The BZR1-EDS1 module regulates plant growth-defense coordination.

Plants have developed sophisticated strategies to coordinate growth and immunity, but our understanding of the underlying mechanism remains limited. In this study, we identified a novel molecular module that regulates plant growth and defense in both compatible and incompatible infections. This module consisted of BZR1, a key transcription factor in brassinosteroid (BR) signaling, and EDS1, an essential positive regulator of plant innate immunity. We found that EDS1 interacts with BZR1 and suppresses its transcriptional activities. Consistently, upregulation of EDS1 function by a virulent Pseudomonas syringae strain or salicylic acid treatment inhibited BZR1-regulated expression of BR-responsive genes and BR-promoted growth. Furthermore, we showed that the cytoplasmic fraction of BZR1 positively regulates effector-triggered immunity (ETI) controlled by the TIR-NB-LRR protein RPS4, which is attenuated by BZR1's nuclear translocation. Mechanistically, cytoplasmic BZR1 facilitated AvrRps4-triggered dissociation of EDS1 and RPS4 by binding to EDS1, thus leading to efficient activation of RPS4-controlled ETI. Notably, transgenic expression of a mutant BZR1 that accumulates exclusively in the cytoplasm improved pathogen resistance without compromising plant growth. Collectively, these results shed new light on plant growth-defense coordination and reveal a previously unknown function for the cytoplasmic fraction of BZR1. The BZR1-EDS1 module may be harnessed for the simultaneous improvement of crop productivity and pathogen resistance.

Prediction and Activity of a Cationic α-Helix Antimicrobial Peptide ZM-804 from Maize.

Antimicrobial peptides (AMPs) are small molecules consisting of less than fifty residues of amino acids. Plant AMPs establish the first barrier of defense in the innate immune system in response to invading pathogens. The purpose of this study was to isolate new AMPs from the Zea mays L. inbred line B73 and investigate their antimicrobial activities and mechanisms against certain essential plant pathogenic bacteria. In silico, the Collection of Anti-Microbial Peptides (CAMPR3), a computational AMP prediction server, was used to screen a cDNA library for AMPs. A ZM-804 peptide, isolated from the Z. mays L. inbred line B73 cDNA library, was predicted as a new cationic AMP with high prediction values. ZM-804 was tested against eleven pathogens of Gram-negative and Gram-positive bacteria and exhibited high antimicrobial activities as determined by the minimal inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs). A confocal laser scanning microscope observation showed that the ZM-804 AMP targets bacterial cell membranes. SEM and TEM images revealed the disruption and damage of the cell membrane morphology of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato (Pst) DC3000 caused by ZM-804. In planta, ZM-804 demonstrated antimicrobial activity and prevented the infection of tomato plants by Pst DC3000. Moreover, four virulent phytopathogenic bacteria were prevented from inducing hypersensitive response (HR) in tobacco leaves in response to low ZM-804 concentrations. ZM-804 exhibits low hemolytic activity against mouse red blood cells (RBCs) and is relatively safe for mammalian cells. In conclusion, the ZM-804 peptide has a strong antibacterial activity and provides an alternative tool for plant disease control. Additionally, the ZM-804 peptide is considered a promising candidate for human and animal drug development.

Pseudomonas syringae pv. phaseolicola Uses Distinct Modes of Stationary-Phase Persistence To Survive Bacteriocin and Streptomycin Treatments.

