Surviving the Potato Stems: Differences in Genes Required for Fitness by Dickeya dadantii and Dickeya dianthicola.

Bacteria belonging to the genus Dickeya cause blackleg and soft rot symptoms on many plant hosts, including potato. Although there is considerable knowledge about the genetic determinants that allow Dickeya to colonize host plants, as well as the genes that contribute to virulence, much is still unknown. To identify the genes important for fitness in potato stems, we constructed and evaluated randomly barcoded transposon mutant (RB-TnSeq) libraries of Dickeya dadantii and Dickeya dianthicola. We identified 169 and 157 genes important for growth in D. dadantii and D. dianthicola in stems, respectively. This included genes related to metabolic pathways, chemotaxis and motility, transcriptional regulation, transport across membranes, membrane biogenesis, detoxification mechanisms, and virulence-related genes, including a potential virulence cluster srfABC, c-di-GMP modulating genes, and pectin degradation genes. When we compared the results of the stem assay with other datasets, we identified genes important for growth in stems versus tubers and in vitro conditions. Additionally, our data showed differences in fitness determinants for D. dadantii and D. dianthicola. These data provide important insights into the mechanisms used by Dickeya when interacting with and colonizing plants and thus might provide targets for management.

Helper NLRs Nrc2 and Nrc3 act codependently with Prf/Pto and activate MAPK signaling to induce immunity in tomato.

Plant intracellular immune receptors, primarily nucleotide-binding, leucine-rich repeat proteins (NLRs), detect pathogen effector proteins and activate NLR-triggered immunity (NTI). Recently, 'sensor' NLRs have been reported to function with 'helper' NLRs to activate immunity. We investigated the role of two helper NLRs, Nrc2 and Nrc3, on immunity in tomato to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) mediated by the sensor NLR Prf and the Pto kinase. An nrc2/nrc3 mutant no longer activated Prf/Pto-mediated NTI to Pst containing the effectors AvrPto and AvrPtoB. An nrc3 mutant showed intermediate susceptibility between wild-type plants and a Prf mutant, while an nrc2 mutant developed only mild disease. These observations indicate that Nrc2 and Nrc3 act additively in Prf-/Pto-mediated immunity. We examined at what point Nrc2 and Nrc3 act in the Prf/Pto-mediated immune response. In the nrc2/3 mutant, programmed cell death (PCD) normally induced by constitutively active variants of AvrPtoB, Pto, or Prf was abolished, but that induced by M3Kα or Mkk2 was not. PCD induced by a constitutively active Nrc3 was also abolished in a Nicotiana benthamiana line with reduced expression of Prf. MAPK activation triggered by expression of AvrPto in the wild-type tomato plants was completely abolished in the nrc2/3 mutant. These results indicate that Nrc2 and Nrc3 act with Prf/Pto and upstream of MAPK signaling. Nrc2 and Nrc3 were not required for PCD triggered by Ptr1, another sensor NLR-mediating Pst resistance, although these helper NLRs do appear to be involved in resistance to certain Pst race 1 strains.

Complete Genome Sequence Resource for Xanthomonas hortorum Isolated from Greek Oregano.

In spring 2019, necrotic leaf spots were detected on Greek oregano (Origanum vulgare var. hirtum) plants in a commercial greenhouse operation. An isolate was recovered from the diseased plants. Partial 16S ribosomal RNA sequencing and multilocus sequence analysis revealed that the isolate was a Xanthomonas sp. but proved insufficient to identify the species with certainty. Therefore, whole-genome sequencing using both Nanopore and Illumina technologies was performed. Here, we report the complete and annotated genome sequence of Xanthomonas hortorum strain 108, which was originally isolated from Greek oregano in Long Island, NY, U.S.A.

Pseudomonas syringae Type III Secretion Protein HrpP Manipulates Plant Immunity To Promote Infection.

