Plant Apoplast Research Articles

Escherichia coli O157:H7 Induces Stronger Plant Immunity than Salmonella enterica Typhimurium SL1344

Consumption of fresh produce contaminated with bacterial human pathogens has resulted in various, sometimes deadly, disease outbreaks. In this study, we assessed plant defense responses induced by the fully pathogenic bacteria Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium SL1344 in both Arabidopsis thaliana and lettuce (Lactuca sativa). Unlike SL1344, O157:H7 induced strong plant immunity at both pre-invasion and post-invasion steps of infection. For instance, O157:H7 triggered stomatal closure even under high relative humidity, an environmental condition that generally weakens plant defenses against bacteria in the field and laboratory conditions. SL1344 instead induced a transient stomatal immunity. We also observed that PR1 gene expression was significantly higher in Arabidopsis leaves infected with O157:H7 compared with SL1344. These results suggest that plants may recognize and respond to some human pathogens more effectively than others. Furthermore, stomatal immunity can diminish the penetration of human pathogens through the leaf epidermis, resulting in low bacterial titers in the plant apoplast and suggesting that additional control measures can be employed to prevent food contamination. The understanding of how plant responses can diminish bacterial contamination is paramount in preventing outbreaks and improving the safety of food supplies.

Hydrophobic proteins secreted into the apoplast may contribute to resistance against Phytophthora infestans in potato

During plant-pathogen interaction, oomycetes secrete effectors into the plant apoplast where they interact with host resistance proteins, which are accumulated after wounding or infection. Previous studies showed that the expression profile of pathogenesis related proteins is proportional to the resistance of different cultivars toward Phytophthora infestans infection. The aim of this work was to analyze the expression pattern of apoplastic hydrophobic proteins (AHPs), after 24h of wounding or infection, in tubers from two potato cultivars with different resistance to P.infestans, Spunta (susceptible) and Innovator (resistant). Intercellular washing fluid (IWF) was extracted from tubers and chromatographed into a PepRPC™ HR5-5 column in FPLC eluted with a linear gradient of 75% acetonitrile. Then, AHPs were analyzed by SDS-PAGE and identified by MALDI-TOF-MS. Innovator cv. showed a higher basal AHP content compared to Spunta cv. In the latter, infection induced accumulation of patatins and protease inhibitors (PIs), whereas in Innovator cv. no changes in PIs accumulation were observed. In response to P.infestans infection, lipoxygenase, enolase, annexin p34 and glutarredoxin/cyclophilin were accumulated in both cultivars. These results suggest that the AHPs content may be related to the protection against the oomycete and with the degree of potato resistance to pathogens. Additionally, a considerable number of the proteins putatively identified lacked the signal peptide and, being SecretomeP positive, suggest unconventional protein secretion.

Apoplastic and cytoplasmic location of harpin protein Hpa1Xoo plays different roles in H2O2 generation and pathogen resistance in Arabidopsis

Harpin proteins secreted by phytopathogenic bacteria have been shown to activate the plant defense pathway, which involves transduction of a hydrogen peroxide (H(2)O(2)) signal generated in the apoplast. However, the way in which harpins are recognized in the pathway and what role the apoplastic H(2)O(2) plays in plant defenses are unclear. Here, we examine whether the cellular localization of Hpa1(Xoo), a harpin protein produced by the rice bacterial leaf blight pathogen, impacts H(2)O(2) production and pathogen resistance in Arabidopsis thaliana. Transformation with the hpa1 (Xoo) gene and hpa1 (Xoo) fused to an apoplastic localization signal (shpa1 (Xoo)) generated h pa1 (Xoo)- and sh pa1 (Xoo)-expressing transgenic A . t haliana (HETAt and SHETAt) plants, respectively. Hpa1(Xoo) was associated with the apoplast in SHETAt plants but localized inside the cell in HETAt plants. In addition, Hpa1(Xoo) localization accompanied H(2)O(2) accumulation in both the apoplast and cytoplasm of SHETAt plants but only in the cytoplasm of HETAt plants. Apoplastic H(2)O(2) production via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) located in the plasma membrane is a common feature of plant defenses. In SHETAt plants, H(2)O(2) was generated in apoplasts in a NOX-dependent manner but accumulated to a greater extent in the cytoplasm than in the apoplast. After being applied to the wild-type plant, Hpa1(Xoo) localized to apoplasts and stimulated H(2)O(2) production as in SHETAt plants. In both plants, inhibiting apoplastic H(2)O(2) generation abrogated both cytoplasmic H(2)O(2) accumulation and plant resistance to bacterial pathogens. These results suggest the possibility that the apoplastic H(2)O(2) is subject to a cytoplasmic translocation for participation in the pathogen defense.

