<Emphasis Type="Italic">Brassica napus</Emphasis> possesses an expanded set of polygalacturonase inhibitor protein genes that are differentially regulated in response to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> infection,wounding and defense hormone treatment

Most plants encode a limited set of polygalacturonase inhibitor (PGIP) genes that may be involved in aspects of plant development, but more importantly in the inactivation of polygalacturonases (PG) secreted by pathogens. Previously, we characterized two Brassica napus PGIP genes, BnPgip1 and BnPgip2, which were differentially expressed in response to pathogen infection and wounding. Here we report that the B. napus genome encodes a set of at least 16 PGIP genes that are similar to BnPgip1 or BnPgip2. This is the largest Pgip gene family reported to date. Comparison of the BnPGIPs revealed several sites within the xxLxLxx region of leucine rich repeats that form beta-sheets along the interacting face of the PGIP that are hypervariable and represent good candidates for generating PGIP diversity. Characterization of the regulatory regions and RT-PCR studies with gene-specific primers revealed that individual genes were differentially responsive to pathogen infection, mechanical wounding and signaling molecules. Many of the BnPgip genes responded to infection by the necrotic pathogen, Sclerotinia sclerotiorum; however, these genes were also induced either by jasmonic acid, wounding and salicylic acid or some combination thereof. The large number of PGIPs and the differential manner in which they are regulated likely ensures that B. napus can respond to attack from a broad spectrum of pathogens and pests.     

Nitric oxide is induced by wounding and influences jasmonic acid signaling in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Nitric oxide (NO) has been associated with plant defense responses during microbial attack, and with induction and/or regulation of programmed cell death. Here, we addressed whether NO participates in wound responses in Arabidopsis thaliana (L.) Heynh.. Real-time imaging by confocal laser-scanning microscopy in conjunction with the NO-selective fluorescence indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) uncovered a strong NO burst after wounding or after treatment with JA. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive responses. Furthermore, we were able to detect NO in plants (here induced by wounding) by means of electron paramagnetic resonance measurements using diethyldithiocarbamate as a spin trap. When plants were treated with NO, Northern analyses revealed that NO strongly induces key enzymes of jasmonic acid (JA) biosynthesis such as allene oxide synthase (AOS) and lipoxygenase (LOX2). On the other hand, wound-induced AOS gene expression was independent of NO. Furthermore, JA-responsive genes such as defensin (PDF1.2) were not induced, and NO induction of JA-biosynthesis enzymes did not result in elevated levels of JA. However, treatment with NO resulted in accumulation of salicylic acid (SA). In transgenic NahG plants (impaired in SA accumulation and/or signaling), NO did induce JA production and expression of JA-responsive genes. Altogether, the presented data demonstrate that wounding in Arabidopsis induces a fast accumulation of NO, and that NO may be involved in JA-associated defense responses and adjustments.Abbreviations AOS Allene oxide synthase - cPTIO Carboxy-2-phenyl-4,4,5,5-tetramethylimidazolinone-3-oxide-1-oxyl - DAF-2 DA 4,5-Diaminofluorescein diacetate - DETC Diethyldithiocarbamate - EPR Electron paramagnetic resonance - iNOS Inducible nitric oxide synthase - JA Jasmonic acid - JIP Jasmonic acid-induced protein - LOX2 Lipoxygenase 2 - NO Nitric oxide - OPR3 12-Oxophytodienoate reductase - PDF1.2 Plant defensin - ROS Reactive oxygen species - SA Salicylic acid - SNP Sodium nitroprusside     

Induction of systemic acquired resistance in Zea mays also enhances the plant’s attractiveness to parasitoids

Plants under attack by caterpillars emit volatile compounds that attract the herbivore’s natural enemies. In maize, the caterpillar-induced production of volatiles involves the phytohormone jasmonic acid (JA). In contrast, pathogen attack usually up-regulates the salicylic acid (SA)-pathway and results in systemic acquired resistance (SAR) against plant diseases. Activation of the SA-pathway has often been found to repress JA-dependent direct defenses, but little is known about the effects of SAR induction on indirect defenses such as volatile emission and parasitoid attraction. We examined if induction of SAR in maize, by chemical elicitation with the SA-mimic benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), attenuates the emission of volatiles induced by Spodoptera littoralis or exogenously applied JA. In addition, we determined how these treatments affected the attractiveness of the plants to the parasitoid Microplitis rufiventris in a six-arm-olfactometer. BTH treatment alone resulted in significant systemic resistance of maize seedlings against the pathogen Setosphaeria turcica, but had no detectable effect on volatile emissions. Induction of SAR significantly reduced the emission rates of two compounds (indole and (E)-β-caryophyllene) in JA-treated plants, whereas no such negative cross-talk was found in caterpillar-damaged plants. Surprisingly, however, BTH treatment prior to caterpillar-feeding made the plants far more attractive to the parasitoid than plants that were only damaged by the herbivore. Control experiments showed that this response was due to plant-mediated effects rather than attractiveness of BTH itself. We conclude that in the studied system, plant protection by SAR activation is compatible with and can even enhance indirect defense against herbivores.     

