Reproducible Quantitative Trait Loci for Resistance to Soft Rot Caused by Dickeya dianthicola Derived from the Wild Potato Solanum microdontum (PI 458355) Are Located on Chromosomes 1, 3, and 5.

The potato wild relative Solanum microdontum is a breeder-friendly source of genetic resistance to soft rot. Our objectives were to (i) identify loci associated with soft rot resistance in S. microdontum germplasm and (ii) develop bi-parental populations in a self-compatible S. tuberosum genetic background to recover segregating F2 progenies, construct a linkage map, and identify quantitative trait loci (QTLs). Under objective (i), tubers from 103 S. microdontum genotypes from the United States Potato Genebank were inoculated with a high virulence strain of Dickeya dianthicola, and lesion size was measured after a 24-h incubation period at 30°C. Association analysis using 3,490 polymorphic Infinium array SNP markers identified soft rot resistance loci on chromosomes 1, 2, 3, 5, 7, 8, 11, and 12. Under objective (ii), a resistant S. microdontum accession PI 458355 was crossed with a highly fertile, self-compatible, diploid S. tuberosum pollen parent (PI 654351) to generate segregating F2 populations. Composite interval mapping was conducted using a genetic linkage map with 970 GBS-based SNP markers. Reproducible QTLs were detected on chromosomes 1, 3, and 5, explaining 11, 13, and 23% of the phenotypic variation, respectively. Homozygous S. microdontum alleles at the QTL on chromosome 5 and heterozygous or homozygous S. microdontum alleles at QTLs on chromosomes 1 and 3 significantly decrease lesion size compared with the homozygous S. tuberosum parent. The germplasm created in these studies provides a resource for studying traits from S. microdontum, and we can use the advanced F2 selections for future potato improvement. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of Cucurbit Yellow Vine Disease Caused by Serratia marcescens on Cucurbits in New York.

Cucurbits are one of the most significant commodities in New York, with a value of $92.3 million in 2021 (NASS-USDA 2021). In August 2021, several acorn squash (Cucurbita pepo) cultivar Turbinate plants at Cornell AgriTech research farm in Geneva, NY, had chlorotic, wilting leaves, and older leaves appeared scorched. The phloem of stems, bisected at the crown, had a honey-brown discoloration. The incidence of symptomatic plants was 22% in a one-acre planting field. Most of the symptomatic plants rapidly declined and died. The following year, similar symptoms were observed on muskmelon (Cucumis melo), acorn squash, and winter squash (C. pepo) cultivar Bush Delicata at the same location. These symptoms were typical of Cucurbit Yellow Vine Disease (CYVD) caused by the Gram-negative bacterium Serratia marcescens (Bruton et al. 1998, 2003). Moreover, a high incidence of squash bugs (vector of CYVD) was observed. To identify the causal agent, 45 stems from the symptomatic Bush delicata plants were collected. Each stem was cut into small pieces (2 to 3 mm), surface sterilized with 70% ethanol for 60 sec, 10% bleach for 60 sec, and rinsed with sterile water. The tissue was macerated in sterile water, and the resultant suspension was streaked on King's B (KB) medium (King et al. 1954). Plates were incubated at 28°C for 24 h, and 11 developed white, round bacterial colonies that were smooth and creamy in appearance. Single colonies were transferred to new KB plates and incubated for 24 h. The genomic DNA of two isolates (22212 and 22213) was extracted with the Wizard® Genomic DNA Purification Kit Protocol (Promega, Madison, WI). PCR was carried out using YV1 and YV4 primers specific to the 16S rDNA region of S. marcescens and 79F/R primers specific for S. marcescens causing CYVD (Zhang et al. 2005). The DNA sequence of each PCR product was obtained using Sanger sequencing and submitted to GenBank. Accessions OQ584799 and OQ584800 for YV1/YV4 (isolates 22212 and 22213, respectively) exhibited 100% identity to S. marcescens (384/384 bp, nearest accession identity: CP083754). Accession numbers OQ693911 and OQ693912 for 79F/R showed 99% identity to S. marcescens isolates (309/313 bp, nearest accession identity: CP033623). To fulfill Koch's postulates, Bush Delicata squash plants were grown for two weeks in a greenhouse, and three plants per isolate were inoculated using S. marcescens 22212 and 22213, three plants with Escherichia coli DH5a as a non-pathogenic control, distilled water as a mock-inoculated control, and a noninoculated control. Inoculation was performed by taking a single bacterial colony with a small pin and puncturing the plant's lower stem four to five times (Bruton et al. 2003). Twenty-eight days after inoculation, three of the six plants inoculated with the two S. marcescens isolates (two from 22212 and one from 22213) developed CYVD symptoms as observed in the field. Isolations were made from the stems of symptomatic plants and the mock-inoculated controls. PCR was conducted using YV1/YV4 primers and 79F/R primers (Zhang et al. 2005). Only isolations from symptomatic plants amplified with these primers and PCR products were sequenced. These sequences were identical to the original isolates. To our knowledge, this is the first report of CYVD and phytopathogenic S. marcescens in New York. The impact of CYVD can be substantial, with losses up to 100% (Zhang et al. 2005). Therefore, more knowledge on S. marcescens is needed to determine its biology and prevalence in New York.