Antimicrobial treatment of bacteria often results in a small population of surviving tolerant cells, or persisters, that may contribute to recurrent infection. Antibiotic persisters are metabolically dormant, but the basis of their persistence in the presence of membrane-disrupting biological compounds is less well understood. We previously found that the model plant pathogen Pseudomonas syringae pv. phaseolicola 1448A (Pph) exhibits persistence to tailocin, a membrane-disrupting biocontrol compound with potential for sustainable disease control. Here, we compared physiological traits associated with persistence to tailocin and to the antibiotic streptomycin and established that both treatments leave similar frequencies of persisters. Microscopic profiling of treated populations revealed that while tailocin rapidly permeabilizes most cells, streptomycin treatment results in a heterogeneous population in the redox and membrane permeability state. Intact cells were sorted into three fractions according to metabolic activity, as indicated by a redox-sensing reporter dye. Streptomycin persisters were cultured from the fraction associated with the lowest metabolic activity, but tailocin persisters were cultured from a fraction associated with an active metabolic signal. Cells from culturable fractions were able to infect host plants, while the nonculturable fractions were not. Tailocin and streptomycin were effective in eliminating all persisters when applied sequentially, in addition to eliminating cells in other viable states. This study identifies distinct metabolic states associated with antibiotic persistence, tailocin persistence, and loss of virulence and demonstrates that tailocin is highly effective in eliminating dormant cells.IMPORTANCE Populations of genetically identical bacteria encompass heterogeneous physiological states. The small fraction of bacteria that are dormant can help the population survive exposure to antibiotics and other stresses, potentially contributing to recurring infection cycles in animal or plant hosts. Membrane-disrupting biological control treatments are effective in killing dormant bacteria, but these treatments also leave persister-like survivors. The current work demonstrates that in Pph, persisters surviving treatment with membrane-disrupting tailocin proteins have an elevated redox state compared to that of dormant streptomycin persisters. Combination treatment was effective in killing both persister types. Culturable persisters corresponded closely with infectious cells in each treated population, whereas the high-redox and unculturable fractions were not infectious. In linking redox states to heterogeneous phenotypes of tailocin persistence, streptomycin persistence, and infection capability, this work will inform the search for mechanisms and markers for each phenotype.

Agromonas: a rapid disease assay for Pseudomonas syringae growth in agroinfiltrated leaves.

The lengthy process to generate transformed plants is a limitation in current research on the interactions of the model plant pathogen Pseudomonas syringae with plant hosts. Here we present an easy method called agromonas, where we quantify P. syringae growth in agroinfiltrated leaves of Nicotiana benthamiana using a cocktail of antibiotics to select P. syringae on plates. As a proof of concept, we demonstrate that transient expression of PAMP receptors reduces bacterial growth, and that transient depletion of a host immune gene and transient expression of a type-III effector increase P. syringae growth in agromonas assays. We show that we can rapidly achieve structure-function analysis of immune components and test the function of immune hydrolases. The agromonas method is easy, fast and robust for routine disease assays with various Pseudomonas strains without transforming plants or bacteria. The agromonas assay offers a reliable approach for further comprehensive analysis of plant immunity.

Multi-Omics Revealed Molecular Mechanisms Underlying Guard Cell Systemic Acquired Resistance.

Systemic Acquired Resistance (SAR) improves immunity of plant systemic tissue after local exposure to a pathogen. Guard cells that form stomatal pores on leaf surfaces recognize bacterial pathogens via pattern recognition receptors, such as Flagellin Sensitive 2 (FLS2). However, how SAR affects stomatal immunity is not known. In this study, we aim to reveal molecular mechanisms underlying the guard cell response to SAR using multi-omics of proteins, metabolites and lipids. Arabidopsis plants previously exposed to pathogenic bacteria Pseudomonas syringae pv. tomato DC3000 (Pst) exhibit an altered stomatal response compared to control plants when they are later exposed to the bacteria. Reduced stomatal apertures of SAR primed plants lead to decreased number of bacteria in leaves. Multi-omics has revealed molecular components of SAR response specific to guard cells functions, including potential roles of reactive oxygen species (ROS) and fatty acid signaling. Our results show an increase in palmitic acid and its derivative in the primed guard cells. Palmitic acid may play a role as an activator of FLS2, which initiates stomatal immune response. Improved understanding of how SAR signals affect stomatal immunity can aid biotechnology and marker-based breeding of crops for enhanced disease resistance.

Distinctiveness of genes contributing to growth of Pseudomonas syringae in diverse host plant species.

A variety of traits are necessary for bacterial colonization of the interior of plant hosts, including well-studied virulence effectors as well as other phenotypes contributing to bacterial growth and survival within the apoplast. High-throughput methods such as transposon sequencing (TnSeq) are powerful tools to identify such genes in bacterial pathogens. However, there is little information as to the distinctiveness of traits required for bacterial colonization of different hosts. Here, we utilize randomly barcoded TnSeq (RB-TnSeq) to identify the genes that contribute to the ability of Pseudomonas syringae strain B728a to grow within common bean (Phaseolus vulgaris), lima bean (Phaseolus lunatus), and pepper (Capsicum annuum); species representing two different plant families. The magnitude of contribution of most genes to apoplastic fitness in each of the plant hosts was similar. However, 50 genes significantly differed in their fitness contributions to growth within these species. These genes encoded proteins in various functional categories including polysaccharide synthesis and transport, amino acid metabolism and transport, cofactor metabolism, and phytotoxin synthesis and transport. Six genes that encoded unannotated, hypothetical proteins also contributed differentially to growth in these hosts. The genetic repertoire of a relatively promiscuous pathogen such as P. syringae may thus be shaped, at least in part, by the conditional contribution of some fitness determinants.

