Antagonistic activity of endophytic actinobacteria from native potatoes (Solanum tuberosum subsp. tuberosum L.) against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum.

BACKGROUND: The native potatoes (Solanum tuberosum subsp. tuberosum L.) grown in Chile (Chiloé) represent a new, unexplored source of endophytes to find potential biological control agents for the prevention of bacterial diseases, like blackleg and soft rot, in potato crops. RESULT: The objective of this study was the selection of endophytic actinobacteria from native potatoes for antagonistic activity against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum, and their potential to suppress tissue maceration symptoms in potato tubers. This potential was determined through the quorum quenching activity using a Chromobacterium violaceaum ATCC 12472 Wild type (WT) bioassay and its colonization behavior of the potato plant root system (S. tuberosum) by means of the Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) targeting technique. The results showed that although Streptomyces sp. TP199 and Streptomyces sp. A2R31 were able to inhibit the growth of the pathogens, only the Streptomyces sp. TP199 isolate inhibited Pectobacterium sp. growth and diminished tissue maceration in tubers (p ≤ 0.05). Streptomyces sp. TP199 had metal-dependent acyl homoserine lactones (AHL) quorum quenching activity in vitro and was able to colonize the root endosphere 10 days after inoculation. CONCLUSIONS: We concluded that native potatoes from southern Chile possess endophyte actinobacteria that are potential agents for the disease management of soft rot and blackleg.

The Role of Pectobacterium atrosepticum Exopolysaccharides in Plant-Pathogen Interactions.

The phytopathogenic bacterium Pectobacterium atrosepticum (Pba), one of the members of the soft rot Pectobacteriaceae, forms biofilm-like structures known as bacterial emboli when colonizing the primary xylem vessels of the host plants. The initial extracellular matrix of the bacterial emboli is composed of the host plant's pectic polysaccharides, which are gradually substituted by the Pba-produced exopolysaccharides (Pba EPS) as the bacterial emboli "mature". No information about the properties of Pba EPS and their possible roles in Pba-plant interactions has so far been obtained. We have shown that Pba EPS possess physical properties that can promote the maintenance of the structural integrity of bacterial emboli. These polymers increase the viscosity of liquids and form large supramolecular aggregates. The formation of Pba EPS aggregates is provided (at least partly) by the acetyl groups of the Pba EPS molecules. Besides, Pba EPS scavenge reactive oxygen species (ROS), the accumulation of which is known to be associated with the formation of bacterial emboli. In addition, Pba EPS act as suppressors of the quantitative immunity of plants, repressing PAMP-induced reactions; this property is partly lost in the deacetylated form of Pba EPS. Overall, our study shows that Pba EPS play structural, protective, and immunosuppressive roles during Pba-plant interactions and thus should be considered as virulence factors of these bacteria.

Use of a Specific Phage Cocktail for Soft Rot Control on Ware Potatoes: A Case Study.

Using bacteriophages (bacterial viruses) to control pathogenic bacteria is a promising approach in horticulture. However, the application of this strategy in real conditions requires compliance with particular technological and environmental restraints. The presented paper concerns the process of phage selection to create a cocktail that is efficient against the circulating causal agents of potato soft rot. The resulting phage cocktail causes a complete lysis of a mixture of circulating pectobacterial strains in vitro. In the context of being used to treat ware potatoes during off-season storage, the protocol of phage application via the humidity maintenance system was designed. The phage cocktail was shown to reduce the population of Pectobacterium spp. 10-12-fold, achieving a population that was below a symptomatic threshold.

Antibacterial activity and mode of action of potassium tetraborate tetrahydrate against soft-rot bacterial plant pathogens.

