Gram-negative Plant Pathogenic Bacteria Research Articles

Antimicrobial potential of essential oil artemisia sieversiana: a medicinal plant from the high altitude nubra valley, Ladakh

Artemisia sieversiana Ehrh. ex Willd. (common name: wormwood; family: Asteraceae) is a medicinal plant thriving at high altitudes. It is extensively utilized in traditional folk medicine by various ethnic communities to alleviate and treat a wide range of health issues. However, despite its widespread use, the chemical characterization and identification of bioactive compounds responsible for these benefits is still incomplete, especially in the context of Trans−Himalayan region. Prior research has demonstrated the substantial efficacy of this plant against various pathogenic bacteria, although these studies utilized essential oil (EO) extracted from different geographic regions. There is a need for further research to explore the antibacterial activity of A. sieversiana EO from the Trans−Himalayan, Ladakh region, as the unique environmental conditions of this region may influence the plant’s chemical composition and biological activity. This research therefore focusses on characterizing the phytochemical composition and evaluating the antibacterial properties of EO extracted from A. sieversiana, specifically from the Nubra valley, Ladakh (India). The antimicrobial potential of A. sieversiana essential oil was tested against a diverse spectrum of gram-negative (Erwinia herbicola and Pseudomaonas syringae) and gram-positive bacteria (Bacillius cereus, B. pumilus, Rhodococcus fasciens, and Streptomyces scabiei) plant and food pathogenic bacteria. Gas Chromatography−Mass Spectrometry (GC−MS) analysis led to the identification of 28 components with camphor (18.8%), borneol (11.2%), and 1,8−cineole (9.7%) as the major constituents. Further, the EO was more active against gram−positive bacteria, with a greater inhibition percentage for all gram−positive bacteria (24.68%−38.90%) than for gram−negative bacteria (0%−50.19%) at 10 µg mL−1. Moreover, the antibacterial activity showed a concentration dependent response, escalating from 24.68% to complete inhibition (100%) as the EO concentration increased from 10 to 160 µg mL−1. Notably, the EO demonstrated strongest activity against B. pumilus, even more than the common antibiotic Rifampicin. Among gram−negative bacteria, Erwinia herbicola showed the highest susceptibility. This observation shows that the essential oil of A. sieversiana exhibits promising antimicrobial activity and highlighted their potential for further exploration and utilization.

Bacterial outer membrane vesicles induce a transcriptional shift in arabidopsis towards immune system activation leading to suppression of pathogen growth in planta.

Gram-negative bacteria form spherical blebs on their cell periphery, which later dissociate from the bacterial cell wall to form extracellular vesicles. These nano scale structures, known as outer membrane vesicles (OMVs), have been shown to promote infection and disease and can induce typical immune outputs in both mammal and plant hosts. To better understand the broad transcriptional change plants undergo following exposure to OMVs, we treated Arabidopsis thaliana (Arabidopsis) seedlings with OMVs purified from the Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. campestris and performed RNA-seq analysis on OMV- and mock-treated plants at 2, 6 and 24h post challenge. The most pronounced transcriptional shift occurred at the first two time points tested, as reflected by the number of differentially expressed genes and the average fold change. OMVs induce a major transcriptional shift towards immune system activation, upregulating a multitude of immune-related pathways including a variety of immune receptors. Comparing the response of Arabidopsis to OMVs and to purified elicitors, revealed that OMVs induce a similar suite of genes and pathways as single elicitors, however, pathways activated by OMVs and not by other elicitors were detected. Pretreating Arabidopsis plants with OMVs and subsequently infecting with a bacterial pathogen led to a significant reduction in pathogen growth. Mutations in the plant elongation factor receptor (EFR), flagellin receptor (FLS2), or the brassinosteroid-insensitive 1-associated kinase (BAK1) co-receptor, did not significantly affect the immune priming effect of OMVs. All together these results show that OMVs induce a broad transcriptional shift in Arabidopsis leading to upregulation of multiple immune pathways, and that this transcriptional change may facilitate resistance to bacterial infection.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue.