Comparative transcriptomic analysis of global gene expression mediated by (p) ppGpp reveals common regulatory networks in Pseudomonas syringae.

BACKGROUND: Pseudomonas syringae is an important plant pathogen, which could adapt many different environmental conditions. Under the nutrient-limited and other stress conditions, P. syringae produces nucleotide signal molecules, i.e., guanosine tetra/pentaphosphate ((p)ppGpp), to globally regulate gene expression. Previous studies showed that (p) ppGpp played an important role in regulating virulence factors in P. syringae pv. tomato DC3000 (PstDC3000) and P. syringae pv. syringae B728a (PssB728a). Here we present a comparative transcriptomic analysis to uncover the overall effects of (p)ppGpp-mediated stringent response in P. syringae. RESULTS: In this study, we investigated global gene expression profiles of PstDC3000 and PssB728a and their corresponding (p)ppGpp0 mutants in hrp-inducing minimal medium (HMM) using RNA-seq. A total of 1886 and 1562 differentially expressed genes (DEGs) were uncovered between the (p)ppGpp0 mutants and the wild-type in PstDC3000 and PssB728a, respectively. Comparative transcriptomics identified 1613 common DEGs, as well as 444 and 293 unique DEGs in PstDC3000 and PssB728a, respectively. Functional cluster analysis revealed that (p) ppGpp positively regulated a variety of virulence-associated genes, including type III secretion system (T3SS), type VI secretion system (T6SS), cell motility, cell division, and alginate biosynthesis, while negatively regulated multiple basic physiological processes, including DNA replication, RNA processes, nucleotide biosynthesis, fatty acid metabolism, ribosome protein biosynthesis, and amino acid metabolism in both PstDC3000 and PssB728a. Furthermore, (p) ppGpp had divergent effects on other processes in PstDC3000 and PssB728a, including phytotoxin, nitrogen regulation and general secretion pathway (GSP). CONCLUSION: In this study, comparative transcriptomic analysis reveals common regulatory networks in both PstDC3000 and PssB728a mediated by (p) ppGpp in HMM. In both P. syringae systems, (p) ppGpp re-allocate cellular resources by suppressing multiple basic physiological activities and enhancing virulence gene expression, suggesting a balance between growth, survival and virulence. Our research is important in that due to similar global gene expression mediated by (p) ppGpp in both PstDC3000 and PssB728a, it is reasonable to propose that (p) ppGpp could be used as a target to develop novel control measures to fight against important plant bacterial diseases.

Pathogenic Bacteria Target Plant Plasmodesmata to Colonize and Invade Surrounding Tissues.

A hallmark of multicellular organisms is their ability to maintain physiological homeostasis by communicating among cells, tissues, and organs. In plants, intercellular communication is largely dependent on plasmodesmata (PD), which are membrane-lined channels connecting adjacent plant cells. Upon immune stimulation, plants close PD as part of their immune responses. Here, we show that the bacterial pathogen Pseudomonas syringae deploys an effector protein, HopO1-1, that modulates PD function. HopO1-1 is required for P. syringae to spread locally to neighboring tissues during infection. Expression of HopO1-1 in Arabidopsis (Arabidopsis thaliana) increases the distance of PD-dependent molecular flux between neighboring plant cells. Being a putative ribosyltransferase, the catalytic activity of HopO1-1 is required for regulation of PD. HopO1-1 physically interacts with and destabilizes the plant PD-located protein PDLP7 and possibly PDLP5. Both PDLPs are involved in bacterial immunity. Our findings reveal that a pathogenic bacterium utilizes an effector to manipulate PD-mediated host intercellular communication for maximizing the spread of bacterial infection.

Pseudomonas bacteriocin syringacin M released upon desiccation suppresses the growth of sensitive bacteria in plant necrotic lesions.