Bacterial soft rot caused by the bacteria Dickeya and Pectobacterium is a destructive disease of vegetables, as well as ornamental plants. Several management options exist to help control these pathogens. Because of the limited success of these approaches, there is a need for the development of alternative methods to reduce losses. In this study, we evaluated the effect of potassium tetraborate tetrahydrate (PTB) on the growth of six Dickeya and Pectobacterium spp. Disc diffusion assays showed that Dickeya spp. and Pectobacterium spp. differ in their sensitivity to PTB. Spontaneous PTB-resistant mutants of Pectobacterium were identified and further investigation of the mechanism of PTB resistance was conducted by full genome sequencing. Point mutations in genes cpdB and supK were found in a single Pectobacterium atrosepticum PTB-resistant mutant. Additionally, point mutations in genes prfB (synonym supK) and prmC were found in two independent Pectobacterium brasiliense PTB-resistant mutants. prfB and prmC encode peptide chain release factor 2 and its methyltransferase, respectively. We propose the disruption of translation activity due to PTB leads to Pectobacterium growth inhibition. The P. atrosepticum PTB-resistant mutant showed altered swimming motility. Disease severity was reduced for P. atrosepticum-inoculated potato stems sprayed with PTB. We discuss the potential risk of selecting for bacterial resistance to this chemical.

Expansin-like Exl1 from Pectobacterium is a virulence factor required for host infection, and induces a defence plant response involving ROS, and jasmonate, ethylene and salicylic acid signalling pathways in Arabidopsis thaliana.

Expansins are encoded by some phytopathogenic bacteria and evidence indicates that they act as virulence factors for host infection. Here we analysed the expression of exl1 by Pectobacterium brasiliense and Pectobacterium atrosepticum. In both, exl1 gene appears to be under quorum sensing control, and protein Exl1 can be observed in culture medium and during plant infection. Expression of exl1 correlates with pathogen virulence, where symptoms are reduced in a Δexl1 mutant strain of P. atrosepticum. As well as Δexl1 exhibiting less maceration of potato plants, fewer bacteria are observed at distance from the inoculation site. However, bacteria infiltrated into the plant tissue are as virulent as the wild type, suggesting that this is due to alterations in the initial invasion of the tissue. Additionally, swarming from colonies grown on MacConkey soft agar was delayed in the mutant in comparison to the wild type. We found that Exl1 acts on the plant tissue, probably by remodelling of a cell wall component or altering the barrier properties of the cell wall inducing a plant defence response, which results in the production of ROS and the induction of marker genes of the JA, ET and SA signalling pathways in Arabidopsis thaliana. Exl1 inactive mutants fail to trigger such responses. This defence response is protective against Pectobacterium brasiliense and Botrytis cinerea in more than one plant species.

Transcriptome of Pectobacterium carotovorum subsp. carotovorum PccS1 infected in calla plants in vivo highlights a spatiotemporal expression pattern of genes related to virulence, adaptation, and host response.

Bacterial pathogens from the genus Pectobacterium cause soft rot in various plants, and result in important economic losses worldwide. We understand much about how these pathogens digest their hosts and protect themselves against plant defences, as well as some regulatory networks in these processes. However, the spatiotemporal expression of genome-wide infection of Pectobacterium remains unclear, although researchers analysed this in some phytopathogens. In the present work, comparing the transcriptome profiles from cellular infection with growth in minimal and rich media, RNA-Seq analyses revealed that the differentially expressed genes (log2 -fold ratio ≥ 1.0) in the cells of Pectobacterium carotovorum subsp. carotovorum PccS1 recovered at a series of time points after inoculation in the host in vivo covered approximately 50% of genes in the genome. Based on the dynamic expression changes in infection, the significantly differentially expressed genes (log2 -fold ratio ≥ 2.0) were classified into five types, and the main expression pattern of the genes for carbohydrate metabolism underlying the processes of infection was identified. The results are helpful to our understanding of the inducement of host plant and environmental adaption of Pectobacterium. In addition, our results demonstrate that maceration caused by PccS1 is due to the depression of callose deposition in the plant for resistance by the pathogenesis-related genes and the superlytic ability of pectinolytic enzymes produced in PccS1, rather than the promotion of plant cell death elicited by the T3SS of bacteria as described in previous work.

The Great Five-an artificial bacterial consortium with antagonistic activity towards Pectobacterium spp. and Dickeya spp.: formulation, shelf life, and the ability to prevent soft rot of potato in storage.