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Recognition of a translocation motif in the regulator HpaA from Xanthomonas euvesicatoria is controlled by the type III secretion chaperone HpaB.

The Gram-negative plant-pathogenic bacterium Xanthomonas euvesicatoria is the causal agent of bacterial spot disease in pepper and tomato plants. Pathogenicity of X. euvesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells and is associated with an extracellular pilus and a translocon in the plant plasma membrane. Effector protein translocation is activated by the cytoplasmic T3S chaperone HpaB which presumably targets effectors to the T3S system. We previously reported that HpaB is controlled by the translocated regulator HpaA which binds to and inactivates HpaB during the assembly of the T3S system. In the present study, we show that translocation of HpaA depends on the T3S substrate specificity switch protein HpaC and likely occurs after pilus and translocon assembly. Translocation of HpaA requires the presence of a translocation motif (TrM) in the N-terminal region. The TrM consists of an arginine-and proline-rich amino acid sequence and is also essential for the in vivo function of HpaA. Mutation of the TrM allowed the translocation of HpaA in hpaB mutant strains but not in the wild-type strain, suggesting that the recognition of the TrM depends on HpaB. Strikingly, the contribution of HpaB to the TrM-dependent translocation of HpaA was independent of the presence of the C-terminal HpaB-binding site in HpaA. We propose that HpaB generates a recognition site for the TrM at the T3S system and thus restricts the access to the secretion channel to effector proteins. Possible docking sites for HpaA at the T3S system were identified by in vivo and in vitro interaction studies and include the ATPase HrcN and components of the predicted cytoplasmic sorting platform of the T3S system. Notably, the TrM interfered with the efficient interaction of HpaA with several T3S system components, suggesting that it prevents premature binding of HpaA. Taken together, our data highlight a yet unknown contribution of the TrM and HpaB to substrate recognition and suggest that the TrM increases the binding specificity between HpaA and T3S system components.

A myxobacterial GH19 lysozyme with bacteriolytic activity on both Gram-positive and negative phytopathogens

Myxobacteria, as predatory bacteria, have good application potential in the biocontrol of pathogenic microorganisms. Extracellular enzymes are thought to play an important role in their predation and also provide resources for discovering new antibacterial molecules. We previously isolated a myxobacterium, Corallococcus silvisoli c25j21 GDMCC 1.1387, which is predatory to plant pathogenic bacteria. In this study, we identified an endolysin-like GH19 glycoside hydrolase, C25GH19B, from the genome of c25j21. After its heterologous expression and purification from E. coli, the enzymatic properties of C25GH19B were characterized. C25GH19B showed lysozyme activity with the optimal reaction conditions at 40 °C and pH 4.5–5.0. Moreover, C25GH19B showed bacteriolytic activity against both Gram-positive and Gram-negative plant pathogenic bacteria. Our research provides not only a candidate enzyme for the development of novel biocontrol agents but also an experimental basis for further study on the function and mechanisms of extracellular enzymes in myxobacterial predation.

Complete Genome Resources for Xylella fastidiosa Strains AlmaEM3 and BB08-1 Reveal Prophage-Associated Structural Variation Among Blueberry-Infecting Strains.

Xylella fastidiosa is a gram-negative plant pathogenic bacterium with wide geographical distribution and host range. X. fastidiosa strains are separated into genetically distinct subspecies, and further categorized into sequence types (ST). Genetic characterization of X. fastidiosa strains infecting blueberry has revealed that strains of subspecies multiplex and fastidiosa are capable of causing bacterial leaf scorch disease of blueberry under field conditions. To better elucidate the relationships among blueberry-infecting X. fastidiosa subsp. multiplex strains, we completed the genomes of an ST 42 strain, AlmaEM3, and a ST 43 strain, BB08-1, using a hybrid assembly approach. Comparison of these assemblies reveals a large (0.95 Mb) chromosomal inversion in BB08-1 relative to AlmaEM3 and the reference strain M12, likely resulting from recombination between prophage regions.