First Report of Pectobacterium brasiliense Causing Bacterial Blackleg and Soft Rot of Potato in Pennsylvania.

Potato (Solanum tuberosum) plants showing blackleg and soft rot symptoms were collected at a commercial vegetable farm near Newmanstown, PA in August 2021 (Fig. S1). The incidence of potato blackleg in the unirrigated field was about 5 to 8%, but approximately 30% in the irrigated field. The diseased stems were cut into 5 cm and surface disinfected. The stem segments were placed into a 50-mL tube containing 15 mL of sterile water for 15 min for bacterial release. The bacterial suspension was streaked on crystal violet polypectate (CVP) (Hélias et al. 2012) plates and incubated at 28°C for 48 h. Three single colonies produced pits on CVP were picked and purified. Genomic DNA of all three isolates were extracted using the FastDNA Spin Kit (MP Biomedicals, Santa Ana, CA). Polymerase chain reaction (PCR) was performed using all three extracted DNAs as a template with the primer pairs gapA 7F/938R (Cigna et al. 2017), recA F/R (Waleron et al. 2001), dnaA F/R (Schneider et al. 2011) and dnaX F/R (Slawiak et al. 2009) targeting the gapA, recA, dnaA and dnaX genes, respectively. Isolate 21PA01 was further studied as a representative isolate. PCR amplicons derived from both forward and reverse primers were sequenced and analyzed using the BLAST algorithm against the NCBI database (https://www.ncbi.nlm.nih.gov). The regions of gapA (GenBank accession No. ON989738), recA (ON989739), dnaA (OP121183), and dnaX (OP121184) had 99.86%, 100%, 98.88%, and 100% identities with Pectobacterium brasiliense strains S1.16.01.3M (MN167062.1), BL-2 (MW721598.1), IPO:4132 (CP059956.1), and BL-2 (MW721603.1), respectively. A phylogenetic maximum-likelihood tree of the concatenated genes with the length of 2551 bp was constructed to visualize the relationship among different species of Dickeya and Pectobacterium. As a result, 21PA01 was in a single monophyletic cluster with other Pectobacterium brasiliense reference strains (Fig. S2 C). To confirm the pathogen, Koch's postulates were performed. Seed pieces of potato 'Lamoka' were planted in potting mix in one-gallon plastic pots in a greenhouse. Three weeks after emergence, the stems of three plants were each injected with 10 µL of bacteria suspension of either 21PA01 at 107 CFU/mL, P. parmentieri ME175 in tryptic soy broth (TSB) at 107 CFU/mL or TSB at 2 cm above the soil line. Seven days after inoculation, stems inoculated with 21PA01 and ME175 showed black and rotten symptoms, whereas the TSB-injected control plants remained symptomless. In addition, 'Lamoka' tubers were inoculated by placing 10 µL 21PA01 and ME175 suspensions at 107 CFU/mL, and TSB in a 1-cm-deep hole poked in a tuber separately and then sealed with petroleum gel, followed by incubation in a moist chamber at 22 °C for 4 d. The 21PA01 and ME175 inoculated tubers showed soft rot symptoms, but the TSB treatment had no symptoms. Bacterial colonies were isolated from the infected stems and confirmed by the DNA sequences as described above. PCR result was negative on control plant samples. Both stem and tuber inoculation trials were repeated two times, and the results were consistent. Thus, 21PA01 was identified as Pectobacterium brasiliense. To our knowledge, this is the first report of P. brasiliense infecting potatoes in Pennsylvania, USA, although it has been reported somewhere else (van der Merwe et al. 2010, Zhao et al. 2018). This could be a new species in Northeastern US.