"The Great Five" (GF) is an artificial bacterial consortium developed to protect potato tubers from soft rot caused by Pectobacterium spp. and Dickeya spp. To investigate the commercialization potential of the GF, we developed liquid and powder formulations of the consortium and of each of the comprising strains (Serratia plymuthica strain A294, Enterobacter amnigenus strain A167, Rahnella aquatilis strain H145, Serratia rubidaea strain H440, and S. rubidaea strain H469). To form powders, the cells were lyophilized using a newly developed lyoprotectant: Reagent PS. The shelf life of the formulations stored at 8 and 22 °C was monitored for a period of 12 months. The longest shelf life was obtained for formulations stored at 8 °C; however, the viability of all formulations was negatively affected at 22 °C. For the consortium, a 2.5 log10 cfu (colony forming units) drop in cell number was recorded for the liquid formulation after 6 months, while in case of powders, the drop remained below 1 log10 cfu following 12 months. The ability of the powder formulations to preserve biocontrol activity of the consortium was tested on potato tubers treated with the formulations and a mixture of the soft rot pathogens. The inoculated tubers were stored for 6 months at 8 °C to mimic commercial storage conditions. Soft rot severity and incidence on potato tubers treated with formulations were significantly reduced (62-75% and 48-61%, respectively) in comparison to positive control with pathogens alone. The potential use of the newly developed formulations of "The Great Five" for the biocontrol of soft rot is discussed. KEY POINTS : • An innovative reagent to protect bacterial cells during lyophilization was developed. • Powder formulations of "The Great Five" prolonged its shelf life. • The powder-formulated "The Great Five" was active against soft rot bacteria on potato tubers.

Analysis of two Mexican Pectobacterium brasiliense strains reveals an inverted relationship between c-di-GMP levels with exopolysaccharide production and swarming motility.

Pectobacterium is a diverse genus of phytopathogenic species from soil and water that cause infection either to restricted or multiple plant hosts. Phylogenetic analysis and metabolic fingerprinting of large numbers of genomes have expanded classification of Pectobacterium members. Pectobacterium brasiliense sp. nov has been elevated to the species level having detached from P. carotovorum. Here we present two P. brasiliense strains BF20 and BF45 isolated in Mexico from Opuntia and tobacco, respectively, which cluster into two different groups in whole genome comparisons with other Pectobacterium. We found that BF20 and BF45 strains are phenotypically different as BF45 showed more severe and rapid symptoms in comparison to BF20 in the host models celery and broccoli. Both strains produced similar levels of the main autoinducers, but BF45 shows an additional low abundant autoinducer compared to strain BF20. The two strains had different levels of c-di-GMP, which regulates the transition from motile to sessile lifestyle. In contrast to BF45, BF20 had the highest levels of c-di-GMP, was more motile (swarming), non-flocculant and less proficient in biofilm formation and exopolysaccharide production. Genomic comparisons revealed that differences in c-di-GMP accumulation and perhaps the associated phenotypes might be due to unique c-di-GMP metabolic genes in these two strains. Our results improve our understanding of the associations between phenotype and genotype and how this has shaped the physiology of Pectobacterium strains.

Compatible Mixture of Bacterial Antagonists Developed to Protect Potato Tubers from Soft Rot Caused by Pectobacterium spp. and Dickeya spp.

