Pseudomonas Syringae Pv Tomato DC3000 Research Articles

NH4+ protects tomato plants against Pseudomonas syringae by activation of systemic acquired acclimation.

NH4 (+) nutrition provokes mild toxicity by enhancing H2O2 accumulation, which acts as a signal activating systemic acquired acclimation (SAA). Until now, induced resistance mechanisms in response to an abiotic stimulus and related to SAA were only reported for exposure to a subsequent abiotic stress. Herein, the first evidence is provided that this acclimation to an abiotic stimulus induces resistance to later pathogen infection, since NH4 (+) nutrition (N-NH4 (+))-induced resistance (NH4 (+)-IR) against Pseudomonas syringae pv tomato DC3000 (Pst) in tomato plants was demonstrated. N-NH4 (+) plants displayed basal H2O2, abscisic acid (ABA), and putrescine (Put) accumulation. H2O2 accumulation acted as a signal to induce ABA-dependent signalling pathways required to prevent NH4 (+) toxicity. This acclimatory event provoked an increase in resistance against later pathogen infection. N-NH4 (+) plants displayed basal stomatal closure produced by H2O2 derived from enhanced CuAO and rboh1 activity that may reduce the entry of bacteria into the mesophyll, diminishing the disease symptoms as well as strongly inducing the oxidative burst upon Pst infection, favouring NH4 (+)-IR. Experiments with inhibitors of Put accumulation and the ABA-deficient mutant flacca demonstrated that Put and ABA downstream signalling pathways are required to complete NH4 (+)-IR. The metabolic profile revealed that infected N-NH4 (+) plants showed greater ferulic acid accumulation compared with control plants. Although classical salicylic acid (SA)-dependent responses against biotrophic pathogens were not found, the important role of Put in the resistance of tomato against Pst was demonstrated. Moreover, this work revealed the cross-talk between abiotic stress acclimation (NH4 (+) nutrition) and resistance to subsequent Pst infection.

Pseudomonas syringae Effector Avirulence Protein E Localizes to the Host Plasma Membrane and Down-Regulates the Expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 Gene Required for Antibacterial Immunity in Arabidopsis.

Many bacterial pathogens of plants and animals deliver effector proteins into host cells to promote infection. Elucidation of how pathogen effector proteins function not only is critical for understanding bacterial pathogenesis but also provides a useful tool in discovering the functions of host genes. In this study, we characterized the Pseudomonas syringae pv tomato DC3000 effector protein Avirulence Protein E (AvrE), the founding member of a widely distributed, yet functionally enigmatic, bacterial effector family. We show that AvrE is localized in the plasma membrane (PM) and PM-associated vesicle-like structures in the plant cell. AvrE contains two physically interacting domains, and the amino-terminal portion contains a PM-localization signal. Genome-wide microarray analysis indicates that AvrE, as well as the functionally redundant effector Hypersensitive response and pathogenicity-dependent Outer Protein M1, down-regulates the expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13) gene in Arabidopsis (Arabidopsis thaliana). Mutational analysis shows that NHL13 is required for plant immunity, as the nhl13 mutant plant displayed enhanced disease susceptibility. Our results defined the action site of one of the most important bacterial virulence proteins in plants and the antibacterial immunity function of the NHL13 gene.

An Overdose of the Arabidopsis Coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 or Its Ectodomain Causes Autoimmunity in a SUPPRESSOR OF BIR1-1-Dependent Manner.

The membrane-bound Brassinosteroid insensitive1-associated receptor kinase1 (BAK1) is a common coreceptor in plants and regulates distinct cellular programs ranging from growth and development to defense against pathogens. BAK1 functions through binding to ligand-stimulated transmembrane receptors and activating their kinase domains via transphosphorylation. In the absence of microbes, BAK1 activity may be suppressed by different mechanisms, like interaction with the regulatory BIR (for BAK1-interacting receptor-like kinase) proteins. Here, we demonstrated that BAK1 overexpression in Arabidopsis (Arabidopsis thaliana) could cause detrimental effects on plant development, including growth arrest, leaf necrosis, and reduced seed production. Further analysis using an inducible expression system showed that BAK1 accumulation quickly stimulated immune responses, even under axenic conditions, and led to increased resistance to pathogenic Pseudomonas syringae pv tomato DC3000. Intriguingly, our study also revealed that the plasma membrane-associated BAK1 ectodomain was sufficient to induce autoimmunity, indicating a novel mode of action for BAK1 in immunity control. We postulate that an excess of BAK1 or its ectodomain could trigger immune receptor activation in the absence of microbes through unbalancing regulatory interactions, including those with BIRs. Consistently, mutation of suppressor of BIR1-1, which encodes an emerging positive regulator of transmembrane receptors in plants, suppressed the effects of BAK1 overexpression. In conclusion, our findings unravel a new role for the BAK1 ectodomain in the tight regulation of Arabidopsis immune receptors necessary to avoid inappropriate activation of immunity.