Epidemiology, diversity, and management of bacterial spot of tomato caused by Xanthomonas perforans.

Tomato is an important crop grown worldwide. Various plant diseases cause massive losses in tomato plants due to diverse biotic agents. Bacterial spot of tomato (BST) is a worldwide disease that results in high losses in processed and fresh tomato. Xanthomonas perforans, an aerobic, single-flagellated, rod-shaped, Gram-negative plant pathogenic bacterium, is one of the leading causes of BST. Over the past three decades, X. perforans has increasingly been reported from tomato-growing regions and became a major bacterial disease. X. perforans thrives under high humidity and high temperature, which is commonplace in tropical and subtropical climates. Distinguishing symptoms of BST are necrotic lesions that can coalesce and cause a shot-hole appearance. X. perforans can occasionally cause fruit symptoms depending on disease pressure during fruit development. Short-distance movement in the field is mainly dependent on wind-driven rain, whereas long distance movement occurs through contaminated seed or plant material. X. perforans harbors a suite of effectors that increase pathogen virulence, fitness, and dissemination. BST management mainly relies on copper-based compounds; however, resistance is widespread. Alternative compounds, such as nanomaterials, are currently being evaluated and show high potential for BST management. Resistance breeding remains difficult to attain due to limited resistant germplasm. While the increased genetic diversity and gain and loss of effectors in X. perforans limits the success of single-gene resistance, the adoption of effector-specific transgenes and quantitative resistance may lead to durable host resistance. However, further research that aims to more effectively implement novel management tools is required to curb disease spread. KEY POINTS: • Xanthomonas perforans causes bacterial spot on tomato epidemics through infected seedlings and movement of plant material. • Genetic diversity plays a major role in shaping populations which is evident in loss and gain of effectors. • Management relies on copper sprays, but nanoparticles are a promising alternative to reduce copper toxicity.

Pectobacterium parmentieri SCC 3193 Mutants with Altered Synthesis of Cell Surface Polysaccharides Are Resistant to N4-Like Lytic Bacteriophage ϕA38 (vB_Ppp_A38) but Express Decreased Virulence in Potato (Solanum tuberosum L.) Plants.

Pectobacterium parmentieri is a Gram-negative plant-pathogenic bacterium able to infect potato (Solanum tuberosum L.). Little is known about lytic bacteriophages infecting P. parmentieri and how phage-resistance influences the environmental fitness and virulence of this species. A lytic phage vB_Ppp_A38 (ϕA38) has been previously isolated and characterized as a potential biological control agent for the management of P. parmentieri. In this study, seven P. parmentieri SCC 3193 Tn5 mutants were identified that exhibited resistance to infection caused by vB_Ppp_A38 (ϕA38). The genes disrupted in these seven mutants encoded proteins involved in the assembly of O-antigen, sugar metabolism, and the production of bacterial capsule exopolysaccharides. The potential of A38-resistant P. parmentieri mutants for plant colonization and pathogenicity as well as other phenotypes expected to contribute to the ecological fitness of P. parmentieri, including growth rate, use of carbon and nitrogen sources, production of pectinolytic enzymes, proteases, cellulases, and siderophores, swimming and swarming motility, presence of capsule and flagella as well as the ability to form biofilm were assessed. Compared to the wild-type P. parmentieri strain, all phage-resistant mutants exhibited a reduced ability to colonize and to cause symptoms in growing potato (S. tuberosum L.) plants. The implications of bacteriophage resistance on the ecological fitness of P. parmentieri are discussed.