Possibilities to protect potato tubers from rotting caused by Soft Rot Pectobacteriaceae (SRP) under disease favoring conditions were investigated using compatible mixtures of bacterial antagonists and tested with a newly developed stepwise efficacy-based screening protocol. Twenty-two bacterial antagonists were evaluated against a combination of five Pectobacterium and Dickeya strains representing species and subspecies most often associated with potato soft rot in Europe. To enable potential synergistic activity, the antagonists were initially tested against the combination of pathogens in 15 random mixtures containing up to 5 antagonists each. Three mixtures (M2, M4, and M14) out of 15 tested reduced tuber tissue maceration due to soft rot. The individual antagonists derived from M2, M4, and M14 mixtures were tested on potato slices and whole tuber injection assays. These five strains (S. plymuthica strain A294, E. amnigenus strain A167, R. aquatilis strain H145, S. rubidaea strain H440, and S. rubidaea strain H469) were combined to develop a tailored biological control mixture against potato soft rot. The new mixture, designated the Great Five (GF), was tested on seed potato tubers vacuum infiltrated with antagonists and subsequently with the combination of five SRP pathogens. In these experiments, the GF mixture provided stable protection of inoculated potato tubers, reducing soft rot by 46% (P = 0.0016) under high disease pressure conditions. The A294, A167, H145, H440, and H469 antagonists were characterized for features important for viable commercial applications including growth at different temperatures, resistance to antibiotics, and potential toxicity toward Caenorhabditis elegans. The implications for control of soft rot caused by SRP with the use of the GF mixture of antagonists are discussed.

Biocontrol of Soft Rot: Confocal Microscopy Highlights Virulent Pectobacterial Communication and Its Jamming by Rhodococcal Quorum-Quenching.

Confocal laser-scanning microscopy was chosen to observe the colonization and damage caused by the soft rot Pectobacterium atrosepticum and the protection mediated by the biocontrol agent Rhodococcus erythropolis. We developed dual-color reporter strains suited for monitoring quorum-sensing and quorum-quenching activities leading to maceration or biocontrol, respectively. A constitutively expressed cyan or red fluorescent protein served as a cell tag for plant colonization, while an inducible expression reporter system based on the green fluorescent protein gene enabled the simultaneous recording of signaling molecule production, detection, or degradation. The dual-colored pathogen and biocontrol strains were used to coinoculate potato tubers. At cellular quorum, images revealed a strong pectobacterial quorum-sensing activity, especially at the plant cell walls, as well as a concomitant rhodococcal quorum-quenching response, at both the single-cell and microcolony levels. The generated biosensors appear to be promising and complementary tools useful for molecular and cellular studies of bacterial communication and interference.

Integration Analysis of Small RNA and Degradome Sequencing Reveals MicroRNAs Responsive to Dickeya zeae in Resistant Rice.

Rice foot rot disease caused by the pathogen Dickeya zeae (formerly known as Erwinia chrysanthemi pv. zeae), is a newly emerging damaging bacterial disease in China and the southeast of Asia, resulting in the loss of yield and grain quality. However, the genetic resistance mechanisms mediated by miRNAs to D. zeae are unclear in rice. In the present study, 652 miRNAs including osa-miR396f predicted to be involved in multiple defense responses to D. zeae were identified with RNA sequencing. A total of 79 differentially expressed miRNAs were detected under the criterion of normalized reads ≥10, including 51 known and 28 novel miRNAs. Degradome sequencing identified 799 targets predicted to be cleaved by 168 identified miRNAs. Among them, 29 differentially expressed miRNA and target pairs including miRNA396f-OsGRFs were identified by co-expression analysis. Overexpression of the osa-miR396f precursor in a susceptible rice variety showed enhanced resistance to D. zeae, coupled with significant accumulation of transcripts of osa-miR396f and reduction of its target the Growth-Regulating Factors (OsGRFs). Taken together, these findings suggest that miRNA and targets including miR396f-OsGRFs have a role in resisting the infections by bacteria D. zeae.

Transcriptome and Comparative Genomics Analyses Reveal New Functional Insights on Key Determinants of Pathogenesis and Interbacterial Competition in Pectobacterium and Dickeya spp.