Verticillium longisporum infection affects the leaf apoplastic proteome, metabolome, and cell wall properties in Arabidopsis thaliana.

Verticillium longisporum (VL) is one of the most devastating diseases in important oil crops from the family of Brassicaceae. The fungus resides for much time of its life cycle in the extracellular fluid of the vascular system, where it cannot be controlled by conventional fungicides. To obtain insights into the biology of VL-plant interaction in the apoplast, the secretome consisting of the extracellular proteome and metabolome as well as cell wall properties were studied in the model Brassicaceae, Arabidopsis thaliana. VL infection resulted in increased production of cell wall material with an altered composition of carbohydrate polymers and increased lignification. The abundance of several hundred soluble metabolites changed in the apoplast of VL-infected plants including signalling and defence compounds such as glycosides of salicylic acid, lignans and dihydroxybenzoic acid as well as oxylipins. The extracellular proteome of healthy leaves was enriched in antifungal proteins. VL caused specific increases in six apoplast proteins (three peroxidases PRX52, PRX34, P37, serine carboxypeptidase SCPL20, α-galactosidase AGAL2 and a germin-like protein GLP3), which have functions in defence and cell wall modification. The abundance of a lectin-like, chitin-inducible protein (CILLP) was reduced. Since the transcript levels of most of the induced proteins were not elevated until late infection time points (>20 dpi), whereas those of CILLP and GLP3 were reduced at earlier time points, our results may suggest that VL enhances its virulence by rapid down-regulation and delay of induction of plant defence genes.

Oxidation of dehydroascorbic acid and 2,3-diketogulonate under plant apoplastic conditions

The rate of l-ascorbate catabolism in plants often correlates positively with the rate of cell expansion. The reason for this correlation is difficult to explore because of our incomplete knowledge of ascorbate catabolism pathways. These involve enzymic and/or non-enzymic oxidation to dehydroascorbic acid (DHA), which may then be hydrolysed to 2,3-diketogulonate (DKG). Both DHA and DKG were susceptible to further oxidation under conditions of pH and H2O2 concentration comparable with the plant apoplast. The kinetics of their oxidation and the identity of some of the products have been investigated here. DHA, whether added in pure form or generated in situ by ascorbate oxidation, was oxidised non-enzymically to yield, almost simultaneously, a monoanion (cyclic-oxalyl-threonate; cOxT) and a dianion (oxalyl-threonate; OxT). The monoanion was resistant to periodate oxidation, showing that it was not oxalic threonic anhydride. The OxT population was shown to be an interconverting mixture of 3-OxT and 4-OxT, differing in pKa. The 3-OxT appeared to be formed earlier than 4-OxT, but the latter predominated at equilibrium. DKG was oxidised by H2O2 to two partially characterised products, one of which was itself further oxidised by H2O2 to yield threonate. The possible occurrence of these reactions in the apoplast in vivo and the biological roles of vitamin C catabolites are discussed.

Comparative evaluation of extraction methods for apoplastic proteins from maize leaves

Proteins in the plant apoplast are essential for many physiological processes. We have analysed and compared six different infiltration solutions for proteins contained in the apoplast to recognize the most suitable method for leaves and to establish proteome maps for each extraction. The efficiency of protocols was evaluated by comparing the protein patterns resolved by 1-DE and 2-DE, and revealed distinct characteristics for each infiltration solution. Nano-LC-ESI-Q-TOF MS analysis of all fractions was applied to cover all proteins differentially extracted by infiltration solutions and led to the identification of 328 proteins in total in apoplast preparations. The predicted subcellular protein localisation distinguished the examined infiltration solutions in those with high or low amounts of intracellular protein contaminations, and with high or low quantities of secreted proteins. All tested infiltration solution extracted different subsets of proteins, and those implications on apoplast-specific studies are discussed.