Widespread distribution of prophages signaling the potential for adaptability and pathogenicity evolution of Ralstonia solanacearum species complex

Integrated bacteriophages (prophages) can impact host cells, affecting their lifestyle, genomic diversity, and fitness. However, many basic aspects of how these organisms affect the host cell remain poorly understood. Ralstonia solanacearum is a gram-negative plant pathogenic bacterium that encompasses a great diversity of ecotypes regarded as a species complex (R. solanacearum Species Complex - RSSC). RSSC genomes have a mosaic structure containing numerous elements, signaling the potential for its evolution through horizontal gene transfer. Here, we analyzed 120 Ralstonia spp. genomes from the public database to identify prophage sequences. In total, 379 prophage-like elements were found in the chromosome and megaplasmid of Ralstonia spp. These elements encode genes related to host fitness, virulence factors, antibiotic resistance, and niche adaptation, which might contribute to RSSC adaptability. Prophage-like elements are widespread into the complex in different species and geographic origins, suggesting that the RSSC phages are ancestrally acquired. Complete prophages belonging to the families Inoviridae, Myoviridae, and Siphoviridae were found, being the members of Inoviridae the most abundant. Analysis of CRISPR-Cas spacer sequences demonstrated the presence of prophages sequences that indicate successive infection events during bacterial evolution. Besides complete prophages, we also demonstrated 14 novel putative prophages integrated into Ralstonia spp. genomes. Altogether, our results provide insights into the diversity of prophages in RSSC genomes and suggest that these elements may deeply affect the virulence and host adaptation and shaping the genomes among the strains of this important pathogen.

Xanthomonas TAL effectors hijack host basal transcription factor IIA α and γ subunits for invasion

The Xanthomonas genus includes Gram-negative plant-pathogenic bacteria, which infect a broad range of crops and wild plant species, cause symptoms with leaf blights, streaks, spots, stripes, necrosis, wilt, cankers and gummosis on leaves, stems and fruits in a wide variety of plants via injecting their effector proteins into the host cell during infection. Among these virulent effectors, transcription activator-like effectors (TALEs) interact with the γ subunit of host transcription factor IIA (TFIIAγ) to activate the transcription of host disease susceptibility genes. Functional TFIIA is a ternary complex comprising α, β and γ subunits. However, whether TALEs recruit TFIIAα, TFIIAβ, or both remains unknown. The underlying molecular mechanisms by which TALEs mediate host susceptibility gene activation require full elucidation. Here, we show that TALEs interact with the α+γ binary subcomplex but not the α+β+γ ternary complex of rice TFIIA (holo-OsTFIIA). The transcription factor binding (TFB) regions of TALEs, which are highly conserved in Xanthomonas species, have a dominant role in these interactions. Furthermore, the interaction between TALEs and the α+γ complex exhibits robust DNA binding activity in vitro. These results collectively demonstrate that TALE-carrying pathogens hijack the host basal transcription factors TFIIAα and TFIIAγ, but not TFIIAβ, to enhance host susceptibility during pathogen infection. The uncovered mechanism widens new insights on host-microbe interaction and provide an applicable strategy to breed high-resistance crop varieties.

An Innovative Approach to Study Ralstonia solanacearum Pathogenicity in 6 to 7 Days Old Tomato Seedlings by Root Dip Inoculation.

Ralstonia solanacearum (F1C1) is a Gram-negative plant pathogenic bacterium that causes lethal wilt disease in a wide range of plant species. This pathogen is very well known for its unpredictable behavior during infection and wilting its host. Because of its mysterious infection behavior, virulence and pathogenicity standardization are still a big challenge in the case of R. solanacearum. Here, we report an innovative pathogenicity assay of R. solanacearum (F1C1) in the early stage of tomato seedlings by root dip inoculation. In this assay, we employed 6-7days old tomato seedlings for infection grown under nutrients free and gnotobiotic condition. After that, pathogenicity assay was performed by maintaining the inoculated seedlings in 1.5 or 2 ml sterile microfuge tubes. During infection, wilting symptom starts appearing from ~48 h post inoculation and the pathogenicity assay gets completed within seven days of post inoculation. This method is rapid, consistent as well as less resource dependent in terms of labor, space and cost to screen large numbers of plants. Hence, this newly developed assay is an easy and useful approach to study pathogen virulence functions and its interaction with the host plant during wilting and disease progression at the seedling stage.