A non-JA producing oxophytodienoate reductase functions in salicylic acid-mediated antagonism with jasmonic acid during pathogen attack

Peroxisome-localized oxo-phytodienoic acid (OPDA) reductases (OPR) are enzymes converting 12-OPDA into jasmonic acid (JA). However, the biochemical and physiological functions of the cytoplasmic non-JA producing OPRs remain largely unknown. Here, we generated Mutator-insertional mutants of the maize OPR2 gene and tested its role in resistance to pathogens with distinct lifestyles. Functional analyses showed that the opr2 mutants were more susceptible to the (hemi)biotrophic pathogens Colletotrichum graminicola and Ustilago maydis, but were more resistant to the necrotrophic fungus Cochliobolus heterostrophus. Hormone profiling revealed that increased susceptibility to C. graminicola was associated with decreased salicylic acid (SA) but increased JA levels. Mutation of the JA-producing lipoxygenase 10 (LOX10) reversed this phenotype in the opr2 mutant background, corroborating the notion that JA promotes susceptibility to this pathogen. Exogenous SA did not rescue normal resistance levels in opr2 mutants, suggesting that this SA-inducible gene is the key downstream component of the SA-mediated defences against C. graminicola. Disease assays of the single and double opr2 and lox10 mutants and the JA-deficient opr7opr8 mutants showed that OPR2 negatively regulates JA biosynthesis, and that JA is required for resistance against C. heterostrophus. Overall, this study uncovers a novel function of a non-JA producing OPR as a major negative regulator of JA biosynthesis during pathogen infection, a function that leads to its contrasting contribution to either resistance or susceptibility depending on pathogen lifestyle.     

Silencing of OPR3 in tomato reveals the role of OPDA in callose deposition during the activation of defense responses against Botrytis cinerea

Cis‐(+)‐12‐oxo‐phytodienoic acid (OPDA) is likely to play signaling roles in plant defense that do not depend on its further conversion to the phytohormone jasmonic acid. To elucidate the role of OPDA in Solanum lycopersicum (tomato) plant defense, we have silenced the 12‐oxophytodienoate reductase 3 (OPR3) gene. Two independent transgenic tomato lines (SiOPR3‐1 and SiOPR3‐2) showed significantly reduced OPR3 expression upon infection with the necrotrophic pathogen Botrytis cinerea. Moreover, SiOPR3 plants are more susceptible to this pathogen, and this susceptibility is accompanied by a significant decrease in OPDA levels and by the production of JA‐Ile being almost abolished. OPR3 silencing also leads to a major reduction in the expression of other genes of the jasmonic acid (JA) synthesis and signaling pathways after infection. These results confirm that in tomato plants, as in Arabidopsis, OPR3 determines OPDA availability for JA biosynthesis. In addition, we show that an intact JA biosynthetic pathway is required for proper callose deposition, as its pathogen‐induced accumulation is reduced in SiOPR3 plants. Interestingly, OPDA, but not JA, treatment restored basal resistance to B. cinerea and induced callose deposition in SiOPR3‐1 and SiOPR3‐2 transgenic plants. These results provide clear evidence that OPDA by itself plays a major role in the basal defense of tomato plants against this necrotrophic pathogen.     