Bacteriocins are regarded as important factors mediating microbial interactions, but their exact role in community ecology largely remains to be elucidated. Here, we report the characterization of a mutant strain, derived from Pseudomonas syringae pv. tomato DC3000 (Pst), that was incapable of growing in plant extracts and causing disease. Results showed that deficiency in a previously unannotated gene saxE led to the sensitivity of the mutant to Ca2+ in leaf extracts. Transposon insertions in the bacteriocin gene syrM, adjacent to saxE, fully rescued the bacterial virulence and growth of the ΔsaxE mutant in plant extracts, indicating that syrM-saxE encode a pair of bacteriocin immunity proteins in Pst. To investigate whether the syrM-saxE system conferred any advantage to Pst in competition with other SyrM-sensitive pathovars, we compared the growth of a SyrM-sensitive strain co-inoculated with Pst strains with or without the syrM gene and observed a significant syrM-dependent growth reduction of the sensitive bacteria on plate and in lesion tissues upon desiccation-rehydration treatment. These findings reveal an important biological role of SyrM-like bacteriocins and help to understand the complex strategies used by P. syringae in adaptation to the phyllosphere niche in the context of plant disease.

Genome-Wide Transposon Screen of a Pseudomonas syringae mexB Mutant Reveals the Substrates of Efflux Transporters.

Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The resistance-nodulation-division (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly expressed and conserved among Pseudomonas species. We exposed barcoded transposon mutant libraries in isogenic wild-type and ΔmexB backgrounds in P. syringae B728a to diverse toxic compounds in vitro to identify mutants with increased susceptibility to these compounds. Mutants with mutations in genes encoding both known and novel redundant transporters but with partially overlapping substrate specificities were observed in a ΔmexB background. Psyr_0228, an uncharacterized member of the major facilitator superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance of acriflavine and berberine. The presence of multiple redundant, genomically encoded efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen performed in a hypersusceptible mutant strain revealed numerous transporters that would otherwise be dispensable under these conditions. Bacterial strains such as P. syringae that likely encounter diverse toxins in their environment, such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility with respect to toleration of novel toxicants.IMPORTANCE Bacteria use protein pumps to remove toxic compounds from the cell interior, enabling survival in diverse environments. These protein pumps can be highly redundant, making their targeted examination difficult. In this study, we exposed mutant populations of Pseudomonas syringae to diverse toxicants to identify pumps that contributed to survival in those conditions. In parallel, we examined pump redundancy by testing mutants of a population lacking the primary efflux transporter responsible for toxin tolerance. We identified partial substrate overlap for redundant transporters, as well as several pumps that appeared more substrate specific. For bacteria that are found in diverse environments, having multiple, partially redundant efflux pumps likely allows flexibility in habitat colonization.

Pseudomonas syringae pv. tomato DC3000 Improves Escherichia coli O157:H7 Survival in Tomato Plants.

Recently, outbreaks of food-borne diseases linked to fresh produce have been an emerging public health concerns worldwide. Previous research has shown that when human pathogens co-exist with plant pathogens, they have improved growth and survival rates. In this study, we have assessed whether Escherichia coli O157:H7 benefits in the existence of a phytopathogenic bacterium and the underlying mechanisms were further investigated. When Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and E. coli O157:H7 were co-inoculated by either dipping or infiltration methods, the populations of E. coli O157:H7 increased; however, no effect was observed when type three secretion system (T3SS) mutants were used instead, suggesting that E. coli O157:H7 benefits from the presence of Pst DC3000. In addition, this study confirmed that the E. coli O157:H7 populations increased when they occupied the tomato leaf intercellular space; this colonization of the interior of the leaves was possible due to the suppression of the PAMP triggered immunity (PTI) by Pst DC3000, in particular with the AvrPto effector. In conclusion, our data supports a plausible model that E. coli O157:H7 benefits from the presence of Pst DC3000 via AvrPto suppression of the PTI resistance.

Re-evaluation of a Tn5::gacA mutant of Pseudomonas syringae pv. tomato DC3000 uncovers roles for uvrC and anmK in promoting virulence.

