.29 g/l yeast extract, and 25.86mM Na-citrate) was very close to the experimental values determined in batch experiments. The high BDW content suggested EHM and Na-citrate as very promising feedstocks for CTX bioproduction by the bacterium studied.

The glochidium larvae of freshwater mussels of the family Unionidae need to find suitable hosts to attach themselves and metamorphose into free-living juveniles. The specificity of the host-parasite relationship was investigated for the Iberian Unio tumidiformis by means of experimental infections and also by analyzing naturally infected fish. The process of encapsulation of glochidia was studied using scanning electron microscopy. Unio tumidiformis has proven to be an unusual host-specific unionid mussel, apparently parasitizing only fish of the genus Squalius. Successful encapsulation or complete metamorphosis was observed in five fish taxa: S. aradensis, S. caroliterti, S. pyrenaicus, S. torgalensis and S. alburnoides complex (only for the nuclear hybrids with at least one copy of the S. pyrenaicus genome). Complete metamorphose was achieved in 6 to 14 days at mean temperatures ranging from 21.8 to 26.1 deg C. The current study provides support for cell migration being the main force of cyst formation and shows the influence of potential host's genome in response to the infection process to determine the success of the metamorphosis.