Soft-rot Enterobacteriaceae (SRE), typified by Pectobacterium and Dickeya genera, are phytopathogenic bacteria inflicting soft-rot disease in crops worldwide. By combining genomic information from 100 SRE with whole-transcriptome data sets, we identified novel genomic and transcriptional associations among key pathogenicity themes in this group. Comparative genomics revealed solid linkage between the type I secretion system (T1SS) and the carotovoricin bacteriophage (Ctv) conserved in 96.7% of Pectobacterium genomes. Moreover, their coactivation during infection indicates a novel functional association involving T1SS and Ctv. Another bacteriophage-borne genomic region, mostly confined to less than 10% of Pectobacterium strains, was found, presumably comprising a novel lineage-specific prophage in the genus. We also detected the transcriptional coregulation of a previously predicted toxin/immunity pair (WHH and SMI1_KNR4 families), along with the type VI secretion system (T6SS), which includes hcp and/or vgrG genes, suggesting a role in disease development as T6SS-dependent effectors. Further, we showed that another predicted T6SS-dependent endonuclease (AHH family) exhibited toxicity in ectopic expression assays, indicating antibacterial activity. Additionally, we report the striking conservation of the group 4 capsule (GFC) cluster in 100 SRE strains which consistently features adjacently conserved serotype-specific gene arrays comprising a previously unknown organization in GFC clusters. Also, extensive sequence variations found in gfcA orthologs suggest a serotype-specific role in the GfcABCD machinery.IMPORTANCE Despite the considerable loss inflicted on important crops yearly by Pectobacterium and Dickeya diseases, investigations on key virulence and interbacterial competition assets relying on extensive comparative genomics are still surprisingly lacking for these genera. Such approaches become more powerful over time, underpinned by the growing amount of genomic information in public databases. In particular, our findings point to new functional associations among well-known genomic themes enabling alternative means of neutralizing SRE diseases through disruption of pivotal virulence programs. By elucidating novel transcriptional and genomic associations, this study adds valuable information on virulence candidates that could be decisive in molecular applications in the near future. The utilization of 100 genomes of Pectobacterium and Dickeya strains in this study is unprecedented for comparative analyses in these taxa, and it provides novel insights on the biology of economically important plant pathogens.

The Changing Face of Bacterial Soft-Rot Diseases.

Bacterial soft rot is a disease complex caused by multiple genera of gram-negative and gram-positive bacteria, with Dickeya and Pectobacterium being the most widely studied soft-rot bacterial pathogens. In addition to soft rot, these bacteria also cause blackleg of potato, foot rot of rice, and bleeding canker of pear. Multiple Dickeya and Pectobacterium species cause the same symptoms on potato, complicating epidemiology and disease resistance studies. The primary pathogen species present in potato-growing regions differs over time and space, further complicating disease management. Genomics technologies are providing new management possibilities, including improved detection and biocontrol methods that may finally allow effective disease management. The recent development of inbred diploid potato lines is also having a major impact on studying soft-rot pathogens because it is now possible to study soft-rot disease in model plant species that produce starchy vegetative storage organs. Together, these new discoveries have changed how we face diseases caused by these pathogens.

Genomic characteristics of vB_PpaP_PP74, a T7-like Autographivirinae bacteriophage infecting a potato pathogen of the newly proposed species Pectobacterium parmentieri.

Bacteriophage vB_PpaP_PP74 (PP74) is a novel virulent phage that infects members of the species Pectobacterium parmentieri, a newly established species of soft-rot-causing bacteria in the family Pectobacteriaceae, derived from potato-specific Pectobacterium wasabiae. vB_PpaP_PP74 was identified as a member of the family Podoviridae by transmission electron microscopy. The phage has a 39,790-bp dsDNA genome containing 50 open reading frames (ORFs). Because of the absence of genes encoding toxins or lysogeny factors, PP74 may be considered a candidate phage for pathogen biocontrol applications. The genome layout is similar to genomes of T7-like phages within the subfamily Autographivirinae, and therefore, functions can be attributed to most of ORFs. However, the closest nucleotide sequence homologs of phage PP74 are unclassified Escherichia phages. Based on phylogenetic analysis, vB_PpaP_PP74 is a sensu lato T7-like phage, but it forms a distant subgenus group together with homologous enterobacterial phages.

Molecular methods as tools to control plant diseases caused by Dickeya and Pectobacterium spp: A minireview.