Pseudomonas syringae is a taxon of plant pathogenic bacteria that can colonize and proliferate within the interior space of leaf tissue. This process requires P. syringae to rapidly upregulate the production of virulence factors including a type III secretion system (T3SS) that suppress host defenses. GacS/A is a two-component system that regulates virulence of many plant and animal pathogenic bacteria including P. syringae. We recently investigated the virulence defect of strain AC811, a Tn5::gacA mutant of P. syringae pv. tomato DC3000 that is less virulent on Arabidopsis. We discovered that decreased virulence of AC811 is not caused by loss of GacA function. Here, we report the molecular basis of the virulence defect of AC811. We show that AC811 possesses a nonsense mutation in anmK, a gene predicted to encode a 1,6-anhydromuramic acid kinase involved in cell wall recycling. Expression of a wild-type allele of anmK partially increased growth of AC811 in Arabidopsis leaves. In addition to the defective anmK allele, we also show that the Tn5 insertion in gacA exerts a polar effect on uvrC, a downstream gene encoding a regulator of DNA damage repair. Expression of the wild-type anmK allele together with increased expression of uvrC fully restored the virulence of AC811 during infection of Arabidopsis. These results demonstrate that defects in anmK and uvrC are together sufficient to account for the decreased virulence of AC811, and suggest caution is warranted in assigning phenotypes to GacA function based on insertional mutagenesis of the gacA-uvrC locus.

Common and unique Arabidopsis proteins involved in stomatal susceptibility to Salmonella enterica and Pseudomonas syringae.

Salmonella enterica is one of the most common pathogens associated with produce outbreaks worldwide; nonetheless, the mechanisms uncovering their interaction with plants are elusive. Previous reports demonstrate that S. enterica ser. Typhimurium (STm), similar to the phytopathogen Pseudomonas syringae pv. tomato (Pst) DC3000, triggers a transient stomatal closure suggesting its ability to overcome this plant defense and colonize the leaf apoplast. In order to discover new molecular players that function in the stomatal reopening by STm and Pst DC3000, we performed an Arabidopsis mutant screening using thermal imaging. Further stomatal bioassay confirmed that the mutant plants exo70h4-3, sce1-3, bbe8, stp1, and lsu2 have smaller stomatal aperture widths than the wild type Col-0 in response to STm 14028s. The mutants bbe8, stp1 and lsu2 have impaired stomatal movement in response to Pst DC3000. These findings indicate that EXO70H4 and SCE1 are involved in bacterial-specific responses, while BBE8, STP1, and LSU2 may be required for stomatal response to a broad range of bacteria. The identification of new molecular components of the guard cell movement induced by bacteria will enable a better understanding of the initial stages of plant colonization and facilitate targeted prevention of leaf contamination with harmful pathogens.

Bactericidal In-Vitro Effect of Zinc Ferrite Nanoparticles and the Orange Wax Extracts on Three Phytopathogen Microorganisms.

Phytopathogenic bacteria affect a wide variety of crops, causing significant economic losses. Natural biocides are the alternative to chemical methods of phytopathogens control. The goal of the present study is the evaluation of the biocidal activity of the following: 1) the extract of orange wax (EOW); 2) zinc ferrite nanoparticles (ZF-NPs); 3) the EOW adsorbed on the ZF-NPs; and 4) the EOW/ZF-NPs washed with 40% ethanol. For the biocidal activity, three phytopathogenic bacteria were used, namely, Xanthomonas axonopodis pv. Vesicatoria (Xav) Erwinia amylovora (Ew), and Pseudomonas syringae pv. Phaseolicola (Psph). For the ZF-NPs, an inhibitory effect higher than 50% ( ) was observed for Xav respect to the antibiotic used as positive control. On the other hand, the ZF-NPs did not show inhibitory effects on both Ew and Psph. In addition, the EOW in dimethyl sulfoxide (DMSO) at 100% caused growth inhibition on Xav, bacteriostatic activity on Ew, and had not biological activity on Psph. To the best of our knowledge, the control of Xav by zinc ferrites and orange wax, and the bacteriostatic effect produced by orange wax extract on Ew have not been reported elsewhere. Orange wax and zinc ferrite nanoparticles show potential in control of phytopathogenic bacteria. However, the bactericidal effect depends on the bacterium, the concentration of treatments, and the method of preparation.

Unusual extracellular appendages deployed by the model strain Pseudomonas fluorescens C7R12.