The bacterial plant pathogen Pseudomonas syringae deploys a type III secretion system (T3SS) to deliver effector proteins into plant cells to facilitate infection, for which many effectors have been characterized for their interactions. However, few T3SS Hrp (hypersensitive response and pathogenicity) proteins from the T3SS secretion apparatus have been studied for their direct interactions with plants. Here, we show that the P. syringae pv. tomato DC3000 T3SS protein HrpP induces host cell death, suppresses pattern-triggered immunity (PTI), and restores the effector translocation ability of the hrpP mutant. The hrpP-transgenic Arabidopsis lines exhibited decreased PTI responses to flg22 and elf18 and enhanced disease susceptibility to P. syringae pv. tomato DC3000. Transcriptome analysis reveals that HrpP sensing activates salicylic acid (SA) signaling while suppressing jasmonic acid (JA) signaling, which correlates with increased SA accumulation and decreased JA biosynthesis. Both yeast two-hybrid and bimolecular fluorescence complementation assays show that HrpP interacts with mitogen-activated protein kinase kinase 2 (MKK2) on the plant membrane and in the nucleus. The HrpP truncation HrpP1-119, rather than HrpP1-101, retains the ability to interact with MKK2 and suppress PTI in plants. In contrast, HrpP1-101 continues to cause cell death and electrolyte leakage. MKK2 silencing compromises SA signaling but has no effect on cell death caused by HrpP. Overall, our work highlights that the P. syringae T3SS protein HrpP facilitates effector translocation and manipulates plant immunity to facilitate bacterial infection. IMPORTANCE The T3SS is required for the virulence of many Gram-negative bacterial pathogens of plants and animals. This study focuses on the sensing and function of the T3SS protein HrpP during plant interactions. Our findings show that HrpP and its N-terminal truncation HrpP1-119 can interact with MKK2, promote effector translocation, and manipulate plant immunity to facilitate bacterial infection, highlighting the P. syringae T3SS component involved in the fine-tuning of plant immunity.

Genomic insights into a Pseudomonas amygdali isolate from Hibiscus rosa-sinensis.

The taxonomy of Pseudomonas has been extensively studied, yet the determination of species is currently difficult because of recent taxonomic changes and the lack of complete genomic sequence data. We isolated a bacterium causing a leaf spot disease on hibiscus (Hibiscus rosa-sinensis). Whole genome sequencing revealed similarity to Pseudomonas amygdali pv. tabaci and pv. lachrymans. The genome of this isolate (referred to as P. amygdali 35-1) shared 4987 genes with P. amygdali pv. hibisci, but possessed 204 unique genes and contained gene clusters encoding putative secondary metabolites and copper resistance determinants. We predicted this isolate's type III secretion effector (T3SE) repertoire and identified 64 putative T3SEs, some of which are present in other P. amygdali pv. hibisci strains. Assays showed that the isolate was resistant to copper at a concentration of 1.6 mM. This study provides an improved understanding of the genomic relatedness and diversity of the P. amygdali species.

Genome-Wide Identification of Genes Important for Growth of Dickeya dadantii and Dickeya dianthicola in Potato (Solanum tuberosum) Tubers.

Dickeya species are causal agents of soft rot diseases in many economically important crops, including soft rot disease of potato (Solanum tuberosum). Using random barcode transposon-site sequencing (RB-TnSeq), we generated genome-wide mutant fitness profiles of Dickeya dadantii 3937, Dickeya dianthicola ME23, and Dickeya dianthicola 67-19 isolates collected after passage through several in vitro and in vivo conditions. Though all three strains are pathogenic on potato, D. dadantii 3937 is a well-characterized model while D. dianthicola strains ME23 and 67-19 are recent isolates. Strain ME23 specifically was identified as a representative strain from a 2014 outbreak on potato. This study generated comparable gene fitness measurements across ecologically relevant conditions for both model and non-model strains. Tubers from the potato cultivars "Atlantic," "Dark Red Norland," and "Upstate Abundance" provided highly similar conditions for bacterial growth. Using the homolog detection software PyParanoid, we matched fitness values for orthologous genes in the three bacterial strains. Direct comparison of fitness among the strains highlighted shared and variable traits important for growth. Bacterial growth in minimal medium required many metabolic traits that were also essential for competitive growth in planta, such as amino acid, carbohydrate, and nucleotide biosynthesis. Growth in tubers specifically required the pectin degradation gene kduD. Disruption in three putative DNA-binding proteins had strain-specific effects on competitive fitness in tubers. Though the Soft Rot Pectobacteriaceae can cause disease with little host specificity, it remains to be seen the extent to which strain-level variation impacts virulence.

Identification of Indole-3-Acetic Acid-Regulated Genes in Pseudomonas syringae pv. tomato Strain DC3000.