Cytoplastic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in Nicotiana benthamiana.

Autophagy as a conserved catabolic pathway can respond to reactive oxygen species (ROS) and plays an important role in degrading oxidized proteins in plants under various stress conditions. However, how ROS regulates autophagy in response to oxidative stresses is largely unknown. Here, we show that autophagy-related protein 3 (ATG3) interacts with the cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs) to regulate autophagy in Nicotiana benthamiana plants. We found that oxidative stress inhibits the interaction of ATG3 with GAPCs. Silencing of GAPCs significantly activates ATG3-dependent autophagy, while overexpression of GAPCs suppresses autophagy in N. benthamiana plants. Moreover, silencing of GAPCs enhances N gene-mediated cell death and plant resistance against both incompatible pathogens Tobacco mosaic virus and Pseudomonas syringae pv tomato DC3000, as well as compatible pathogen P. syringae pv tabaci. These results indicate that GAPCs have multiple functions in the regulation of autophagy, hypersensitive response, and plant innate immunity.

Bacillus spp. from rainforest soil promote plant growth under limited nitrogen conditions.

The aim of this study was to evaluate effects of PGPR (plant growth-promoting rhizobacteria) isolated from rainforest soil on different plants under limited nitrogen conditions. Bacterial isolates from a Peruvian rainforest soil were screened for plant growth-promoting effects on Arabidopsis (Col-0). Four selected isolates including one Bacillus subtilis, two B. atrophaeus and one B.pumilus significantly promoted growth of Zea mays L. and Solanum lycopersicum under greenhouse conditions. Moreover, the PGPRs significantly promoted growth of S.lycopersicum in both low and nitrogen-amended soil conditions. These PGPR strains were further studied to obtain insights into possible mechanisms of plant growth promotion. Volatile chemicals from those isolates promoted Arabidopsis growth, and the expression of genes related to IAA production was induced in the Arabidopsis plants treated with PGPRs. Further, selected PGPR strains triggered induced systemic resistance (ISR) against Pseudomonas syringae pv tomato DC3000 in Arabidopsis. PGPR strains isolated from the rainforest soil promoted the plant growth of Arabidopsis, corn and tomato. New PGPR that have wider adaptability to different crops, soils and environmental conditions are needed to decrease our reliance on agricultural amendments derived from fossil-based fuels. The PGPRs isolated from a nonagricultural site constitute new plant growth-promoting strains that could be developed for agricultural uses.

The Arabidopsis transcription factor BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 is a direct substrate of MITOGEN-ACTIVATED PROTEIN KINASE6 and regulates immunity.

Pathogen-associated molecular patterns (PAMPs) are recognized by plant pattern recognition receptors to activate PAMP-triggered immunity (PTI). Mitogen-activated protein kinases (MAPKs), as well as other cytoplasmic kinases, integrate upstream immune signals and, in turn, dissect PTI signaling via different substrates to regulate defense responses. However, only a few direct substrates of these signaling kinases have been identified. Here, we show that PAMP perception enhances phosphorylation of BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1), a transcription factor involved in brassinosteroid (BR) signaling pathway, through pathogen-induced MAPKs in Arabidopsis (Arabidopsis thaliana). BES1 interacts with MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and is phosphorylated by MPK6. bes1 loss-of-function mutants display compromised resistance to bacterial pathogen Pseudomonas syringae pv tomato DC3000. BES1 S286A/S137A double mutation (BES1(SSAA)) impairs PAMP-induced phosphorylation and fails to restore bacterial resistance in bes1 mutant, indicating a positive role of BES1 phosphorylation in plant immunity. BES1 is phosphorylated by glycogen synthase kinase3 (GSK3)-like kinase BR-insensitive2 (BIN2), a negative regulator of BR signaling. BR perception inhibits BIN2 activity, allowing dephosphorylation of BES1 to regulate plant development. However, BES1(SSAA) does not affect BR-mediated plant growth, suggesting differential residue requirements for the modulation of BES1 phosphorylation in PTI and BR signaling. Our study identifies BES1 as a unique direct substrate of MPK6 in PTI signaling. This finding reveals MAPK-mediated BES1 phosphorylation as another BES1 modulation mechanism in plant cell signaling, in addition to GSK3-like kinase-mediated BES1 phosphorylation and F box protein-mediated BES1 degradation.