Complete Genome Sequence Resource for a Recently Isolated Potato Ring Rot Pathogen, Clavibacter sepedonicus K496.

Potato ring rot caused by Clavibacter sepedonicus has been a devastating disease in the U.S. since 1930. In this study, we isolated a recent C. sepedonicus strain, K496, from potato tubers showing discolorations of the vascular cylinder or pith tissues. We de novo assembled the genome sequence of K496 with 1,924,544,313 bp of Nanopore reads (N50 = 13,785 bp) using Flye v2.9 and polished it with 2 × 150 bp paired-end Illumina reads (855,788,703 bp in total). The resulting genome of K496 consists of a single circular chromosome 3,266,016 bp long and a linear plasmid of 135,489 bp. Using the NCBI PGAP v5.3, this genome was predicted to have 3,301 genes, encompassing 3,247 protein-coding genes, 90 pseudogenes, two 5S rRNA-coding, two 16S rRNA-coding, two 23S rRNA-coding sequences, 45 tRNAs, and three noncoding RNAs. The chromosome and plasmid sequences have been deposited at the NCBI GenBank database under the accession numbers CP088266 and CP088267, respectively.

First Report of Rhizopus arrhizus (syn. R. oryzae) Causing Garlic Bulb Soft Rot in Hebei Province, China.

Rhizopus soft rot occurs on the succulent tissues of vegetables, fruits, and ornamental plants throughout the world (Cui et al. 2019). When the garlic is in the seedling stage in the fields (Fig. S1) in November 2021, a disease outbreak on garlic bulbs suspected as Rhizopus soft rot occurred in Daming County, Handan City, Hebei Province of China (N 36°17', E 115° 13'). This disease symptom was first found in the garlic seedling stage in China. Disease incidence was 10% to 30% in cultivated garlic bulbs. There were soft water-soaked lesions on the surface of diseased garlic bulbs and the interiors were brown and soft. In the disease severe field, white to gray mycelia were observed on the diseased garlic bulbs. Infected garlic bulbs were sampled to isolate and determine the identity of the disease-causing organism. Symptomatic bulbs were surface sterilized with 1% NaClO for 2 min, dipped in 75% ethanol for 3 min and rinsed three times with autoclaved distilled water. Small pieces of the inner decayed tissue were removed and cultured on potato dextrose agar (PDA) at 28°C for 2 to 3 days. Five white colonies grew on PDA and then they became brownish gray to blackish-gray mycelium. The fungal strains were purified by hyphal-tip isolation method. To determine the identity of the five isolated fungi, we analyzed their internal transcribed spacer (ITS) region sequences (Jung et al. 2012). BLAST analysis of the ITS sequences from DSF-0-2 (accession no. ON706022), DSF-0-3 (accession no. ON706021), DSF-0-4 (accession no. ON706020), DSF-0-5 (accession no. ON706019) and DSF-0-6 (accession no. ON706018) were all 100% identical with Rhizopus arrhizus (syn. Rhizopus oryzae). Phylogenetic trees were constructed using the neighbor-joining method of MEGA11 based on the sequences of ITS rRNA gene (Walther et al. 2013). Phylogenetic trees indicated that isolates were most likely Rhizopus arrhizus (syn. Rhizopus oryzae) (Fig. S2). We selected one isolated strain, DSF-0-2, for characterize the morphology and test its ability to cause garlic bulb soft rot. Under the microscope, nonseptate rhizoids, sporangia, and sporangiospores were observed (Fig. S1). Sporangiospores were unequal, subglobose, numerous irregular, or oval, and 9.7 (6.2 - 12.5) × 6.5 (4.1 - 8.5) µm (n = 50) in diameter. The sporangia were globose, black, 121.5 (65 - 198) µm (n = 50) in diameter. Based on the rDNA-ITS sequencing and the morphological characteristics, the DSF-0-2 isolate was identified as Rhizopus arrhizus (syn. Rhizopus oryzae) (Zheng et al. 2007; Abeywickrama et al. 2020). To complete Koch's postulates, surface-sterilized healthy garlic bulbs were inoculated with R. arrhizus isolate DSF-0-2. A 1.0-ml sterile syringe was used to inject 50 µl of a 106 conidia/ml suspension into each of five healthy bulbs. As a control, garlic bulbs were treated with sterile distilled water. The inoculated and control bulbs were incubated at 28°C for 7 days. The bulbs inoculated with R. arrhizus DSF-0-2 showed symptoms of water soaking, and the tissues were brown and soft throughout the bulb at 7 days (Fig. S1). Results of the three trials were the same. No symptoms were observed in the control group. R. arrhizus was reisolated from the symptomatic garlic bulb and confirmed as such based-on colony and sporangia morphology and ITS sequence. There were some reports that R. arrhizus infects cassava tubers and potato tubers (Amadioha and Markson 2007; Cui et al. 2019). To our knowledge, this is the first report of R. arrhizus (syn. Rhizopus oryzae) associated with soft rot on garlic bulb in the seedling stage in China. This disease has posed a potential threat during garlic seedling stage in the field. Management measures should be considered before this disease outbreaks widely. Garlic bulbs died in the seedling stage, which caused production reduction, serious economic loss and soil pollution. This finding may help to take effective control measures for this disease.