Investigation of a His‐rich arabinogalactan‐protein for micronutrient biofortification of cereal grain

The micronutrient content of most cereal grains is low and responsible for malnutrition deficiencies in millions of people who rely on grains as their primary food source. Any strategy that can increase the micronutrient content of grain will have significant benefits to world health. We identified a gene from barley encoding a cell wall protein with multiple histidine (His)-rich motifs interspersed with short arabinogalactan-protein (AGP) domains and have called it Hordeum vulgare His-rich AGP (HvHRA1). Sequence analysis shows that His-rich AGPs are rare in plants and that the number of His-rich and AGP domains differ between cereals and dicots. The barley and wheat encoded proteins have more than 13 His-rich domains, whereas the putative rice orthologue has only 5 His-rich regions. His-rich motifs are well-established metal-binding motifs; therefore, we developed transgenic (Tx) rice plants that constitutively overexpress barley HvHRA1. There was no significant effect on plant growth or grain yield in Tx plants. Purification of AGPs from wild-type and Tx plants showed that only Tx plants contained detectable levels of a His-rich AGP. Calcein assay shows that the AGP fraction from Tx plants had increased binding affinity for Cu(2+) . Micronutrient analysis of brown and white rice showed that the grain nutrient yield for Fe, Zn and Cu was higher in two Tx lines compared to their respective nulls, although the differences were not statistically significant. This approach highlights the potential of the plant apoplast (cell wall) for storage of key nutrients through overexpression of genes for metal-binding proteins.

Identification of NaCl stress-responsive apoplastic proteins in rice shoot stems by 2D-DIGE

Plants have evolved sophisticated systems to cope with adverse environmental conditions such as cold, drought, and salinity. Although a number of stress response networks have been proposed, the role of plant apoplast in plant stress response has been ignored. To investigate the role of apoplastic proteins in the salt stress response, 10-day old rice plants were treated with 200mM NaCl for 1, 6 or 12h, and the soluble apoplast proteins of rice shoot stems were extracted for differential analysis, compared with untreated controls, by 2-D DIGE saturation labeling techniques. One hundred twenty-two significantly changed spots were identified by LC-MS/MS, and 117 spots representing 69 proteins have been identified. Of these proteins, 37 are apoplastic proteins according to the bioinformatic analysis. These proteins are mainly involved in the processes of carbohydrate metabolism, oxido-reduction, and protein processing and degradation. According to their functional categories and cluster analysis, a stress response model of apoplastic proteins has been proposed. These data indicate that the apoplast is important in plant stress signal reception and response.

The hrpZ Gene of Pseudomonas syringae pv. phaseolicola Enhances Resistance to Rhizomania Disease in Transgenic Nicotiana benthamiana and Sugar Beet

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZPsph). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZPsph was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZPsph showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZPsph developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.

Carbon-use efficiency in green sinks is increased when a blend of apoplastic fructose and glucose is available for uptake.

Understanding how green sink strength is regulated in planta poses a difficult problem because non-structural carbohydrate (NSC) levels can have integrated, simultaneous feedback effects on photosynthesis, sugar uptake, and respiration that depend on specific NSC moieties. Photosynthetic gametophytes of the fern Ceratopteris richardii provide a simple land plant model to assess how different NSCs imported from the apoplast of intact plants affect green sink strength. Sink strength was quantified as the amount of exogenous sugar that plants grown in low light depleted from their liquid media, and the relative contributions of carbon assimilation by photosynthesis and sugar uptake was estimated from stable isotope analysis of plant dry mass. Gametophytes absorbed fructose and glucose with equal affinity when cultured on either hexose alone, or in the presence of an equimolar blend of both sugars. Plants also depleted sucrose from the surrounding media, although a portion of this disaccharide that was hydrolysed into fructose and glucose by putative cell wall invertase activity remained in the media. The δ13C in plant dry masses harvested from sugar treatments were all close to –18‰, indicating that 25–39% of total plant carbon was from C3 photosynthesis (δ13C=–29‰) and 61–75% was from uptake of exogenous sugars (δ13C=–11‰). Carbon-use efficiency (i.e. carbon accumulated/carbon depleted) was significantly improved when plants had a blend of exogenous sugars available compared with plants grown in a single hexose alone. Plants avoided complete down-regulation of photosynthesis even though a large excess of exogenous carbon fluxed through their cells.