Dickeya spp. and Pectobacterium spp. are etiological agents of soft rot on crops, vegetables, and ornamentals. They also cause blackleg on potato. These pectinolytic phytopathogens are responsible for significant economic losses, mostly within the potato production sector. Importantly, there are no methods to eradicate these microorganisms once they have infected plant material. Solely preventive measures remain, including early detection and identification of the pathogens, monitoring of their spread in addition to planting certified seed material tested for latent infections. As proper identification of the causative agent allows for efficient limitation of disease spread, numerous detection and differentiation methods have been developed. Most commonly followed procedures involve: isolation of viable bacterial cells (alternatively post-enrichment) on semi-selective media, identification to species level by PCR (single, multiplex, Real time), serology or fatty acids profiling. Differentiation of the isolates is often accomplished by sequencing the housekeeping genes or molecular fingerprinting. In view of lowering total costs of next-generation sequencing (NGS), a huge amount of generated data reveals subtle differences between strains that have proven to be potentially useful for the establishment of specific novel detection pipelines. Successful implementation of molecular diagnostic methods is exemplified by 20-year studies on the populations of pectinolytic bacteria on potatoes in Poland. The presented work aims to gather the characteristics of Dickeya spp. and Pectobacterium spp. important for the identification process in addition to providing an overview of modern and newly developed specific, rapid, high-throughput and cost-effective screening methods for the detection and identification of these phytopathogens.

Plant phenolic volatiles inhibit quorum sensing in pectobacteria and reduce their virulence by potential binding to ExpI and ExpR proteins.

Quorum sensing (QS) is a population density-dependent regulatory system in bacteria that couples gene expression to cell density through accumulation of diffusible signaling molecules. Pectobacteria are causal agents of soft rot disease in a range of economically important crops. They rely on QS to coordinate their main virulence factor, production of plant cell wall degrading enzymes (PCWDEs). Plants have evolved an array of antimicrobial compounds to anticipate and cope with pathogens, of which essential oils (EOs) are widely recognized. Here, volatile EOs, carvacrol and eugenol, were shown to specifically interfere with QS, the master regulator of virulence in pectobacteria, resulting in strong inhibition of QS genes, biofilm formation and PCWDEs, thereby leading to impaired infection. Accumulation of the signal molecule N-acylhomoserine lactone declined upon treatment with EOs, suggesting direct interaction of EOs with either homoserine lactone synthase (ExpI) or with the regulatory protein (ExpR). Homology models of both proteins were constructed and docking simulations were performed to test the above hypotheses. The resulting binding modes and docking scores of carvacrol and eugenol support potential binding to ExpI/ExpR, with stronger interactions than previously known inhibitors of both proteins. The results demonstrate the potential involvement of phytochemicals in the control of Pectobacterium.

pH-Dependent DNA Distortion and Repression of Gene Expression by Pectobacterium atrosepticum PecS.

Transcriptional activity is exquisitely sensitive to changes in promoter DNA topology. Transcription factors may therefore control gene activity by modulating the relative positioning of -10 and -35 promoter elements. The plant pathogen Pectobacterium atrosepticum, which causes soft rot in potatoes, must alter gene expression patterns to ensure growth in planta. In the related soft-rot enterobacterium Dickeya dadantii, PecS functions as a master regulator of virulence gene expression. Here, we report that P. atrosepticum PecS controls gene activity by altering promoter DNA topology in response to pH. While PecS binds the pecS promoter with high affinity regardless of pH, it induces significant DNA distortion only at neutral pH, the pH at which the pecS promoter is repressed in vivo. At pH ∼8, DNA distortions are attenuated, and PecS no longer represses the pecS promoter. A specific histidine (H142) located in a crevice between the dimerization- and DNA-binding regions is required for pH-dependent changes in DNA distortion and repression of gene activity, and mutation of this histidine renders the mutant protein incapable of repressing the pecS promoter. We propose that protonated PecS induces a DNA conformation at neutral pH in which -10 and -35 promoter elements are suboptimally positioned for RNA polymerase binding; on deprotonation of PecS, binding is no longer associated with significant changes in DNA conformation, allowing gene expression. We suggest that this mode of gene regulation leads to differential expression of the PecS regulon in response to alkalinization of the plant apoplast.