Identification of Pectobacterium versatile Causing Blackleg of Potato in New York State.

Soft rot bacteria classified in the Pectobacteriaceae (SRP), including Pectobacterium and Dickeya spp., are responsible for soft rot and blackleg diseases of potato. Since 2014, blackleg outbreaks caused by D. dianthicola have increased in the United States and Canada. Our previous study found that the most abundant causal organisms of blackleg disease in New York State were P. parmentieri and D. dianthicola, with the latter being the only Dickeya species reported. In the present study, we identified and characterized pathogenic SRP bacteria from 19 potato samples collected in New York State during the 2017 growing season. We used genome sequence comparison to determine the pathogens' species. We found eight P. versatile, one P. atrosepticum, two P. carotovorum, two P. parmentieri, and six D. dianthicola isolates in our 2017 SRP collection. This is the first time that P. versatile has been reported to cause potato blackleg disease in New York State. We determined the phylogenetic relationships between the SRP strains by using 151 single-copy orthologous gene sequences shared among the set of bacteria in our analysis, which provided better resolution than phylogenies constructed with the dnaX gene.

AlgU, a Conserved Sigma Factor Regulating Abiotic Stress Tolerance and Promoting Virulence in Pseudomonas syringae.

Pseudomonas syringae can rapidly deploy specialized functions to deal with abiotic and biotic stresses. Host niches pose specific sets of environmental challenges driven, in part, by immune defenses. Bacteria use a "just-in-time" strategy of gene regulation, meaning that they only produce the functions necessary for survival as needed. Extracytoplasmic function (ECF) sigma factors transduce a specific set of environmental signals and change gene expression patterns by altering RNA polymerase promoter specificity, to adjust bacterial physiology, structure, or behavior, singly or in combination, to improve chances of survival. The broadly conserved ECF sigma factor AlgU affects virulence in both animal and plant pathogens. Pseudomonas syringae AlgU controls expression of more than 800 genes, some of which contribute to suppression of plant immunity and bacterial fitness in plants. This review discusses AlgU activation mechanisms, functions controlled by AlgU, and how these functions contribute to P. syringae survival in plants.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.

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.

Pseudomonas syringae AlgU Downregulates Flagellin Gene Expression, Helping Evade Plant Immunity.