The auxin indole-3-acetic acid (IAA) is a plant hormone that not only regulates plant growth and development but also plays important roles in plant-microbe interactions. We previously reported that IAA alters expression of several virulence-related genes in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000). To learn more about the impact of IAA on regulation of PtoDC3000 gene expression, we performed a global transcriptomic analysis of bacteria grown in culture, in the presence or absence of exogenous IAA. We observed that IAA repressed expression of genes involved in the type III secretion (T3S) system and motility and promoted expression of several known and putative transcriptional regulators. Several of these regulators are orthologs of factors known to regulate stress responses and accordingly expression of several stress response-related genes was also upregulated by IAA. Similar trends in expression for several genes were also observed by quantitative reverse transcription PCR. Using an Arabidopsis thaliana auxin receptor mutant that accumulates elevated auxin, we found that many of the P. syringae genes regulated by IAA in vitro were also regulated by auxin in planta. Collectively the data indicate that IAA modulates many aspects of PtoDC3000 biology, presumably to promote both virulence and survival under stressful conditions, including those encountered in or on plant leaves. IMPORTANCE Indole-3-acetic acid (IAA), a form of the plant hormone auxin, is used by many plant-associated bacteria as a cue to sense the plant environment. Previously, we showed that IAA can promote disease in interactions between the plant pathogen Pseudomonas syringae strain PtoDC000 and one of its hosts, Arabidopsis thaliana. However, the mechanisms by which IAA impacts the biology of PtoDC3000 and promotes disease are not well understood. Here, we demonstrate that IAA is a signal molecule that regulates gene expression in PtoDC3000. The presence of exogenous IAA affects expression of over 700 genes in the bacteria, including genes involved in type III secretion and genes involved in stress response. This work offers insight into the roles of auxin-promoting pathogenesis.

Complete Genome Sequence Resource for the Necrotrophic Plant-Pathogenic Bacterium Dickeya dianthicola 67-19 Isolated From New Guinea Impatiens.

New Guinea impatiens (NGI, Impatiens hawkeri) are popular bedding plants that can be affected by a number of pathogens. Using 16S rDNA sequencing and genus-specific PCR, we identified the first Dickeya dianthicola strain isolated from NGI presented with blackleg symptoms, herein designated as D. dianthicola 67-19. Here, we report a high-quality complete and annotated genome sequence of D. dianthicola 67-19. The 4,851,809 bp genome was assembled with Nanopore reads and polished with Illumina reads, yielding 422× and 105× coverage, respectively. This closed genome provides a resource for future research on comparative genomics and biology of D. dianthicola, which could translate to improved detection and disease management.

Complete Genome Sequence Resource for the Necrotrophic Plant-Pathogenic Bacterium Pectobacterium carotovorum WPP14.

Pectobacterium spp. are a major cause of loss in vegetable and ornamental plant production. One of these species, Pectobacterium carotovorum, can cause soft rot disease on many plants, particularly potato. These diseases lead to significant economic loss and pose food security threats by reducing crop yields in the field, in transit, and during storage. The Gram-negative enterobacterium P. carotovorum WPP14 is a particularly virulent strain for which there is no available closed genome, limiting the molecular research for this important pathogen. Here, we report a high-quality complete and annotated genome sequence of P. carotovorum WPP14. The 4,892,225-bp genome was assembled with Nanopore reads and polished with Illumina reads, yielding 394× and 164× coverage, respectively. This closed genome provides a resource for research on improved detection and biology of P. carotovorum, which could translate into improved disease management.

First Report of Dickeya dianthicola causing blackleg on New Guinea Impatiens (Impatiens hawkeri) in New York State, USA.