The quest for alternatives to microbial cellulase mix production: corn stover-produced heterologous multi-cellulases readily deconstruct lignocellulosic biomass into fermentable sugars

BACKGROUND: Production of cellulosic ethanol is still expensive compared with corn (maize) grain ethanol due to the high costs of bulk production of microbial cellulases. At least three cellulases including endo-cellulase, exo-cellulase and cellobiase are needed to convert cellulosic biomass into fermentable sugars. All these cellulases could be self-produced within cells of transgenic bio-energy crops. The production of heterologous Acidothermus cellulolyticus (E1) endo-cellulase in endoplasmic reticulum and mitochondria of green tissues of transgenic corn plants was recently reported, and it was confirmed that the heterologous E1 converts cellulose into fermentable sugars. RESULTS: Biologically active A. cellulolyticus E1, Trichoderma reesei 1,4-β-cellobiohydrolases I (CBH I) exo-cellulase and bovine rumen Butyrivibrio fibrisolvens cellobiase were expressed in corn plant endoplasmic reticulum (ER), apoplast (cell wall areas) and vacuole respectively. Results show that the ratio 1:4:1 (E1:CBH I:cellobiase) of crude heterologous cellulases is ideal for converting ammonia fiber explosion (AFEX) pretreated corn stover into fermentable sugars. CONCLUSIONS: Corn plants that express all three biologically active heterologous cellulases within their cellulosic biomass to facilitate conversion of pretreated corn stover into fermentable sugars is a step forward in the quest for alternatives to the present microbial cellulase mix production for cellulosic biofuels. Copyright © 2011 Society of Chemical Industry

Comparative Proteomics Analysis of the Root Apoplasts of Rice Seedlings in Response to Hydrogen Peroxide

BackgroundPlant apoplast is the prime site for signal perception and defense response, and of great importance in responding to environmental stresses. Hydrogen peroxide (H2O2) plays a pivotal role in determining the responsiveness of cells to stress. However, how the apoplast proteome changes under oxidative condition is largely unknown. In this study, we initiated a comparative proteomic analysis to explore H2O2-responsive proteins in the apoplast of rice seedling roots.Methodology/Principal Findings14-day-old rice seedlings were treated with low concentrations (300 and 600 µM) of H2O2 for 6 h and the levels of relative electrolyte leakage, malondialdehyde and H2O2 were assayed in roots. The modified vacuum infiltration method was used to extract apoplast proteins of rice seedling roots, and then two-dimensional electrophoresis gel analysis revealed 58 differentially expressed protein spots under low H2O2 conditions. Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H2O2-responsive proteins. Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs. These proteins identified are involved in a variety of processes, including redox homeostasis, cell wall modification, signal transduction, cell defense and carbohydrate metabolism, indicating a complex regulative network in the apoplast of seedling roots under H2O2 stress.Conclusions/SignificanceThe present study is the first apoplast proteome investigation of plant seedlings in response to H2O2 and may be of paramount importance for the understanding of the plant network to environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H2O2 response.

An efficient petiole-feeding bioassay for introducing aqueous solutions into dicotyledonous plants

Introducing bioactive molecules into plants helps establish their roles in plant growth and development. Here we describe a simple and effective petiole-feeding protocol to introduce aqueous solutions into the vascular stream and apoplast of dicotyledonous plants. This 'intravenous feeding' procedure has wide applicability to plant physiology, specifically with regard to the analysis of source-sink allocations, long-distance signaling, hormone biology and overall plant development. In comparison with existing methods, this technique allows the continuous feeding of aqueous solutions into plants without the need for constant monitoring. Findings are provided from experiments using soybean plants fed with a range of aqueous solutions containing tracer dyes, small metabolites, radiolabeled chemicals and biologically active plant extracts controlling nodulation. Typically, feeding experiments consist of (i) generating samples to feed (extracts, solutions and so on); (ii) growing recipient plants; (iii) setting up the feeding apparatus; and (iv) feeding sample solutions into the recipient plants. When the plants are ready, the feeding procedure can take 1-3 h to set up depending on the size of experiment (not including preparation of materials). The petiole-feeding technique also works with other plant species, including tomato, chili pepper and cabbage plants, as demonstrated here.