The TAL Effector AvrBs3 from Xanthomonas campestris pv. vesicatoria Contains Multiple Export Signals and Can Enter Plant Cells in the Absence of the Type III Secretion Translocon.

Pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. Effector protein delivery is controlled by the T3S chaperone HpaB, which presumably escorts effector proteins to the secretion apparatus. One intensively studied effector is the transcription activator-like (TAL) effector AvrBs3, which binds to promoter sequences of plant target genes and activates plant gene expression. It was previously reported that type III-dependent delivery of AvrBs3 depends on the N-terminal protein region. The signals that control T3S and translocation of AvrBs3, however, have not yet been characterized. In the present study, we show that T3S and translocation of AvrBs3 depend on the N-terminal 10 and 50 amino acids, respectively. Furthermore, we provide experimental evidence that additional signals in the N-terminal 30 amino acids and the region between amino acids 64 and 152 promote translocation of AvrBs3 in the absence of HpaB. Unexpectedly, in vivo translocation assays revealed that AvrBs3 is delivered into plant cells even in the absence of HrpF, which is the predicted channel-forming component of the T3S translocon in the plant plasma membrane. The presence of HpaB- and HrpF-independent transport routes suggests that the delivery of AvrBs3 is initiated during early stages of the infection process, presumably before the activation of HpaB or the insertion of the translocon into the plant plasma membrane.

Dissecting virulence function from recognition: cell deathsuppression in Nicotiana benthamiana by XopQ/HopQ1-family effectors relies on EDS1-dependent immunity.

Many Gram-negative plant pathogenic bacteria express effector proteins of the XopQ/HopQ1 family which are translocated into plant cells via the type III secretion system during infection. In Nicotiana benthamiana, recognition of XopQ/HopQ1 proteins induces an effector-triggered immunity (ETI) reaction which is not associated with strong cell death but renders plants immune against Pseudomonas syringae and Xanthomonas campestris pv. vesicatoria strains. Additionally, XopQ suppresses cell death in N.benthamiana when transiently co-expressed with cell death inducers. Here, we show that representative XopQ/HopQ1 proteins are recognized similarly, likely by a single resistance protein of the TIR-NB-LRR class. Extensive analysis of XopQ derivatives indicates the recognition of structural features. We performed Agrobacterium-mediated protein expression experiments in wild-type and EDS1-deficient (eds1) N.benthamiana leaves, not recognizing XopQ/HopQ1. XopQ recognition limits multiplication of Agrobacterium and attenuates levels of transiently expressed proteins. Remarkably, XopQ fails to suppress cell death reactions induced by different effectors in eds1 plants. We conclude that XopQ-mediated cell death suppression in N.benthamiana is due to the attenuation of Agrobacterium-mediated protein expression rather than the cause of the genuine XopQ virulence activity. Thus, our study expands our understanding of XopQ recognition and function, and also challenges the commonly used co-expression assays for elucidation of in planta effector activities, at least under conditions of ETI induction.

Penicillin V acylases from gram-negative bacteria degrade N-acylhomoserine lactones and attenuate virulence in Pseudomonas aeruginosa.

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation of AHLs leading to attenuation of virulence (quorum quenching) could pave the way for the development of new antibacterials. Penicillin V acylases (PVAs) belong to the Ntn hydrolase superfamily, together with AHL acylases. PVAs are exploited widely in the pharmaceutical industry, but their role in the natural physiology of their native microbes is not clearly understood. This report details the characterization of AHL degradation activity by homotetrameric PVAs from two gram-negative plant pathogenic bacteria, Pectobacterium atrosepticum (PaPVA) and Agrobacterium tumefaciens (AtPVA). Both the PVAs exhibited substrate specificity for degrading long-chain AHLs. Exogenous addition of these enzymes into Pseudomonas aeruginosa greatly diminished the production of elastase and pyocyanin and biofilm formation and increased the survival rate in an insect model of acute infection. Subtle structural differences in the PVA active site that regulate specificity for acyl chain length have been characterized, which could reflect the evolution of AHL-degrading acylases in relation to the environment of the bacteria that produce them and also provide strategies for enzyme engineering. The potential for using these enzymes as therapeutic agents in clinical applications and a few ideas about their possible significance in microbial physiology have also been discussed.