Activation tagging of ATHB13 in Arabidopsis thaliana confers broad-spectrum disease resistance.

Powdery mildew species Oidium neolycopersici (On) can cause serious yield losses in tomato production worldwide. Besides on tomato, On is able to grow and reproduce on Arabidopsis. In this study we screened a collection of activation-tagged Arabidopsis mutants and identified one mutant, 3221, which displayed resistance to On, and in addition showed a reduced stature and serrated leaves. Additional disease tests demonstrated that the 3221 mutant exhibited resistance to downy mildew (Hyaloperonospora arabidopsidis) and green peach aphid (Myzus persicae), but retained susceptibility to bacterial pathogen Pseudomonas syringae pv tomato DC3000. The resistance trait and morphological alteration were mutually linked in 3221. Identification of the activation tag insertion site and microarray analysis revealed that ATHB13, a homeodomain-leucine zipper (HD-Zip) transcription factor, was constitutively overexpressed in 3221. Silencing of ATHB13 in 3221 resulted in the loss of both the morphological alteration and resistance, whereas overexpression of the cloned ATHB13 in Col-0 and Col-eds1-2 backgrounds resulted in morphological alteration and resistance. Microarray analysis further revealed that overexpression of ATHB13 influenced the expression of a large number of genes. Previously, it was reported that ATHB13-overexpressing lines conferred tolerance to abiotic stress. Together with our results, it appears that ATHB13 is involved in the crosstalk between abiotic and biotic stress resistance pathways.

ATP-dependent binding cassette transporter G family member 16 increases plant tolerance to abscisic acid and assists in basal resistance against Pseudomonas syringae DC3000.

Plants have been shown previously to perceive bacteria on the leaf surface and respond by closing their stomata. The virulent bacterial pathogen Pseudomonas syringae pv tomato DC3000 (PstDC3000) responds by secreting a virulence factor, coronatine, which blocks the functioning of guard cells and forces stomata to reopen. After it is inside the leaf, PstDC3000 has been shown to up-regulate abscisic acid (ABA) signaling and thereby suppress salicylic acid-dependent resistance. Some wild plants exhibit resistance to PstDC3000, but the mechanisms by which they achieve this resistance remain unknown. Here, we used genome-wide association mapping to identify an ATP-dependent binding cassette transporter gene (ATP-dependent binding cassette transporter G family member16) in Arabidopsis (Arabidopsis thaliana) that contributes to wild plant resistance to PstDC3000. Through microarray analysis and β-glucuronidase reporter lines, we showed that the gene is up-regulated by ABA, bacterial infection, and coronatine. We also used a green fluorescent protein fusion protein and found that transporter is more likely to localize on plasma membranes than in cell walls. Transferred DNA insertion lines exhibited consistent defective tolerance of exogenous ABA and reduced resistance to infection by PstDC3000. Our conclusion is that ATP-dependent binding cassette transporter G family member16 is involved in ABA tolerance and contributes to plant resistance against PstDC3000. This is one of the first examples, to our knowledge, of ATP-dependent binding cassette transporter involvement in plant resistance to infection by a bacterial pathogen. It also suggests a possible mechanism by which plants reduce the deleterious effects of ABA hijacking during pathogen attack. Collectively, these results improve our understanding of basal resistance in Arabidopsis and offer unique ABA-related targets for improving the innate resistance of plants to bacterial infection.