Pseudomonas fluorescens is considered to be a typical plant-associated saprophytic bacterium with no pathogenic potential. Indeed, some P. fluorescens strains are well-known rhizobacteria that promote plant growth by direct stimulation, by preventing the deleterious effects of pathogens, or both. Pseudomonas fluorescens C7R12 is a rhizosphere-competent strain that is effective as a biocontrol agent and promotes plant growth and arbuscular mycorrhization. This strain has been studied in detail, but no visual evidence has ever been obtained for extracellular structures potentially involved in its remarkable fitness and biocontrol performances. On transmission electron microscopy of negatively stained C7R12 cells, we observed the following appendages: multiple polar flagella, an inducible putative type three secretion system typical of phytopathogenic Pseudomonas syringae strains and densely bundled fimbria-like appendages forming a broad fractal-like dendritic network around single cells and microcolonies. The deployment of one or other of these elements on the bacterial surface depends on the composition and affinity for the water of the microenvironment. The existence, within this single strain, of machineries known to be involved in motility, chemotaxis, hypersensitive response, cellular adhesion and biofilm formation, may partly explain the strong interactions of strain C7R12 with plants and associated microflora in addition to the type three secretion system previously shown to be implied in mycorrhizae promotion.

Gene networks underlying the early regulation of Paraburkholderia phytofirmans PsJN induced systemic resistance in Arabidopsis.

Plant defense responses to biotic stresses are complex biological processes, all governed by sophisticated molecular regulations. Induced systemic resistance (ISR) is one of these defense mechanisms where beneficial bacteria or fungi prime plants to resist pathogens or pest attacks. In ISR, the defense arsenal in plants remains dormant and it is only triggered by an infection, allowing a better allocation of plant resources. Our group recently described that the well-known beneficial bacterium Paraburkholderia phytofirmans PsJN is able to induce Arabidopsis thaliana resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 through ISR, and that ethylene, jasmonate and salicylic acid are involved in this protection. Nevertheless, the molecular networks governing this beneficial interaction remain unknown. To tackle this issue, we analyzed the temporal changes in the transcriptome of PsJN-inoculated plants before and after being infected with Pst DC3000. These data were used to perform a gene network analysis to identify highly connected transcription factors. Before the pathogen challenge, the strain PsJN regulated 405 genes (corresponding to 1.8% of the analyzed genome). PsJN-inoculated plants presented a faster and stronger transcriptional response at 1-hour post infection (hpi) compared with the non-inoculated plants, which presented the highest transcriptional changes at 24 hpi. A principal component analysis showed that PsJN-induced plant responses to the pathogen could be differentiated from those induced by the pathogen itself. Forty-eight transcription factors were regulated by PsJN at 1 hpi, and a system biology analysis revealed a network with four clusters. Within these clusters LHY, WRKY28, MYB31 and RRTF1 are highly connected transcription factors, which could act as hub regulators in this interaction. Concordantly with our previous results, these clusters are related to jasmonate, ethylene, salicylic, acid and ROS pathways. These results indicate that a rapid and specific response of PsJN-inoculated plants to the virulent DC3000 strain could be the pivotal element in the protection mechanism.

Survival and ice nucleation activity of Pseudomonas syringae strains exposed to simulated high-altitude atmospheric conditions.

Pseudomonas syringae produces highly efficient biological ice nuclei (IN) that were proposed to influence precipitation by freezing water in clouds. This bacterium may be capable of dispersing through the atmosphere, having been reported in rain, snow, and cloud water samples. This study assesses its survival and maintenance of IN activity under stressing conditions present at high altitudes, such as UV radiation within clouds. Strains of the pathovars syringae and garcae were compared to Escherichia coli. While UV-C effectively inactivated these cells, the Pseudomonas were much more tolerant to UV-B. The P. syringae strains were also more resistant to radiation from a solar simulator, composed of UV-A and UV-B, while only one of them suffered a decline in IN activity at -5 °C after long exposures. Desiccation at different relative humidity values also affected the IN, but some activity at -5 °C was always maintained. The pathovar garcae tended to be more resistant than the pathovar syringae, particularly to desiccation, though its IN were found to be generally more sensitive. Compared to E. coli, the P. syringae strains appear to be better adapted to survival under conditions present at high altitudes and in clouds.

Comparative genomics reveal pathogenicity-related loci in Pseudomonas syringae pv. actinidiae biovar 3.