Non-host Resistance Induced by the Xanthomonas Effector XopQ Is Widespread within the Genus Nicotiana and Functionally Depends on EDS1.

Most Gram-negative plant pathogenic bacteria translocate effector proteins (T3Es) directly into plant cells via a conserved type III secretion system, which is essential for pathogenicity in susceptible plants. In resistant plants, recognition of some T3Es is mediated by corresponding resistance (R) genes or R proteins and induces effector triggered immunity (ETI) that often results in programmed cell death reactions. The identification of R genes and understanding their evolution/distribution bears great potential for the generation of resistant crop plants. We focus on T3Es from Xanthomonas campestris pv. vesicatoria (Xcv), the causal agent of bacterial spot disease on pepper and tomato plants. Here, 86 Solanaceae lines mainly of the genus Nicotiana were screened for phenotypical reactions after Agrobacterium tumefaciens-mediated transient expression of 21 different Xcv effectors to (i) identify new plant lines for T3E characterization, (ii) analyze conservation/evolution of putative R genes and (iii) identify promising plant lines as repertoire for R gene isolation. The effectors provoked different reactions on closely related plant lines indicative of a high variability and evolution rate of potential R genes. In some cases, putative R genes were conserved within a plant species but not within superordinate phylogenetical units. Interestingly, the effector XopQ was recognized by several Nicotiana spp. lines, and Xcv infection assays revealed that XopQ is a host range determinant in many Nicotiana species. Non-host resistance against Xcv and XopQ recognition in N. benthamiana required EDS1, strongly suggesting the presence of a TIR domain-containing XopQ-specific R protein in these plant lines. XopQ is a conserved effector among most xanthomonads, pointing out the XopQ-recognizing RxopQ as candidate for targeted crop improvement.

SYNTHESIS, ANTI-PHYTOPATHOGENIC AND DPPH RADICAL SCAVENGING ACTIVITIES OF C-PRENYLATED ACETOPHENONES AND BENZALDEHYDES

The syntheses of six prenylated acetophenone and benzaldehyde derivatives and their anti-phytopathogenic and antioxidant activities are reported. These com­pounds were obtained by electrophilic aromatic substitution (SEAr) of the corresponding arenes and 3-methyl-2-buten-1-ol using ZnCl2 as a Lewis acid catalyst in ethyl acetate. Reasonable to good yields were obtained based on unrecovered aromatic starting material (45-73%). All the synthesized compounds were evaluated against phytopathogenic gram-negative bacteria Agrobacterium tumefaciens , Pseudomonas syringae and Erwinia carotovora and plant fungal pathogens Botrytis cinerea , Phytophthora cinnamomi and Gibberella fujikuroi . The antioxidant activity was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scav­enging activity assay and expressed as half maximal inhibitory concentration (IC50) values in μM concentrations. The antioxidant activity went from 27.20 μM to >100 μM. Compound 11 showed statistically significant inhibition of the growth of Botrytis cinerea , and compounds 13 and 15 showed statistically significant inhibition of the growth of Phytophthora cinnamomi , with respect to negative control fungal growth. All six compounds showed bacteriostatic effects against gram-negative plant pathogenic bacteria with IC50 values between 250 and <3.9 μM.

Behind the lines-actions of bacterial type III effector proteins in plant cells.

Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed.

Overexpression of a harpin-encoding gene popW from Ralstonia solanacearum primed antioxidant defenses with enhanced drought tolerance in tobacco plants.