Role of two UDP-Glycosyltransferases from the L group of arabidopsis in resistance against pseudomonas syringae

The role of the salicylic acid (SA) glycosides SA 2-O-β-D-glucose (SAG), SA glucose ester (SGE) and the glycosyl transferases UGT74F1 and UGT74F2 in the establishment of basal resistance of Arabidopsis against Pseudomonas syringae pv tomato DC3000 (Pst) was investigated. Both mutants altered in the corresponding glycosyl transferases (ugt74f1 and ugt74f2) were affected in their basal resistance against Pst. The mutant ugt74f1 showed enhanced susceptibility, while ugt74f2 showed enhanced resistance against the same pathogen. Both mutants have to some extent, altered levels of SAG and SGE compared to wild type plants, however, in response to the infection, ugt74f2 accumulated higher levels of free SA until 24 hpi compared to wild type plants while ugt74f1 accumulated lower SA levels. These SA levels correlated well with reduced expression in PR1 and EDS1 in ugt74f1. In contrast, ugt74f2 has enhanced expression of Enhanced Disease Susceptibility 1 (EDS1) but a strong reduction in the expression of several jasmonate (JA)-dependent genes. Bacterial infection interfered with the expression of Fatty Acid Desaturase (FAD), Lipoxygenase2 (LOX2), carboxyl methyltransferase1 (BSMT1) and 9-cis-epoxycarotenoid dioxygenase (NCED3) genes in ugt74f1, thus promoting an antagonistic effect with SA-signalling and leading to enhanced bacterial growth. UGT74F2 might be a target for bacterial effectors since bacterial mutants affected in effector synthesis were impaired in inducing UGT74F2 expression. These results suggest that UGT74F2 negatively influences the accumulation of free SA, hence leading to an increased susceptibility due to reduced SA levels and increased expression of the JA and ABA markers LOX-2, FAD and NCED-3.

Scavenging Iron: A Novel Mechanism of Plant Immunity Activation by Microbial Siderophores

Siderophores are specific ferric iron chelators synthesized by virtually all microorganisms in response to iron deficiency. We have previously shown that they promote infection by the phytopathogenic enterobacteria Dickeya dadantii and Erwinia amylovora. Siderophores also have the ability to activate plant immunity. We have used complete Arabidopsis transcriptome microarrays to investigate the global transcriptional modifications in roots and leaves of Arabidopsis (Arabidopsis thaliana) plants after leaf treatment with the siderophore deferrioxamine (DFO). Physiological relevance of these transcriptional modifications was validated experimentally. Immunity and heavy-metal homeostasis were the major processes affected by DFO. These two physiological responses could be activated by a synthetic iron chelator ethylenediamine-di(o-hydroxyphenylacetic) acid, indicating that siderophores eliciting activities rely on their strong iron-chelating capacity. DFO was able to protect Arabidopsis against the pathogenic bacterium Pseudomonas syringae pv tomato DC3000. Siderophore treatment caused local modifications of iron distribution in leaf cells visible by ferrocyanide and diaminobenzidine-H₂O₂ staining. Metal quantifications showed that DFO causes a transient iron and zinc uptake at the root level, which is presumably mediated by the metal transporter iron regulated transporter1 (IRT1). Defense gene expression and callose deposition in response to DFO were compromised in an irt1 mutant. Consistently, plant susceptibility to D. dadantii was increased in the irt1 mutant. Our work shows that iron scavenging is a unique mechanism of immunity activation in plants. It highlights the strong relationship between heavy-metal homeostasis and immunity.

Circadian regulation of plant immunity to pathogens.

The plant circadian clock primes the immune response of Arabidopsis thaliana to infection with the bacterial pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000) such that there is a more robust response during the subjective day than subjective night. Thus Pst DC3000 growth in plants infected with the same initial titre of bacteria varies depending on the time of day of infection (Bhardwaj et al., PLoS One 6: e26968, 2011; Zhang et al., PLoS Pathog 9:e1003370, 2013). We describe here a protocol for assaying bacterial leaf titres following pressure infiltration or spray inoculation of Arabidopsis thaliana with Pst DC3000. We also describe a method for assaying plant susceptibility to infection with the necrotrophic fungal pathogen Botrytis cinerea. These methods can be used in studying the circadian clock regulation of signal transduction pathways controlling plant defense responses.

Light regulates motility, attachment and virulence in the plant pathogen Pseudomonas syringae pv tomato DC3000

Pseudomonas syringae pv tomato DC3000 (Pto) is the causal agent of the bacterial speck of tomato, which leads to significant economic losses in this crop. Pto inhabits the tomato phyllosphere, where the pathogen is highly exposed to light, among other environmental factors. Light represents a stressful condition and acts as a source of information associated with different plant defence levels. Here, we analysed the presence of both blue and red light photoreceptors in a group of Pseudomonas. In addition, we studied the effect of white, blue and red light on Pto features related to epiphytic fitness. While white and blue light inhibit motility, bacterial attachment to plant leaves is promoted. Moreover, these phenotypes are altered in a blue-light receptor mutant. These light-controlled changes during the epiphytic stage cause a reduction in virulence, highlighting the relevance of motility during the entry process to the plant apoplast. This study demonstrated the key role of light perception in the Pto phenotype switching and its effect on virulence.