New Guinea impatiens (NGI), Impatiens hawkeri, has a $54-million wholesale market value in the United States (National Agricultural Statistics Service, 2019) and is highly resistant to Impatiens downy mildew (Plasmopara obducens) according to growers' experience (Warfield, 2011). In March 2019, NGI cv. Petticoat White in a New York greenhouse showed wilting, black stem streaks and vascular discoloration, with a 20% disease incidence. Symptomatic tissue pieces were added to sterile water in a test tube and streaks made on potato dextrose agar (PDA). After incubation at 26oC for two days, the most abundant colony type (mucoid, pale yellow) was transferred to PDA. One representative colony was selected and labeled as isolate 67-19. A single colony of isolate 67-19 was transferred to lysogeny broth (LB) (Bertani, 1951) and cultured at 28oC. Genomic DNA was extracted and polymerase chain reaction (PCR) performed using the 16S rRNA gene universal primers fD2 and rP1 resulting in a partial 16S rRNA amplicon (Weisburg et al., 1991). Basic Local Alignment Search Tool (BLASTn) analysis (Altschul et al., 1990) showed 99% identity with sequences of species belonging to Dickeya. Different primer sets have been developed to detect and identify the genus Dickeya and its various species (Pritchard et al., 2013). The primer sets used for genus identification, dnaX (Slawiak et al., 2009), Df/Dr (Laurila et al., 2010) and ADE1/ADE2 (Nassar et al., 1996), resulted in 500-bp, 133-bp, and 420-bp amplicons, respectively. Results suggested the bacterium was a Dickeya sp. To determine whether the species could be D. dianthicola, the specific primer set DIA-A was used (Pritchard et al., 2013) and the expected product of 150-bp was obtained. BLASTn results showed that the partial dnaX sequence (GenBank accession MT895847) of isolate 67-19 had 99% identity with the sequence of D. dianthicola strain RNS04.9 isolated in 2004 from potato (Solanum tuberosum) in France (GenBank accession CP017638.1). Therefore, this isolate 67-19 was designated as D. dianthicola. The complete genome of D. dianthicola strain 67-19 was generated using Nanopore and Illumina sequencing (GenBank accession CP051429) (Liu et al., 2020). Average nucleotide identity (ANI) determined by FastANI (v1.1) (Jain et al., 2018) showed 97.43% identity between the genome of D. dianthicola strain 67-19 and that of D. dianthicola strain NCPPB 453 (GenBank accession GCA_000365305.1), isolated in 1957 from carnation (Dianthus caryophyllus) in the UK. The pathogenicity of D. dianthicola strain 67-19 was shown on NGI cultivars Petticoat White and Tamarinda White. In July 2020, sterile toothpicks were used to make wounds and to transfer bacteria from a 48-hr PDA culture of D. dianthicola strain 67-19 to the stems of four plants of each cultivar. Four plants of each cultivar were mock inoculated similarly and all wound sites were wrapped with Parafilm before placing plants on a greenhouse bench. Ten days later, stems inoculated with D. dianthicola strain 67-19 showed necrotic lesions similar to the original symptoms, while control plants did not show symptoms. One month after inoculation, bacteria were re-isolated from all symptomatic stems. PCR was performed on the re-isolated bacteria as described. The dnaX sequence (GenBank accession MT895847) was confirmed to match that of D. dianthicola strain 67-19 (GenBank accession CP051429) 100% and fragments of the expected size were amplified (Liu et al., 2020). Stab inoculations of strain 67-19 into potato stems and tubers also resulted in blackleg and soft rot symptoms at the sites of inoculation, while mock-inoculated stem and tuber showed no symptoms. The sequence of the dnaX gene of the re-isolated bacterium from inoculated potatoes was confirmed to match that of D. dianthicola strain 67-19. To our knowledge, this is the first report of blackleg of New Guinea impatiens caused by D. dianthicola in the United States and worldwide. Since the disease caused by D. dianthicola poses a significant threat to the ornamentals and potato industries (Charkowski et al., 2020), further research on genome biology, epidemiology and management options is needed. LITERATURE CITED Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local alignment search tool. Journal of Molecular Biology 215:403-410. Bertani, G. 1951. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. Journal of Bacteriology 62:293-300. Charkowski, A., Sharma, K., Parker, M.L., Secor, G.A., and Elphinstone, J. 2020. Bacterial diseases of potato. Pages 351-388 in: The Potato Crop: Its Agricultural, Nutritional and Social Contribution to Humankind, H. Campos and O. Ortiz, eds. Springer International Publishing, Cham. Jain, C., Rodriguez-R, L.M., Phillippy, A.M., Konstantinidis, K.T., and Aluru, S. 2018. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nature Communications 9:5114. Laurila, J., Hannukkala, A., Nykyri, J., Pasanen, M., Hélias, V., Garlant, L., and Pirhonen, M. 2010. Symptoms and yield reduction caused by Dickeya spp. strains isolated from potato and river water in Finland. European Journal of Plant Pathology 126:249-262. Liu, Y., Helmann, T., Stodghill, P., and Filiatrault, M. 2020. Complete genome sequence resource for the necrotrophic plant-pathogenic bacterium Dickeya dianthicola 67-19 isolated from New Guinea Impatiens. Plant Disease. https://doi.org/10.1094/PDIS-09-20-1968-A. Nassar, A., Darrasse, A., Lemattre, M., Kotoujansky, A., Dervin, C., Vedel, R., and Bertheau, Y. 1996. Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes. Applied and Environmental Microbiology 62:2228-2235. National Agricultural Statistics Service. 2019. Floriculture crops 2018 summary. ISSN: 1949-0917. https://downloads.usda.library.cornell.edu/usda-esmis/files/0p0966899/rr1728124/76537c134/floran19.pdf Pritchard, L., Humphris, S., Saddler, G.S., Parkinson, N.M., Bertrand, V., Elphinstone, J.G., and Toth, I.K. 2013. Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences. Plant Pathology 62:587-596. Slawiak, M., van Beckhoven, J.R.C.M., Speksnijder, A.G.C.L., Czajkowski, R., Grabe, G., and van der Wolf, J.M. 2009. Biochemical and genetical analysis reveal a new clade of biovar 3 Dickeya spp. strains isolated from potato in Europe. European Journal of Plant Pathology 125:245-261. Warfield, C.Y. (2011). Downy Mildew of Impatiens. In GrowerTalks. https://www.growertalks.com/Article/?articleid=18921 Weisburg, W.G., Barns, S.M., Pelletier, D.A., and Lane, D.J. 1991. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173:697-703.