HvCEBiP, a gene homologous to rice chitin receptor CEBiP, contributes to basal resistance of barley to Magnaporthe oryzae

BackgroundRice CEBiP recognizes chitin oligosaccharides on the fungal cell surface or released into the plant apoplast, leading to the expression of plant disease resistance against fungal infection. However, it has not yet been reported whether CEBiP is actually required for restricting the growth of fungal pathogens. Here we evaluated the involvement of a putative chitin receptor gene in the basal resistance of barley to the ssd1 mutant of Magnaporthe oryzae, which induces multiple host defense responses.ResultsThe mossd1 mutant showed attenuated pathogenicity on barley and appressorial penetration was restricted by the formation of callose papillae at attempted entry sites. When conidial suspensions of mossd1 mutant were spotted onto the leaves of HvCEBiP-silenced plants, small brown necrotic flecks or blast lesions were produced but these lesions did not expand beyond the inoculation site. Wild-type M. oryzae also produced slightly more severe symptoms on the leaves of HvCEBiP-silenced plants. Cytological observation revealed that these lesions resulted from appressorium-mediated penetration into plant epidermal cells.ConclusionsThese results suggest that HvCEBiP is involved in basal resistance against appressorium-mediated infection and that basal resistance might be triggered by the recognition of chitin oligosaccharides derived from M. oryzae.

Functional characterization of the Xcs and Xps type II secretion systems from the plant pathogenic bacterium Xanthomonas campestris pv vesicatoria

*Type II secretion (T2S) systems of many plant-pathogenic bacteria often secrete cell wall-degrading enzymes into the plant apoplast. *Here, we show that the Xps-T2S system from the plant pathogen Xanthomonas campestris pv vesicatoria (Xcv) promotes disease and contributes to the translocation of effector proteins that are delivered into the plant cell by the type III secretion (T3S) system. *The Xcs-T2S system instead lacks an obvious virulence function. However, individual xcs genes can partially complement mutants in homologous xps genes, indicating that they encode functional components of T2S systems. Enzyme activity assays showed that the Xps system contributes to secretion of proteases and xylanases. We identified the virulence-associated xylanase XynC as a substrate of the Xps system. However, homologs of known T2S substrates from other Xanthomonas spp. are not secreted by the T2S systems from Xcv. Thus, T2S systems from Xanthomonas spp. appear to differ significantly in their substrate specificities. *Transcript analyses revealed that expression of xps genes in Xcv is activated by HrpG and HrpX, key regulators of the T3S system. By contrast, expression of xynC and extracellular protease and xylanase activities are repressed by HrpG and HrpX, suggesting that components and substrates of the Xps system are differentially regulated.

A Prominent Role of the Flagellin Receptor FLAGELLIN-SENSING2 in Mediating Stomatal Response to Pseudomonas syringae pv tomato DC3000 in Arabidopsis

The FLAGELLIN-SENSING2 (FLS2) receptor kinase recognizes bacterial flagellin and initiates a battery of downstream defense responses to reduce bacterial invasion through stomata in the epidermis and bacterial multiplication in the apoplast of infected plants. Recent studies have shown that during Pseudomonas syringae pv tomato (Pst) DC3000 infection of Arabidopsis (Arabidopsis thaliana), FLS2-mediated immunity is actively suppressed by effector proteins (such as AvrPto and AvrPtoB) secreted through the bacterial type III secretion system (T3SS). We provide evidence here that T3SS effector-based suppression does not appear to be sufficient to overcome FLS2-based immunity during Pst DC3000 infection, but that the phytotoxin coronatine (COR) produced by Pst DC3000 also plays a critical role. COR-deficient mutants of Pst DC3000 are severely reduced in virulence when inoculated onto the leaf surface of wild-type Columbia-0 plants, but this defect was rescued almost fully in fls2 mutant plants. Although bacteria are thought to carry multiple microbe-associated molecular patterns, stomata of fls2 plants are completely unresponsive to COR-deficient mutant Pst DC3000 bacteria. The responses of fls2 plants were similar to those of the Arabidopsis G-protein alpha subunit1-3 mutant, which is defective in abscisic acid-regulated stomatal closure, but were distinct from those of the Arabidopsis non-expressor of PR genes1 mutant, which is defective in salicylic acid-dependent stomatal closure and apoplast defense. Epistasis analyses show that salicylic acid signaling acts upstream of abscisic acid signaling in bacterium-triggered stomatal closure. Taken together, these results suggest a particularly important role of FLS2-mediated resistance to COR-deficient mutant Pst DC3000 bacteria, and nonredundant roles of COR and T3SS effector proteins in the suppression of FLS2-mediated resistance in the Arabidopsis-Pst DC3000 interaction.