Flagella power bacterial movement through liquids and over surfaces to access or avoid certain environmental conditions, ultimately increasing a cell's probability of survival and reproduction. In some cases, flagella and chemotaxis are key virulence factors enabling pathogens to gain entry and attach to suitable host tissues. However, flagella are not always beneficial; both plant and animal immune systems have evolved receptors to sense the proteins that make up flagellar filaments as signatures of bacterial infection. Microbes poorly adapted to avoid or counteract these immune functions are unlikely to be successful in host environments, and this selective pressure has driven the evolution of diverse and often redundant pathogen compensatory mechanisms. We tested the role of AlgU, the Pseudomonas extracytoplasmic function sigma factor σE/σ22 ortholog, in regulating flagellar expression in the context of Pseudomonas syringae-plant interactions. We found that AlgU is necessary for downregulating bacterial flagellin expression in planta and that this results in a corresponding reduction in plant immune elicitation. This AlgU-dependent regulation of flagellin gene expression is beneficial to bacterial growth in the course of plant infection, and eliminating the plant's ability to detect flagellin makes this AlgU-dependent function irrelevant for bacteria growing in the apoplast. Together, these results add support to an emerging model in which P. syringae AlgU functions at a key control point that serves to optimize the expression of bacterial functions during host interactions, including minimizing the expression of immune elicitors and concomitantly upregulating beneficial virulence functions.IMPORTANCE Foliar plant pathogens, like Pseudomonas syringae, adjust their physiology and behavior to facilitate host colonization and disease, but the full extent of these adaptations is not known. Plant immune systems are triggered by bacterial molecules, such as the proteins that make up flagellar filaments. In this study, we found that during plant infection, AlgU, a gene expression regulator that is responsive to external stimuli, downregulates expression of fliC, which encodes the flagellin protein, a strong elicitor of plant immune systems. This change in gene expression and resultant change in behavior correlate with reduced plant immune activation and improved P. syringae plant colonization. The results of this study demonstrate the proximate and ultimate causes of flagellar regulation in a plant-pathogen interaction.

Complete Genome Sequence of Dickeya dianthicola ME23, a Pathogen Causing Blackleg and Soft Rot Diseases of Potato.

In 2014, an outbreak of potato blackleg and soft rot disease emerged in North America and continues to impact potato production. Here, we report the annotated genome sequence of Dickeya dianthicola ME23, a strain hypothesized to be representative of the bacterial population responsible for this disease outbreak.

Pectobacterium and Dickeya Responsible for Potato Blackleg Disease in New York State in 2016.

Beginning in 2014, outbreaks of blackleg disease compromised potato (Solanum tuberosum) production in the northeastern United States. Disease severity was atypical for plantings with certified seed. During 2016, 43 samples with blackleg symptoms were analyzed, originating from more than 20 farms operating in New York State. A combination of techniques was employed to identify the blackleg pathogens: isolation in vitro, diagnostic PCR assays for Pectobacterium and Dickeya sp., pathogenicity assays, and DNA sequencing. Twenty-three bacterial isolates were obtained, the majority of which were designated D. dianthicola or P. parmentieri; two of the isolates were designated P. atrosepticum. All isolates were pathogenic in stem lesion and tuber soft rot assays and exhibited pectin degrading activity (pitting) in crystal violet pectate agar medium. Phylogenetic analyses of dnaX gene sequences placed all but one of the isolates into clades corresponding to D. dianthicola, P. parmentieri, or P. atrosepticum. One atypical isolate clustered with P. carotovorum subspecies. Data are consistent with the hypothesis that D. dianthicola from New York and the northeast are part of a single clade, and at least three different soft rot bacteria were associated with blackleg during 2016 in New York.

An AlgU-Regulated Antisense Transcript Encoded within the Pseudomonas syringae fleQ Gene Has a Positive Effect on Motility.

Production of bacterial flagella is controlled by a multitiered regulatory system that coordinates the expression of 40 to 50 subunits and ordered assembly of these elaborate structures. Flagellar expression is environmentally controlled, presumably to optimize the benefits and liabilities of having these organelles on cell growth and survival. We recently reported a global survey of AlgU-dependent regulation and binding in Pseudomonas syringae pv. tomato DC3000 that included evidence for strong downregulation of many flagellar and chemotaxis motility genes. Here, we returned to those data to look for other AlgU-dependent influences on the flagellar regulatory network. We identified an AlgU-dependent antisense transcript expressed from within the fleQ gene, the master regulator of flagellar biosynthesis in Pseudomonas We tested whether expression of this antisense RNA influenced bacterial behavior and found that it reduces AlgU-dependent downregulation of motility. Importantly, this antisense expression influenced motility only under conditions in which AlgU was expressed. Comparative sequence analysis of the locus containing the antisense transcript's AlgU-dependent promoter in over 300 Pseudomonas genomes revealed sequence conservation in most strains that encode AlgU. This suggests that the antisense transcript plays an important role that is conserved across most of the genus PseudomonasIMPORTANCEPseudomonas syringae is a globally distributed host-specific bacterial pathogen that causes disease in a wide-range of plants. An elaborate gene expression regulation network controls flagellum production, which is important for proper flagellum assembly and a key aspect of certain lifestyle transitions. P. syringae pv. tomato DC3000 uses flagellum-powered motility in the early stages of host colonization and adopts a sessile lifestyle after entering plant tissues, but the regulation of this transition is not understood. Our work demonstrates a link between regulation of motility and global transcriptional control that facilitates bacterial growth and disease in plants. Additionally, sequence comparisons suggest that this regulation mechanism is conserved in most members of the genus Pseudomonas.