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.

Complete Genome Sequence of a Gram-Positive Bacterium, Leifsonia sp. Strain PS1209, a Potato Endophyte.

We report the complete and annotated genome sequence of a Gram-positive bacterium, Leifsonia sp. strain PS1209, a potato endophyte that was isolated from apparently healthy tubers of potato cultivar NY166. The circular genome is 4,091,164 bp long, with a GC content of 69.08%, containing 3,926 genes.

Complete Genome Sequence of the Necrotrophic Plant-Pathogenic Bacterium Pectobacterium brasiliense 1692.

We report here the complete and annotated genome sequence of Pectobacterium brasiliense 1692, a Gram-negative enterobacterium that can cause soft rot disease in many plant hosts.

Prevention of Surface-Associated Calcium Phosphate by the Pseudomonas syringae Two-Component System CvsSR.

CvsSR is a Ca2+-induced two-component system (TCS) in the plant pathogen Pseudomonas syringae pv. tomato DC3000. Here, we discovered that CvsSR is induced by Fe3+, Zn2+, and Cd2+ However, only supplementation of Ca2+ to medium resulted in rugose, opaque colonies in ΔcvsS and ΔcvsR strains. This phenotype corresponded to formation of calcium phosphate precipitation on the surface of ΔcvsS and ΔcvsR colonies. CvsSR regulated swarming motility in P. syringae pv. tomato in a Ca2+-dependent manner, but swarming behavior was not influenced by Fe3+, Zn2+, or Cd2+ We hypothesized that reduced swarming displayed by ΔcvsS and ΔcvsR strains was due to precipitation of calcium phosphate on the surface of ΔcvsS and ΔcvsR cells grown on agar medium supplemented with Ca2+ By reducing the initial pH or adding glucose to the medium, calcium precipitation was inhibited, and swarming was restored to ΔcvsS and ΔcvsR strains, suggesting that calcium precipitation influences swarming ability. Constitutive expression of a CvsSR-regulated carbonic anhydrase and a CvsSR-regulated putative sulfate major facilitator superfamily transporter in ΔcvsS and ΔcvsR strains inhibited formation of calcium precipitates and restored the ability of ΔcvsS and ΔcvsR bacteria to swarm. Lastly, we found that glucose inhibited Ca2+-based induction of CvsSR. Hence, CvsSR is a key regulator that controls calcium precipitation on the surface of bacterial cells.IMPORTANCE Bacteria are capable of precipitating and dissolving minerals. We previously reported the characterization of the two-component system CvsSR in the plant-pathogenic bacterium Pseudomonas syringae CvsSR responds to the presence of calcium and is important for causing disease. Here, we show that CvsSR controls the ability of the bacterium to prevent calcium phosphate precipitation on the surface of cells. We also identified a carbonic anhydrase and transporter that modulate formation of surface-associated calcium precipitates. Furthermore, our results demonstrate that the ability of the bacterium to swarm is controlled by the formation and dissolution of calcium precipitates on the surface of cells. Our study describes new mechanisms for microbially induced mineralization and provides insights into the role of mineral deposits on bacterial physiology. The discoveries may lead to new technological and environmental applications.