The Chinese wild grapevine (Vitis pseudoreticulata) E3 ubiquitin ligase Erysiphe necator‐induced RING finger protein 1 (EIRP1) activates plant defense responses by inducing proteolysis of the VpWRKY11 transcription factor

Ubiquitin-mediated regulation responds rapidly to specific stimuli; this rapidity is particularly important for defense responses to pathogen attack. Here, we investigated the role of the E3 ubiquitin ligase Erysiphe necator-induced RING finger protein 1 (EIRP1) in the defense response of Chinese wild grapevine Vitis pseudoreticulata. The regulatory function of E3 ubiquitin ligase EIRP1 was investigated using molecular, genetic and biochemical approaches. EIRP1 encodes a C3HC4-type Really Interesting New Gene (RING) finger protein that harbors E3 ligase activity. This activity requires the conserved RING domain, and VpWRKY11 also interacts with EIRP1 through the RING domain. VpWRKY11 localizes to the nucleus and activates W-box-dependent transcription in planta. EIRP1 targeted VpWRKY11 in vivo, resulting in VpWRKY11 degradation. The expression of EIRP1 and VpWRKY11 responds rapidly to powdery mildew in Vitis pseudoreticulata grapevine; also, overexpression of EIRP1 in Arabidopsis confers enhanced resistance to the pathogens Golovinomyces cichoracearum and Pseudomonas syringae pv tomato DC3000. Our data suggest that the EIRP1 E3 ligase positively regulates plant disease resistance by mediating proteolysis of the negative regulator VpWRKY11 via degradation by the 26S proteasome.

Overexpression of a moso bamboo (Phyllostachys edulis) transcription factor gene PheWRKY1 enhances disease resistance in transgenic Arabidopsis thaliana

Members of plant WRKY transcription factor families are implicated in defense responses and various other physiological processes. To study the function of WRKY transcription factors in moso bamboo (Phyllostachys edulis (Carrière) J. Houz.), a PheWRKY1 gene (GenBank accession number: GU944762) was cloned by reverse transcription polymerase chain reaction. An overexpression vector for this gene was constructed and transformed into Arabidopsis thaliana (L.) Heynh. by an agrobacterium-mediated method. To determine the effect of PheWRKY1 gene overexpression on disease resistance in A. thaliana, the photosynthetic parameters and the expression of pathogenesis-related (PR) genes were examined after Pseudomonas syringae PV Tomato DC3000 (Pst DC3000) infection. Compared to wild-type plants, most PheWRKY1-overexpressing plants did not exhibit spreading maceration to systemic leaves and had a better photosynthetic capacity after Pst DC3000 infection. Furthermore, PheWRKY1-overexpressing plants accumulated significantly more PR1, PR2, PR5, and nonexpressor of PR genes (NPR1) transcripts. These results demonstrated that transgenic Arabidopsis overexpressing PheWRKY1 gene exhibited improved resistance to bacterial infection.

Transient Transcriptional Regulation of theCYS-C1Gene and Cyanide Accumulation upon Pathogen Infection in the Plant Immune Response

Cyanide is produced concomitantly with ethylene biosynthesis. Arabidopsis (Arabidopsis thaliana) detoxifies cyanide primarily through the enzyme β-cyanoalanine synthase, mainly by the mitochondrial CYS-C1. CYS-C1 loss of function is not toxic for the plant and leads to an increased level of cyanide in cys-c1 mutants as well as a root hairless phenotype. The classification of genes differentially expressed in cys-c1 and wild-type plants reveals that the high endogenous cyanide content of the cys-c1 mutant is correlated with the biotic stress response. Cyanide accumulation and CYS-C1 gene expression are negatively correlated during compatible and incompatible plant-bacteria interactions. In addition, cys-c1 plants present an increased susceptibility to the necrotrophic fungus Botrytis cinerea and an increased tolerance to the biotrophic Pseudomonas syringae pv tomato DC3000 bacterium and Beet curly top virus. The cys-c1 mutation produces a reduction in respiration rate in leaves, an accumulation of reactive oxygen species, and an induction of the alternative oxidase AOX1a and pathogenesis-related PR1 expression. We hypothesize that cyanide, which is transiently accumulated during avirulent bacterial infection and constitutively accumulated in the cys-c1 mutant, uncouples the respiratory electron chain dependent on the cytochrome c oxidase, and this uncoupling induces the alternative oxidase activity and the accumulation of reactive oxygen species, which act by stimulating the salicylic acid-dependent signaling pathway of the plant immune system.

