Diversity and Abundance of Ice Nucleating Strains of Pseudomonas syringae in a Freshwater Lake in Virginia, USA

The bacterium Pseudomonas syringae is found in a variety of terrestrial and aquatic environments. Some strains of P. syringae express an ice nucleation protein (hereafter referred to as Ice+) allowing them to catalyze the heterogeneous freezing of water. Though P. syringae has been sampled intensively from freshwater sources in France, little is known about the genetic diversity of P. syringae in natural aquatic habitats in North America. We collected samples of freshwater from three different depths in Claytor Lake, Virginia, USA between November 2015 and June 2016. Samples were plated on non-selective medium (TSA) and on medium selective for Pseudomonas (KBC) and closely related species to estimate the total number of culturable bacteria and of Pseudomonas, respectively. A droplet freezing assay was used to screen colonies for the Ice+ phenotype. Ice+ colonies were then molecularly identified based on the cts (citrate synthase) gene and the 16S rDNA gene. Phylogenetic analysis of cts sequences showed a surprising diversity of phylogenetic subgroups of P. syringae. Frequencies of Ice+ isolates on P. syringae selective medium ranged from 0 to 15% per sample with the highest frequency being found in spring. Our work shows that freshwater lakes can be a significant reservoir of Ice+ P. syringae. Future work is needed to determine the contribution of P. syringae from freshwater lakes to the P. syringae populations present in the atmosphere and on plants and, in particular, if freshwater lakes could be an inoculum source of P. syringae-caused plant disease outbreaks

Genome-enabled phylogeographic investigation of the quarantine pathogen Ralstonia solanacearum race 3 biovar 2 and screening for sources of resistance against its core effectors.

Phylogeographic studies inform about routes of pathogen dissemination and are instrumental for improving import/export controls. Genomes of seventeen isolates of the bacterial wilt and potato brown rot pathogen Ralstonia solanacearum race 3 biovar 2 (R3bv2), a select agent in the USA, were thus analyzed to get insight into the phylogeography of this pathogen. Thirteen of fourteen isolates from Europe, Africa, and Asia were found to belong to a single clonal lineage while isolates from South America were genetically diverse and carried ancestral alleles at the analyzed genomic loci consistent with a South American origin of R3bv2. The R3bv2 isolates share a core repertoire of thirty-one type III-secreted effector genes representing excellent candidates to be targeted with resistance genes in breeding programs to develop durable disease resistance. Towards this goal, 27 R3bv2 effectors were tested in eggplant, tomato, pepper, tobacco, and lettuce for induction of a hypersensitive-like response indicative of recognition by cognate resistance receptors. Fifteen effectors, eight of them core effectors, triggered a response in one or more plant species. These genotypes may harbor resistance genes that could be identified and mapped, cloned and expressed in tomato or potato, for which sources of genetic resistance to R3bv2 are extremely limited.National Science Foundatio

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

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.Many bacterial pathogens are well characterized but, in some cases, relatively 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.Caroline L. Monteil received support from INRA and the European Union, in the framework of the Marie-Curie FP7 COFUND People Programme, through the award of an AgreenSkills’ fellowship (under grant agreement n° 267196). Research in Boris A. Vinatzer’s laboratory and genome sequencing was funded by the National Science Foundation of the USA (grants IOS-1354215 and DEB-1241068). Funding for work in the Vinatzer laboratory was also provided in part by the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture. Work carried out in the Sheppard laboratory was supported by the Medical Research Council (MRC) grant MR/L015080/1, and the Wellcome Trust grant 088786/C/09/Z. GM was supported by a NISCHR Health Research Fellowship (HF-14-13)

Comprehensive characterization of an aspen (<i>Populus tremuloides</i>) leaf litter sample that maintained ice nucleation activity for 48 years

Decaying vegetation was determined to be a potentially important source of atmospheric ice nucleation particles (INPs) in the early 1970s. The bacterium Pseudomonas syringae was the first microorganism with ice nucleation activity (INA) isolated from decaying leaf litter in 1974. However, the ice nucleation characteristics of P. syringae are not compatible with the characteristics of leaf litter-derived INPs since the latter were found to be sub-micron in size, while INA of P. syringae depends on much larger intact bacterial cells. Here we determined the cumulative ice nucleation spectrum and microbial community composition of the historic leaf litter sample 70-S-14 collected in 1970 that conserved INA for 48 years. The majority of the leaf litter-derived INPs were confirmed to be sub-micron in size and to be sensitive to boiling. Culture-independent microbial community analysis only identified Pseudomonas as potential INA. Culture-dependent analysis identified one P. syringae isolate, two isolates of the bacterial species Pantoea ananatis, and one fungal isolate of Mortierella alpina as having INA among 1170 bacterial colonies and 277 fungal isolates, respectively. Both Pa. ananatis and M. alpina are organisms that produce heat-sensitive sub-micron INPs. They are thus both likely sources of the INPs present in sample 70-S-14 and may represent important terrestrial sources of atmospheric INPs, a conclusion that is in line with other recent results obtained in regard to INPs from soil, precipitation, and the atmosphere.</p