Bacterial canker of kiwifruit, is a severe global disease caused by Pseudomonas syringae pv. actinidiae (Psa). Here, we found that Psa biovar 3 (Psa3) was the only biovar consisting of three widely distributed clades in the largest Chinese kiwifruit cultivated area. Comparative genomics between the three clades revealed 13 polymorphic genes, each of which had multiple intra-clade variations. For instance, we confirmed that the polymorphic copA gene, which encodes a periplasmic protein CopA that is translocated by the Twin-arginine targeting (Tat) system, was involved in copper tolerance. We also found extensive variation in pathogenicity amongst strains within each genetically monomorphic clade. Accordingly, the pathogenic determinants of Psa3 were identified via a genomic comparison of phenotypically different strains within each clade. A case study of the high- and low-virulence strains in the clade 2 of Psa3 revealed that an hfq variant involved in in vitro growth and virulence, while a conserved locus 930 bp upstream of the hrpR gene in the Type III secretion system (T3SS) cluster was required for full pathogenicity on kiwifruit and elicitation of the hypersensitivity response on non-host Nicotiana benthamiana. The '-930' locus is involved in transcriptional regulation of hrpR/S and modulates T3SS function via the hierarchical 'HrpR/S-HrpL-T3SS/effector' regulatory cascade in Psa. Our results provide insights into the molecular basis underlying the genetic diversification and evolution of pathogenicity in Psa3 since kiwifruit canker emerged in China in the 1980s.

Visualization and characterization of Pseudomonas syringae pv. tomato DC3000 pellicles.

Cellulose, whose production is controlled by c-di-GMP, is a commonly found exopolysaccharide in bacterial biofilms. Pseudomonas syringae pv. tomato (Pto) DC3000, a model organism for molecular studies of plant-pathogen interactions, carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose. The high intracellular levels of the second messenger c-di-GMP induced by the overexpression of the heterologous diguanylate cyclase PleD stimulate cellulose production and enhance air-liquid biofilm (pellicle) formation. To characterize the mechanisms involved in Pto DC3000 pellicle formation, we studied this process using mutants lacking flagella, biosurfactant or different extracellular matrix components, and compared the pellicles produced in the absence and in the presence of PleD. We have discovered that neither alginate nor the biosurfactant syringafactin are needed for their formation, whereas cellulose and flagella are important but not essential. We have also observed that the high c-di-GMP levels conferred more cohesion to Pto cells within the pellicle and induced the formation of intracellular inclusion bodies and extracellular fibres and vesicles. Since the pellicles were very labile and this greatly hindered their handling and processing for microscopy, we have also developed new methods to collect and process them for scanning and transmission electron microscopy. These techniques open up new perspectives for the analysis of fragile biofilms in other bacterial strains.

Comparative transcriptome analysis of resistant and susceptible kiwifruits in response to Pseudomonas syringae pv. Actinidiae during early infection.

Kiwifruit bacterial canker is a devastating disease threatening kiwifruit production. To clarify the defense mechanism in response to Pseudomonas syringae pv. actinidiae (Psa), we observed phenotypic changes in resistant Huate (HT) and susceptible Hongyang (HY) kiwifruit varieties at 0, 12, 24, 48, 96, and 144 hour after inoculation (hai) with Psa. Brown lesions appeared in the inoculation areas 12 hai in HY shoots, and the lesion length gradually increased from 24 to 144 h. In contrast, no lesions were found in HT shoots at any time points. Furthermore, RNA-seq analysis showed significantly more differentially expressed genes between HT and HY at 12 hai than at any other time point. According to weighted gene co-expression network analysis, five modules were notably differentially expressed between HT and HY; pathway mapping using the Kyoto Encyclopedia of Gene and Genomes database was performed for the five modules. In MEgreenyellow and MEyellow modules, pathways related to"plant-pathogen interaction", "Endocytosis", "Glycine, serine and threonine metabolism", and "Carbon fixation in photosynthetic organisms" were enriched, whereas in the MEblack module, pathways related to "protein processing in endoplasmic reticulum", "plant-pathogen interaction", and "Glycolysis / Gluconeogenesis" were enriched. In particular, the Pti1 and RPS2 encoding effector receptors, and the NPR1, TGA, and PR1 genes involved in the salicylic acid signaling pathway were significantly up-regulated in HT compared with HY. This indicates that the effector-triggered immunity response was stronger and that the salicylic acid signaling pathway played a pivotal role in the Psa defense response of HT. In addition, we identified other important genes, involved in phenylpropanoid biosynthesis and Ca2+ internal flow, which were highly expressed in HT. Taken together, these results provide important information to elucidate the defense mechanisms of kiwifruit during Psa infection.