Physiological and genetic characterization of calcium phosphate precipitation by Pseudomonas species.

Microbial biomineralization is a widespread phenomenon. The ability to induce calcium precipitation around bacterial cells has been reported in several Pseudomonas species but has not been thoroughly tested. We assayed 14 Pseudomonas strains representing five different species for the ability to precipitate calcium. Calcium phosphate precipitated adjacent to the colonies of all the Pseudomonas strains tested and also precipitated on the surface of colonies for several of the Pseudomonas strains assayed. The precipitate was commonly precipitated as amorphous calcium phosphate, however seven of the 14 Pseudomonas strains tested precipitated amorphous apatite in agar adjacent to the colonies. Out of the seven Pseudomonas strains that precipitated amorphous apatite, six are plant pathogenic. The formation of amorphous apatite was commonly observed in the area of the agar where amorphous calcium phosphate had previously formed. A transposon mutagenesis screen in Pseudomonas syringae pv. tomato DC3000 revealed genes involved in general metabolism, lipopolysaccharide and cell wall biogenesis, and in regulation of virulence play a role in calcium precipitation. These results shed light on the common ability of Pseudomonas species to perform calcium precipitation and the underlying genetic regulation involved in biomineralization.

Transcriptomic Profiling Suggests That Promysalin Alters the Metabolic Flux, Motility, and Iron Regulation in Pseudomonas putida KT2440.

Promysalin, a secondary metabolite produced by P. putida RW10S1, is a narrow-spectrum antibiotic that targets P. aeruginosa over other Pseudomonas spp. P. putida KT2440, a nonproducing strain, displays increased swarming motility and decreased pyoverdine production in the presence of exogenous promysalin. Herein, proteomic and transcriptomic experiments were used to provide insight about how promysalin elicits responses in PPKT2440 and rationalize its species selectivity. RNA-sequencing results suggest that promysalin affects PPKT2440 by (1) increasing swarming in a flagella-independent manner; (2) causing cells to behave as if they were experiencing an iron-deficient environment, and (3) shifting metabolism away from glucose conversion to pyruvate via the Entner-Doudoroff pathway. These findings highlight nature's ability to develop small molecules with specific targets, resulting in exquisite selectivity.

Ca2+-Induced Two-Component System CvsSR Regulates the Type III Secretion System and the Extracytoplasmic Function Sigma Factor AlgU in Pseudomonas syringae pv. tomato DC3000.

Two-component systems (TCSs) of bacteria regulate many different aspects of the bacterial life cycle, including pathogenesis. Most TCSs remain uncharacterized, with no information about the signal(s) or regulatory targets and/or role in bacterial pathogenesis. Here, we characterized a TCS in the plant-pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 composed of the histidine kinase CvsS and the response regulator CvsR. CvsSR is necessary for virulence of P. syringae pv. tomato DC3000, since ΔcvsS and ΔcvsR strains produced fewer symptoms than the wild type (WT) and demonstrated reduced growth on multiple hosts. We discovered that expression of cvsSR is induced by Ca2+ concentrations found in leaf apoplastic fluid. Thus, Ca2+ can be added to the list of signals that promote pathogenesis of P. syringae pv. tomato DC3000 during host colonization. Through chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and global transcriptome analysis (RNA-seq), we discerned the CvsR regulon. CvsR directly activated expression of the type III secretion system regulators, hrpR and hrpS, that regulate P. syringae pv. tomato DC3000 virulence in a type III secretion system-dependent manner. CvsR also indirectly repressed transcription of the extracytoplasmic sigma factor algU and production of alginate. Phenotypic analysis determined that CvsSR inversely regulated biofilm formation, swarming motility, and cellulose production in a Ca2+-dependent manner. Overall, our results show that CvsSR is a key regulatory hub critical for interaction with host plants.IMPORTANCE Pathogenic bacteria must be able to react and respond to the surrounding environment, make use of available resources, and avert or counter host immune responses. Often, these abilities rely on two-component systems (TCSs) composed of interacting proteins that modulate gene expression. We identified a TCS in the plant-pathogenic bacterium Pseudomonas syringae that responds to the presence of calcium, which is an important signal during the plant defense response. We showed that when P. syringae is grown in the presence of calcium, this TCS regulates expression of factors contributing to disease. Overall, our results provide a better understanding of how bacterial pathogens respond to plant signals and control systems necessary for eliciting disease.