Differential inducible defense mechanisms against bacterial speck pathogen in Arabidopsis thaliana by plant-growth-promoting-fungus Penicillium sp. GP16-2 and its cell free filtrate

Although a wealth of information is available regarding resistance induced by plant growth-promoting rhizobacteria (PGPR), not much is known about plant growth-promoting fungi (PGPF). Hence, the goal of the present research was to provide more information on this matter. In Arabidopsis thaliana L., root colonizing PGPF Penicillium sp. GP16-2 or its cell free filtrate (CF) elicited an induced systemic resistance (ISR) against infection by Pseudomonas syringae pv. tomato DC3000 (Pst), leading to a restriction of pathogen growth and disease development. We demonstrate that signal transduction leading to GP16-2-mediated ISR requires responsiveness to JA and ET in a NPR1-dependent manner, while CF-mediated ISR shows dispensability of SA, JA, ET and NPR1-dependent signaling (at least individually). In addition, root colonization by GP16-2 is not associated with a direct effect on expression of known defense-related genes, but potentiates the activation of JA/ET-inducible ChitB, which only becomes apparent after infection by Pst. However, CF-mediated ISR was partly associated with the direct activation of marker genes responsive to both SA and JA/ET signaling pathways and partly associated with priming, leading to activation of JA-/ET-inducible ChitB and Hel genes. These suggest that CF may contain one or more elicitors that induce resistance by way where at least SA, JA and ET may play a role in defense signaling in Arabidopsis. Therefore, defense gene changes and underlying signaling pathways induced by Penicillium sp. GP16-2 root colonization and its CF application are not the same and only partially overlap.     

Jasmonates induce Nod factor production by Bradyrhizobium japonicum

Jasmonates are signaling molecules involved in induced systemic resistance, wounding and stress responses of plants. We have previously demonstrated that jasmonates can induce nod genes of Bradyrhizobium japonicum when measured by beta-galactosidase activity. In order to test whether jasmonates can effectively induce the production and secretion of Nod factors (lipo-chitooligosaccharides, LCOs) from B. japonicum, we induced two B. japonicum strains, 532C and USDA3, with jasmonic acid (JA), methyl jasmonate (MeJA) and genistein (Ge). As genistein is well characterized as an inducer of nod genes it was used a positive control. The high-performance liquid chromatography (HPLC) profile of LCOs isolated following treatment with jasmonates or genistein showed that both JA and MeJA effectively induced nod genes and caused production of LCOs from bacterial cultures. JA and MeJA are more efficacious inducers of LCO production than genistein. Genistein plus JA or MeJA resulted in greater LCO production than either alone. A soybean root hair deformation assay showed that jasmonate induced LCOs were as effective as those induced by genistein. This is the first report that jasmonates induce Nod factor production by B. japonicum. This report establishes the role of jasmonates as a new class of signaling molecules in the Bradyrhizobium-soybean symbiosis.     

Interacting signal pathways control defense gene expression in Arabidopsis in response to cell wall-degrading enzymes from Erwinia carotovora

We have characterized the role of salicylic acid (SA)-independent defense signaling in Arabidopsis thaliana in response to the plant pathogen Erwinia carotovora subsp. carotovora. Use of pathway-specific target genes as well as signal mutants allowed us to elucidate the role and interactions of ethylene, jasmonic acid (JA), and SA signal pathways in this response. Gene expression studies suggest a central role for both ethylene and JA pathways in the regulation of defense gene expression triggered by the pathogen or by plant cell wall-degrading enzymes (CF) secreted by the pathogen. Our results suggest that ethylene and JA act in concert in this regulation. In addition, CF triggers another, strictly JA-mediated response inhibited by ethylene and SA. SA does not appear to have a major role in activating defense gene expression in response to CF. However, SA may have a dual role in controlling CF-induced gene expression, by enhancing the expression of genes synergistically induced by ethylene and JA and repressing genes induced by JA alone.     

<Emphasis Type="Italic">Pythium</Emphasis> infection activates conserved plant defense responses in mosses

The moss Physcomitrella patens (P. patens) is a useful model to study abiotic stress responses since it is highly tolerant to drought, salt and osmotic stress. However, very little is known about the defense mechanisms activated in this moss after pathogen assault. In this study, we show that P. patens activated multiple and similar responses against Pythium irregulare and Pythium debaryanum, including the reinforcement of the cell wall, induction of the defense genes CHS, LOX and PAL, and accumulation of the signaling molecules jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (OPDA). However, theses responses were not sufficient and infection could not be prevented leading to hyphae colonization of moss tissues and plant decay. Pythium infection induced reactive oxygen species production and caused cell death of moss tissues. Taken together, these data indicate that Pythium infection activates in P. patens common responses to those previously characterized in flowering plants. Microscopic analysis also revealed intracellular relocation of chloroplasts in Pythium-infected tissues toward the infection site. In addition, OPDA, JA and its methyl ester methyl jasmonate induced the expression of PAL. Our results show for the first time JA and OPDA accumulation in a moss and suggest that this defense pathway is functional and has been maintained during the evolution of plants. Authors Juan Pablo Oliver and Alexandra Castro contributed equally to this work.