Pathogen-induced conditioning of the primary xylem vessels - a prerequisite for the formation of bacterial emboli by Pectobacterium atrosepticum.

Representatives of Pectobacterium genus are some of the most harmful phytopathogens in the world. In the present study, we have elucidated novel aspects of plant-Pectobacterium atrosepticum interactions. This bacterium was recently demonstrated to form specific 'multicellular' structures - bacterial emboli in the xylem vessels of infected plants. In our work, we showed that the process of formation of these structures includes the pathogen-induced reactions of the plant. The colonisation of the plant by P. atrosepticum is coupled with the release of a pectic polysaccharide, rhamnogalacturonan I, into the vessel lumen from the plant cell wall. This polysaccharide gives rise to a gel that serves as a matrix for bacterial emboli. P. atrosepticum-caused infection involves an increase of reactive oxygen species (ROS) levels in the vessels, creating the conditions for the scission of polysaccharides and modification of plant cell wall composition. Both the release of rhamnogalacturonan I and the increase in ROS precede colonisation of the vessels by bacteria and occur only in the primary xylem vessels, the same as the subsequent formation of bacterial emboli. Since the appearance of rhamnogalacturonan I and increase in ROS levels do not hamper the bacterial cells and form a basis for the assembly of bacterial emboli, these reactions may be regarded as part of the susceptible response of the plant. Bacterial emboli thus represent the products of host-pathogen integration, since the formation of these structures requires the action of both partners.

Alternative scenarios of starvation-induced adaptation in Pectobacterium atrosepticum.

Bacteria have high adaptive potential that ensures their survival during various environmental challenges. To adapt, bacteria activate a physiological program of stress response that makes them able to persist under adverse conditions. The present study sought to examine the ability of a particular bacterial species to induce a stress response in alternative scenarios. Cells of the phytopathogenic microorganism Pectobacterium atrosepticum were taken as a model. The cells were exposed to starvation in different physiological states (actively growing exponential phase and stationary phase cells), and the resulting starving cultures were monitored using CFU counting, quantitative PCR and electron microscopy. When exponential phase cells were subjected to starvation, the nucleoids of the cells became condensed and their DNA was detected by qPCR less effectively than that of cells growing in nutrient-rich medium, or stationary phase cells after starvation. Exponential phase cells subjected to starvation showed increased expression of genes encoding DNA binding histone-like proteins, whereas, in cultures inoculated by stationary phase cells, cell-wall-deficient forms that were inefficient at colony forming and that had a non-culturable phenotype were formed. The cell-wall-deficient forms displayed reduced expression of genes encoding synthases of cell wall components.

GRP-3 and KAPP, encoding interactors of WAK1, negatively affect defense responses induced by oligogalacturonides and local response to wounding.

Conserved microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs) act as danger signals to activate the plant immune response. These molecules are recognized by surface receptors that are referred to as pattern recognition receptors. Oligogalacturonides (OGs), DAMPs released from the plant cell wall homogalacturonan, have also been proposed to act as local signals in the response to wounding. The Arabidopsis Wall-Associated Kinase 1 (WAK1), a receptor of OGs, has been described to form a complex with a cytoplasmic plasma membrane-localized kinase-associated protein phosphatase (KAPP) and a glycine-rich protein (GRP-3) that we find localized mainly in the cell wall and, in a small part, on the plasma membrane. By using Arabidopsis plants overexpressing WAK1, and both grp-3 and kapp null insertional mutant and overexpressing plants, we demonstrate a positive function of WAK1 and a negative function of GRP-3 and KAPP in the OG-triggered expression of defence genes and the production of an oxidative burst. The three proteins also affect the local response to wounding and the basal resistance against the necrotrophic pathogen Botrytis cinerea. GRP-3 and KAPP are likely to function in the phasing out of the plant immune response.