The ECF sigma factor, PSPTO_1043, in Pseudomonas syringae pv. tomato DC3000 is induced by oxidative stress and regulates genes involved in oxidative stress response.

The bacterial plant pathogen Pseudomonas syringae adapts to changes in the environment by modifying its gene expression profile. In many cases, the response is mediated by the activation of extracytoplasmic function (ECF) sigma factors that direct RNA polymerase to transcribe specific sets of genes. In this study we focus on PSPTO_1043, one of ten ECF sigma factors in P. syringae pv. tomato DC3000 (DC3000). PSPTO_1043, together with PSPTO_1042, encode an RpoERsp/ChrR-like sigma/anti-sigma factor pair. Although this gene pair is unique to the P. syringae group among the pseudomonads, homologous genes can be found in photosynthetic genera such as Rhodospirillum, Thalassospira, Phaeospirillum and Parvibaculum. Using ChIP-Seq, we detected 137 putative PSPTO_1043 binding sites and identified a likely promoter motif. We characterized 13 promoter candidates, six of which regulate genes that appear to be found only in P. syringae. PSPTO_1043 responds to the presence of singlet oxygen (1O2) and tert-butyl hydroperoxide (tBOOH) and several of the genes regulated by PSPTO_1043 appear to be involved in response to oxidative stress.

Virulence of Pseudomonas syringae pv. tomato DC3000 Is Influenced by the Catabolite Repression Control Protein Crc.

Pseudomonas syringae infects diverse plant species and is widely used as a model system in the study of effector function and the molecular basis of plant diseases. Although the relationship between bacterial metabolism, nutrient acquisition, and virulence has attracted increasing attention in bacterial pathology, it is largely unexplored in P. syringae. The Crc (catabolite repression control) protein is a putative RNA-binding protein that regulates carbon metabolism as well as a number of other factors in the pseudomonads. Here, we show that deletion of crc increased bacterial swarming motility and biofilm formation. The crc mutant showed reduced growth and symptoms in Arabidopsis and tomato when compared with the wild-type strain. We have evidence that the crc mutant shows delayed hypersensitive response (HR) when infiltrated into Nicotiana benthamiana and tobacco. Interestingly, the crc mutant was more susceptible to hydrogen peroxide, suggesting that, in planta, the mutant may be sensitive to reactive oxygen species generated during pathogen-associated molecular pattern-triggered immunity (PTI). Indeed, HR was further delayed when PTI-induced tissues were challenged with the crc mutant. The crc mutant did not elicit an altered PTI response in plants compared with the wild-type strain. We conclude that Crc plays an important role in growth and survival during infection.

The conserved hypothetical protein PSPTO_3957 is essential for virulence in the plant pathogen Pseudomonas syringae pv. tomato DC3000.

The plant pathogen Pseudomonas syringae accounts for substantial crop losses and is considered an important agricultural issue. To better manage disease in the field, it is important to have an understanding of the underlying genetic mechanisms that mediate virulence. There are a substantial number of genes in sequenced bacterial genomes, including P. syringae, that encode for conserved hypothetical proteins; some of these have been functionally characterized in other Pseudomonads and have been demonstrated to play important roles in disease. PSPTO_3957 encodes a conserved hypothetical protein of unknown function. To evaluate the role of PSPTO_3957 in P. syringae pv. tomato DC3000, a PSPTO_3957 deletion mutant was constructed. Here, we show that PSPTO_3957 does not influence growth on rich media, motility or biofilm formation but is necessary for nitrate assimilation and full virulence in P. syringae. Our results have revealed an important role for PSPTO_3957 in the biology of P. syringae. Given the conservation of this protein among many bacteria, this protein might serve as an attractive target for disease management of this and other bacterial plant pathogens.