Glutamate Receptor-Like Channel3.3 Is Involved in Mediating Glutathione-Triggered Cytosolic Calcium Transients, Transcriptional Changes, and Innate Immunity Responses in Arabidopsis

The tripeptide reduced glutathione (GSH; γ-glutamate [Glu]-cysteine [Cys]-glycine) is a major endogenous antioxidant in both animal and plant cells. It also functions as a neurotransmitter mediating communication among neurons in the central nervous system of animals through modulating specific ionotropic Glu receptors (GLRs) in the membrane. Little is known about such signaling roles in plant cells. Here, we report that transient rises in cytosolic calcium triggered by exogenous GSH in Arabidopsis (Arabidopsis thaliana) leaves were sensitive to GLR antagonists and abolished in loss-of-function atglr3.3 mutants. Like the GSH biosynthesis-defective mutant PHYTOALEXIN DEFICIENT2, atglr3.3 showed enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Pathogen-induced defense marker gene expression was also decreased in atglr3.3 mutants. Twenty-seven percent of genes that were rapidly responsive to GSH treatment of seedlings were defense genes, most of which were dependent on functional AtGLR3.3, while GSH suppressed pathogen propagation through the AtGLR3.3-dependent pathway. Eight previously identified putative AtGLR3.3 ligands, GSH, oxidized glutathione, alanine, asparagine, Cys, Glu, glycine, and serine, all elicited the AtGLR3.3-dependent cytosolic calcium transients, but only GSH and Cys induced the defense response, with the Glu-induced AtGLR3.3-dependent transcription response being much less apparent than that triggered by GSH. Together, these observations suggest that AtGLR3.3 is required for several signaling effects mediated by extracellular GSH, even though these effects may not be causally related.

CrcZ and CrcX regulate carbon source utilization in Pseudomonas syringae pathovar tomato strain DC3000

Small non-coding RNAs (ncRNAs) are important components of many regulatory pathways in bacteria and play key roles in regulating factors important for virulence. Carbon catabolite repression control is modulated by small RNAs (crcZ or crcZ and crcY) in Pseudomonas aeruginosa and Pseudomonas putida. In this study, we demonstrate that expression of crcZ and crcX (formerly designated psr1 and psr2, respectively) is dependent upon RpoN together with the two-component system CbrAB, and is influenced by the carbon source present in the medium in the model plant pathogen Pseudomonas syringae pv tomato DC3000. The distribution of the members of the Crc ncRNA family was also determined by screening available genomic sequences of the Pseudomonads. Interestingly, variable numbers of the Crc family members exist in Pseudomonas genomes. The ncRNAs are comprised of three main subfamilies, named CrcZ, CrcX and CrcY. Most importantly the CrcX subfamily appears to be unique to all P. syringae strains sequenced to date.

Involvement of the Salicylic Acid Signaling Pathway in the Systemic Resistance Induced in Arabidopsis by Plant Growth-Promoting Fungus Fusarium equiseti GF19-1

Plant growth-promoting fungi (PGPF) are effective biocontrol agents for a number of soil-borne diseases and are known for their ability to trigger induced systemic resistance (ISR). In this study, we investigated the mechanisms triggered by PGPF Fusarium equiseti GF19-1, which is known to increase pathogen resistance in plants, by using GF19-1 spores and the culture filtrate (CF) to treat the roots of Arabidopsis thaliana. Subsequently, the leaves were challenged with Pseudomonas syringae pv tomato DC3000 (Pst) bacteria. Arabidopsis plants treated with GF19-1 spores or the CF elicited ISR against the Pst pathogen, resulting in a restriction of disease severity and suppression of pathogen proliferation. Examination of ISR in various signaling mutants and transgenic plants showed that GF19-1-induced protection was observed in the jasmonate response mutant jar1 and the ethylene response mutant etr1, whereas it was blocked in Arabidopsis plants expressing the NahG transgene or demonstrating a disruption of the NPR1 gene (npr1). Analysis of systemic gene expression revealed that GF19-1 modulates the expression of salicylic acid (SA)-responsive PR-1, PR-2, and PR-5 genes. Moreover, transient accumulation of SA was observed in GF19-1-treated plant, whereas the level was further enhanced after Pst infection of GF19-1-pretreated plants, indicating that accumulation of SA was potentiated when Arabidopsis plants were primed for disease resistance by GF19-1. In conclusion, these findings imply that the induced protective effect conferred by F. equiseti GF19-1 against the leaf pathogen Pst requires responsiveness to an SA-dependent pathway.