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.

Comparative genomics of Pseudomonas syringae pathovar tomato reveals novel chemotaxis pathways associated with motility and plant pathogenicity.

The majority of bacterial foliar plant pathogens must invade the apoplast of host plants through points of ingress, such as stomata or wounds, to replicate to high population density and cause disease. How pathogens navigate plant surfaces to locate invasion sites remains poorly understood. Many bacteria use chemical-directed regulation of flagellar rotation, a process known as chemotaxis, to move towards favorable environmental conditions. Chemotactic sensing of the plant surface is a potential mechanism through which foliar plant pathogens home in on wounds or stomata, but chemotactic systems in foliar plant pathogens are not well characterized. Comparative genomics of the plant pathogen Pseudomonas syringae pathovar tomato (Pto) implicated annotated chemotaxis genes in the recent adaptations of one Pto lineage. We therefore characterized the chemosensory system of Pto. The Pto genome contains two primary chemotaxis gene clusters, che1 and che2. The che2 cluster is flanked by flagellar biosynthesis genes and similar to the canonical chemotaxis gene clusters of other bacteria based on sequence and synteny. Disruption of the primary phosphorelay kinase gene of the che2 cluster, cheA2, eliminated all swimming and surface motility at 21 °C but not 28 °C for Pto. The che1 cluster is located next to Type IV pili biosynthesis genes but disruption of cheA1 has no observable effect on twitching motility for Pto. Disruption of cheA2 also alters in planta fitness of the pathogen with strains lacking functional cheA2 being less fit in host plants but more fit in a non-host interaction.

AlgU Controls Expression of Virulence Genes in Pseudomonas syringae pv. tomato DC3000.

UNLABELLED: Plant-pathogenic bacteria are able to integrate information about their environment and adjust gene expression to provide adaptive functions. AlgU, an extracytoplasmic function (ECF) sigma factor encoded by Pseudomonas syringae, controls expression of genes for alginate biosynthesis and genes involved with resisting osmotic and oxidative stress. AlgU is active while these bacteria are associated with plants, where its presence supports bacterial growth and disease symptoms. We found that AlgU is an important virulence factor for P. syringae pv. tomato DC3000 but that alginate production is dispensable for disease in host plants. This implies that AlgU regulates additional genes that facilitate bacterial pathogenesis. We used transcriptome sequencing (RNA-seq) to characterize the AlgU regulon and chromatin immunoprecipitation sequencing (ChIP-seq) to identify AlgU-regulated promoters associated with genes directly controlled by this sigma factor. We found that in addition to genes involved with alginate and osmotic and oxidative stress responses, AlgU regulates genes with known virulence functions, including components of the Hrp type III secretion system, virulence effectors, and the hrpL and hrpRS transcription regulators. These data suggest that P. syringae pv. tomato DC3000 has adapted to use signals that activate AlgU to induce expression of important virulence functions that facilitate survival and disease in plants. IMPORTANCE: Plant immune systems produce antimicrobial and bacteriostatic conditions in response to bacterial infection. Plant-pathogenic bacteria are adapted to suppress and/or tolerate these conditions; however, the mechanisms controlling these bacterial systems are largely uncharacterized. The work presented here provides a mechanistic explanation for how P. syringae pv. tomato DC3000 coordinates expression of multiple genetic systems, including those dedicated to pathogenicity, in response to environmental conditions. This work demonstrates the scope of AlgU regulation in P. syringae pv. tomato DC3000 and characterizes the promoter sequence regulated by AlgU in these bacteria.

Population-genomic insights into emergence, crop adaptation and dissemination of Pseudomonas syringae pathogens.