In Vivo Evolution of Tumor-Derived Endothelial Cells

The growth of a malignant tumor beyond a certain, limited size requires that it first develop an independent blood supply. In addition to providing metabolic support, this neovasculature also allows tumor cells to access the systemic circulation, thus facilitating metastatic dissemination. The neovasculature may originate either from normal blood vessels in close physical proximity to the tumor and/or from the recruitment of bone marrow-derived endothelial cell (EC) precursors. Recent studies have shown that human tumor vasculature ECs may also arise directly from tumor cells themselves and that the two populations have highly similar or identical karyotypes. We now show that, during the course of serial in vivo passage, these tumor-derived ECs (TDECs) progressively acquire more pronounced EC-like properties. These include higher-level expression of EC-specific genes and proteins, a greater capacity for EC-like behavior in vitro, and a markedly enhanced propensity to incorporate into the tumor vasculature. In addition, both vessel density and size are significantly increased in neoplasms derived from mixtures of tumor cells and serially passaged TDECs. A comparison of early- and late-passage TDECs using whole-genome single nucleotide polymorphism profiling showed the latter cells to have apparently evolved by a process of clonal expansion of a population with a distinct pattern of interstitial chromosomal gains and losses affecting a relatively small number of genes. The majority of these have established roles in vascular development, tumor suppression or epithelial-mesenchymal transition. These studies provide direct evidence that TDECs have a strong evolutionary capacity as a result of their inherent genomic instability. Consequently such cells might be capable of escaping anti-angiogenic cancer therapies by generating resistant populations

Deletions in the Repertoire of Pseudomonas syringae pv. tomato DC3000 Type III Secretion Effector Genes Reveal Functional Overlap among Effectors

The γ-proteobacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 uses the type III secretion system to inject ca. 28 Avr/Hop effector proteins into plants, which enables the bacterium to grow from low inoculum levels to produce bacterial speck symptoms in tomato, Arabidopsis thaliana, and (when lacking hopQ1-1) Nicotiana benthamiana. The effectors are collectively essential but individually dispensable for the ability of the bacteria to defeat defenses, grow, and produce symptoms in plants. Eighteen of the effector genes are clustered in six genomic islands/islets. Combinatorial deletions involving these clusters and two of the remaining effector genes revealed a redundancy-based structure in the effector repertoire, such that some deletions diminished growth in N. benthamiana only in combination with other deletions. Much of the ability of DC3000 to grow in N. benthamiana was found to be due to five effectors in two redundant-effector groups (REGs), which appear to separately target two high-level processes in plant defense: perception of external pathogen signals (AvrPto and AvrPtoB) and deployment of antimicrobial factors (AvrE, HopM1, HopR1). Further support for the membership of HopR1 in the same REG as AvrE was gained through bioinformatic analysis, revealing the existence of an AvrE/DspA/E/HopR effector superfamily, which has representatives in virtually all groups of proteobacterial plant pathogens that deploy type III effectors

Deep Sequencing of Small RNAs in Tomato for Virus and Viroid Identification and Strain Differentiation

Small RNAs (sRNA), including microRNAs (miRNA) and small interfering RNAs (siRNA), are produced abundantly in plants and animals and function in regulating gene expression or in defense against virus or viroid infection. Analysis of siRNA profiles upon virus infection in plant may allow for virus identification, strain differentiation, and de novo assembly of virus genomes. In the present study, four suspected virus-infected tomato samples collected in the U.S. and Mexico were used for sRNA library construction and deep sequencing. Each library generated between 5–7 million sRNA reads, of which more than 90% were from the tomato genome. Upon in-silico subtraction of the tomato sRNAs, the remaining highly enriched, virus-like siRNA pools were assembled with or without reference virus or viroid genomes. A complete genome was assembled for Potato spindle tuber viroid (PSTVd) using siRNA alone. In addition, a near complete virus genome (98%) also was assembled for Pepino mosaic virus (PepMV). A common mixed infection of two strains of PepMV (EU and US1), which shared 82% of genome nucleotide sequence identity, also could be differentially assembled into their respective genomes. Using de novo assembly, a novel potyvirus with less than 60% overall genome nucleotide sequence identity to other known viruses was discovered and its full genome sequence obtained. Taken together, these data suggest that the sRNA deep sequencing technology will likely become an efficient and powerful generic tool for virus identification in plants and animals