Microbe-Associated Molecular Patterns-Triggered Root Responses Mediate Beneficial Rhizobacterial Recruitment in Arabidopsis

This study demonstrated that foliar infection by Pseudomonas syringae pv tomato DC3000 induced malic acid (MA) transporter (ALUMINUM-ACTIVATED MALATE TRANSPORTER1 [ALMT1]) expression leading to increased MA titers in the rhizosphere of Arabidopsis (Arabidopsis thaliana). MA secretion in the rhizosphere increased beneficial rhizobacteria Bacillus subtilis FB17 (hereafter FB17) titers causing an induced systemic resistance response in plants against P. syringae pv tomato DC3000. Having shown that a live pathogen could induce an intraplant signal from shoot-to-root to recruit FB17 belowground, we hypothesized that pathogen-derived microbe-associated molecular patterns (MAMPs) may relay a similar response specific to FB17 recruitment. The involvement of MAMPs in triggering plant innate immune response is well studied in the plant's response against foliar pathogens. In contrast, MAMPs-elicited plant responses on the roots and the belowground microbial community are not well understood. It is known that pathogen-derived MAMPs suppress the root immune responses, which may facilitate pathogenicity. Plants subjected to known MAMPs such as a flagellar peptide, flagellin22 (flg22), and a pathogen-derived phytotoxin, coronatine (COR), induced a shoot-to-root signal regulating ALMT1 for recruitment of FB17. Micrografts using either a COR-insensitive mutant (coi1) or a flagellin-insensitive mutant (fls2) as the scion and ALMT1(pro):β-glucuronidase as the rootstock revealed that both COR and flg22 are required for a graft transmissible signal to recruit FB17 belowground. The data suggest that MAMPs-induced signaling to regulate ALMT1 is salicylic acid and JASMONIC ACID RESISTANT1 (JAR1)/JASMONATE INSENSITIVE1 (JIN1)/MYC2 independent. Interestingly, a cell culture filtrate of FB17 suppressed flg22-induced MAMPs-activated root defense responses, which are similar to suppression of COR-mediated MAMPs-activated root defense, revealing a diffusible bacterial component that may regulate plant immune responses. Further analysis showed that the biofilm formation in B. subtilis negates suppression of MAMPs-activated defense responses in roots. Moreover, B. subtilis suppression of MAMPs-activated root defense does require JAR1/JIN1/MYC2. The ability of FB17 to block the MAMPs-elicited signaling pathways related to antibiosis reflects a strategy adapted by FB17 for efficient root colonization. These experiments demonstrate a remarkable strategy adapted by beneficial rhizobacteria to suppress a host defense response, which may facilitate rhizobacterial colonization and host-mutualistic association.

Oxidative Stress Acts on Special Membrane Proteins To Reduce the Viability of Pseudomonas syringae pv tomato

Reactive oxygen species (ROS) play a vital role in reducing the viability of invading pathogens during plant-pathogen interactions. To understand how oxidative stress caused by ROS reduces cell viability, it is important to identify the proteins affected by ROS. In the present study, we investigated the changes in the expression of proteins from the outer and inner membrane fractions in Pseudomonas syringae pv tomato DC3000 under oxidative stress through membrane subproteomics. A total number of 17 differentially expressed proteins from the outer and inner membrane fractions were identified, among which 11 proteins belong to transporters, such as porins and ABC transporters. Their abundance was all decreased under oxidative stress, indicating that transporters are likely to be affected by oxidative stress. The function of two identified transporters was further characterized by constructing their gene mutant and overexpression strains. We found that mutation of one transporter gene PSPTO_1720 rendered Pseudomonas more sensitive to oxidative stress, whereas overexpression of this gene made the strain more resistant. By comparison, the mutant and overexpression strains of another transporter gene PSPTO_2152 exhibited the same sensitivity to oxidative stress compared with the wild-type. Our data suggest that oxidative stress reduces the viability of bacterial cells by acting on special transporters.