Many bacterial pathogens are well characterized but, in some cases, little is known about the populations from which they emerged. This limits understanding of the molecular mechanisms underlying disease. The crop pathogen Pseudomonas syringae sensu lato has been widely isolated from the environment, including wild plants and components of the water cycle, and causes disease in several economically important crops. Here, we compared genome sequences of 45 P. syringae crop pathogen outbreak strains with 69 closely related environmental isolates. Phylogenetic reconstruction revealed that crop pathogens emerged many times independently from environmental populations. Unexpectedly, differences in gene content between environmental populations and outbreak strains were minimal with most virulence genes present in both. However, a genome-wide association study identified a small number of genes, including the type III effector genes hopQ1 and hopD1, to be associated with crop pathogens, but not with environmental populations, suggesting that this small group of genes may play an important role in crop disease emergence. Intriguingly, genome-wide analysis of homologous recombination revealed that the locus Psyr 0346, predicted to encode a protein that confers antibiotic resistance, has been frequently exchanged among lineages and thus may contribute to pathogen fitness. Finally, we found that isolates from diseased crops and from components of the water cycle, collected during the same crop disease epidemic, form a single population. This provides the strongest evidence yet that precipitation and irrigation water are an overlooked inoculum source for disease epidemics caused by P. syringae.

Pseudomonas syringae pv. tomato DC3000 Type III Secretion Effector Polymutants Reveal an Interplay between HopAD1 and AvrPtoB.

The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered plant innate immune system by injecting a complex repertoire of type III secretion effector (T3E) proteins. Beyond redundancy and interplay, individual T3Es may interact with multiple immunity-associated proteins, rendering their analysis challenging. We constructed a Pst DC3000 polymutant lacking all 36 T3Es and restored individual T3Es or their mutants to explore the interplay among T3Es. The weakly expressed T3E HopAD1 was sufficient to elicit immunity-associated cell death in Nicotiana benthamiana. HopAD1-induced cell death was suppressed partially by native AvrPtoB and completely by AvrPtoBM3, which has mutations disrupting its E3 ubiquitin ligase domain and two known domains for interacting with immunity-associated kinases. AvrPtoBM3 also gained the ability to interact with the immunity-kinase MKK2, which is required for HopAD1-dependent cell death. Thus, AvrPtoB has alternative, competing mechanisms for suppressing effector-triggered plant immunity. This approach allows the deconvolution of individual T3E activities.

Global analysis of the HrpL regulon in the plant pathogen Pseudomonas syringae pv. tomato DC3000 reveals new regulon members with diverse functions.

The type III secretion system (T3SS) is required for virulence in the gram-negative plant pathogen Pseudomonas syringae pv. tomato DC3000. The alternative sigma factor HrpL directly regulates expression of T3SS genes via a promoter sequence, often designated as the "hrp promoter." Although the HrpL regulon has been extensively investigated in DC3000, it is not known whether additional regulon members remain to be found. To systematically search for HrpL-regulated genes, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and bulk mRNA sequencing (RNA-Seq) to identify HrpL-binding sites and likely hrp promoters. The analysis recovered 73 sites of interest, including 20 sites that represent new hrp promoters. The new promoters lie upstream of a diverse set of genes encoding potential regulators, enzymes and hypothetical proteins. PSPTO_5633 is the only new HrpL regulon member that is potentially an effector and is now designated HopBM1. Deletions in several other new regulon members, including PSPTO_5633, PSPTO_0371, PSPTO_2130, PSPTO_2691, PSPTO_2696, PSPTO_3331, and PSPTO_5240, in either DC3000 or ΔhopQ1-1 backgrounds, do not affect the hypersensitive response or in planta growth of the resulting strains. Many new HrpL regulon members appear to be unrelated to the T3SS, and orthologs for some of these can be identified in numerous non-pathogenic bacteria. With the identification of 20 new hrp promoters, the list of HrpL regulon members is approaching saturation and most likely includes all DC3000 effectors.

RecTE(Psy)-mediated recombineering in Pseudomonas syringae.

A recently developed Pseudomonas syringae recombineering system simplifies the procedure for installing specific mutations at a chosen genomic locus. The procedure involves transforming P. syringae cells expressing recombineering functions with a PCR product that contains desired changes flanked by sequences homologous to a target location. Cells transformed with the substrate undergo homologous recombination between the genomic DNA and the recombineering substrate. The recombinants are found by selection for traits carried by the recombineering substrate, usually antibiotic resistance.