The tobacco plants transformed with popW gene showed enhanced drought tolerance, and the mechanism was found with primed antioxidant defenses and reduced drought stress damages in the transgenic lines. Harpin proteins are elicitors produced by several gram-negative plant pathogenic bacteria, triggering multiple beneficial responses in plants, such as induction of defense response against diverse pathogens and insects, growth promotion, and drought tolerance. In this study, the harpin-encoding gene popW derived from Ralstonia solanacearum ZJ3721 was transferred to tobacco. We examined the tolerance of transgenic tobacco plants toward drought stress under greenhouse conditions and analyzed the molecular mechanisms underlying the enhanced drought tolerance. The results revealed that the transgenic lines primed antioxidant defenses and reduced drought stress damages. In addition, they displayed lower malondialdehyde and relative electrical conductivity, while higher relative water content and recovery intension than the tobacco plants transformed with empty vector pBI121 and the wild-type (WT) plants under drought stress. Furthermore, the transgenic lines displayed a significant increase in peroxidase, superoxide dismutase, catalase activities, and ascorbic acid content compared with control plants under drought stress, and these levels were up to 1.95, 1.68, 1.34, and 1.43 times higher than those of WT plants, respectively. Overexpression of popW in tobacco also significantly enhanced the relative transcript levels of oxidative stress-responsive genes NtAPX, NtCAT1, NtGST, and NtCu/Zn-SOD under drought stress. The relative transcript levels of these genes in the transgenic line PW12 were up to 1.94, 2.36, 5.24, and 3.62 times higher than those of WT plants, respectively. These results confirmed that the popW gene, which was transformed into tobacco primed antioxidant responses, increased tolerance to drought stress in tobacco plants.

Using Ecology, Physiology, and Genomics to Understand Host Specificity in Xanthomonas.

How pathogens coevolve with and adapt to their hosts are critical to understanding how host jumps and/or acquisition of novel traits can lead to new disease emergences. The Xanthomonas genus includes Gram-negative plant-pathogenic bacteria that collectively infect a broad range of crops and wild plant species. However, individual Xanthomonas strains usually cause disease on only a few plant species and are highly adapted to their hosts, making them pertinent models to study host specificity. This review summarizes our current understanding of the molecular basis of host specificity in the Xanthomonas genus, with a particular focus on the ecology, physiology, and pathogenicity of the bacterium. Despite our limited understanding of the basis of host specificity, type III effectors, microbe-associated molecular patterns, lipopolysaccharides, transcriptional regulators, and chemotactic sensors emerge as key determinants for shaping host specificity.

Plant Pathogenic Microbial Communication Affected by Elevated Temperature in Pectobacterium carotovorum subsp. carotovorum.

Gram-negative plant pathogenic bacteria regulate specific gene expression in a population density-dependent manner by sensing level of Acyl-Homoserine Lactone (HSL) molecules which they produce and liberate to the environment, called Quorum Sensing (QS). The production of virulence factors (extracellular enzyme viz. cellulase, pectinase, etc.) in Pectobacterium carotovorum subsp. carotovorum (Pcc) is under strong regulation of QS. The QS signal molecule, N-(3-oxohexanoyl)-L-Homoserine Lactone (OHHL) was found as the central regulatory system for the virulence factor production in Pcc and is also under strict regulation of external environmental temperature. Under seven different incubation temperatures (24, 26, 28, 30, 33, 35, and 37 °C) in laboratory condition, highest amount of OHHL (804 violacein unit) and highest (79 %) Disease Severity Index (DSI) were measured at 33 °C. The OHHL production kinetics showed accumulation of highest concentration of OHHL at late log phase of the growth but diminution in the concentration occurred during stationary phase onwards to death phase. At higher temperature (35 and 37 °C) exposure, OHHL was not at detectable range. The effect of temperature on virulence factor production is the concomitant effect of HSL production and degradation which justifies less disease severity index in cross-inoculated tomato fruits incubated at 35 and 37 °C. The nondetection of the OHHL in the elevated temperature may because of degradation as these signal molecules are quite sensitive and prone to get degraded under different physical factors. This result provides the rationale behind the highest disease severity up to certain elevated temperature and leaves opportunities for investigation on mutation, co-evolution of superior plant pathogen with more stable HSL signals-mediated pathogenesis under global warming context.