Effectors, effectors et toujours des effectors

Effectors, effectors <i>et toujours des</i> effectors

A novel high-affinity sucrose transporter is required for virulence of the plant pathogen Ustilago maydis.

Author SummaryThe plant parasitic fungus Ustilago maydis is a biotrophic pathogen that depends on live plant tissue for development. It is highly adapted to maize (Zea mays), where it causes the corn smut disease. Fungal cells growing within the plant apoplast are surrounded by the host plasma membrane at all growth stages, thereby establishing tight interaction zones with the host cells that assure optimal access to host-derived nutrients, including organic carbon sources. Here, we focus on the previously unknown feeding mechanisms of this plant pathogen within its host plant. We identified a fungal plasma membrane transporter, Srt1, that is expressed exclusively after plant infection and that turns out to be essential for virulence development of Ustilago in infected plants. Srt1 is the first characterized fungal transporter that allows direct utilization of sucrose without extracellular hydrolysis into monosaccharides, the carbon form more commonly taken up by pathogenic fungi. It is highly specific for sucrose, and its affinity largely exceeds that of equivalent plant transporters. This not only provides advantages for the carbon acquisition by the pathogen, but quite likely also offers a mechanism to prevent induction of plant defense responses known to occur upon apoplastic sucrose hydrolysis.

Expression profiling of a complex thaumatin-like protein family in western white pine

The protein content in the plant apoplast is believed to change dramatically as a result of host defense response upon infection with various pathogens. In this study, six novel thaumatin-like proteins (TLPs) were identified in western white pine (Pinus monticola) needle apoplast by a proteomic strategy using two-dimensional protein electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sequent cDNA cloning found that ten P. monticola TLP genes (PmTLP-L1 to -L6 and -S1 to -S4) were expressed in various tissues. Phylogenetic analysis demonstrated that these PmTLP genes belong to a large, complex, and highly diverse plant TLP family. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) using gene-specific primer pairs showed that each PmTLP gene exhibited a characteristic pattern of mRNA expression based on their unique organ distribution, seasonal regulation, and response to abiotic and biotic stresses. A time-course analysis at the early stages of infection by white pine blister rust pathogen Cronartium ribicola revealed that a coordinated upregulation of multiple PmTLP genes was involved in P. monticola major gene (Cr2) resistance. The structural and expressional differentiations suggest that the PmTLP family may contribute to host defense as well as other mechanism.

Bacterial chemoattraction towards jasmonate plays a role in the entry of Dickeya dadantii through wounded tissues.

Jasmonate is a key signalling compound in plant defence that is synthesized in wounded tissues. In this work, we have found that this molecule is also a strong chemoattractant for the phythopathogenic bacteria Dickeya dadantii (ex-Erwinia chysanthemi). Jasmonic acid induced the expression of a subset of bacterial genes possibly involved in virulence/survival in the plant apoplast and bacterial cells pre-treated with jasmonate showed increased virulence in chicory and Saintpaulia leaves. We also showed that tissue wounding induced bacterial spread through the leaf surface. Moreover, the jasmonate-deficient aos1 Arabidopsis thaliana mutant was more resistant to bacterial invasion by D. dadantii than wild-type plants. These results are consistent with the hypothesis that sensing jasmonic acid by this bacterium helps the pathogen to ingress inside plant tissues.