Cytosolic phospholipase A2-α expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours

BACKGROUND: The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A(2)α (cPLA(2)α) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA(2)α expression correlated with EGFR/HER2 over-expression in a small number of breast cancer cell lines. METHODS: The importance of differential cPLA(2)α activity in clinical breast cancer was established by relating the expression of cPLA(2)α in tissue samples from breast cancer patients, and two microarray-based gene expression datasets to different clinicopathological and therapeutic parameters. RESULTS: High cPLA(2)α mRNA expression correlated with clinical parameters of poor prognosis, which are characteristic of highly invasive tumours of the HER2-positive and basal-like subtype, including low oestrogen receptor expression and high EGFR expression. High cPLA(2)α expression decreased overall survival in patients with luminal cancers, and correlated with a reduced effect of tamoxifen treatment. The cPLA(2)α expression was an independent predictive parameter of poor response to endocrine therapy in the first 5 years of follow-up. CONCLUSION: This study shows a role of cPLA(2)α in luminal breast cancer progression, in which the enzyme could represent a novel therapeutic target and a predictive marker

A FRUITFULL-like gene is associated with genetic variation for fruit flesh firmness in apple (Malus domestica Borkh.)

The FRUITFULL (FUL) and SHATTERPROOF (SHP) genes are involved in regulating fruit development and dehiscence in Arabidopsis. We tested the hypothesis that this class of genes are also involved in regulating the development of fleshy fruits, by exploring genetic and phenotypic variation within the apple (Malus domestica) gene pool. We isolated and characterised the genomic sequences of two candidate orthologous FUL-like genes, MdMADS2.1 and MdMADS2.2. These were mapped using the reference population ‘Prima x Fiesta’ to loci on Malus linkage groups LG14 and LG06, respectively. An additional MADS-box gene, MdMADS14, shares high amino acid identity with the Arabidopsis SHATTERPROOF1/2 genes and was mapped to Malus linkage group LG09. Association analysis between quantitative fruit flesh firmness estimates of ‘Prima x Fiesta’ progeny and the MdMADS2.1, MdMADS2.2 and MdMADS14 loci was carried out using a mixed model analysis of variance. This revealed a significant association (P < 0.01) between MdMADS2.1 and fruit flesh firmness. Further evidence for the association between MdMADS2.1 and fruit flesh firmness was obtained using a case–control population-based genetic association approach. For this, a polymorphic repeat, (AT)n, in the 3′ UTR of MdMADS2.1 was used as a locus-specific marker to screen 168 apple accessions for which historical assessments of fruit texture attributes were available. This analysis revealed a significant association between the MdMADS2.1 and fruit flesh firmness at both allelic (χ 2 = 34, df = 9, P < 0.001) and genotypic (χ 2 = 57, df = 32, P < 0.01) levels

Genome Sequence Analyses of Pseudomonas savastanoi pv. glycinea and Subtractive Hybridization-Based Comparative Genomics with Nine Pseudomonads

Bacterial blight, caused by Pseudomonas savastanoi pv. glycinea (Psg), is a common disease of soybean. In an effort to compare a current field isolate with one isolated in the early 1960s, the genomes of two Psg strains, race 4 and B076, were sequenced using 454 pyrosequencing. The genomes of both Psg strains share more than 4,900 highly conserved genes, indicating very low genetic diversity between Psg genomes. Though conserved, genome rearrangements and recombination events occur commonly within the two Psg genomes. When compared to each other, 437 and 163 specific genes were identified in B076 and race 4, respectively. Most specific genes are plasmid-borne, indicating that acquisition and maintenance of plasmids may represent a major mechanism to change the genetic composition of the genome and even acquire new virulence factors. Type three secretion gene clusters of Psg strains are near identical with that of P. savastanoi pv. phaseolicola (Pph) strain 1448A and they shared 20 common effector genes. Furthermore, the coronatine biosynthetic cluster is present on a large plasmid in strain B076, but not in race 4. In silico subtractive hybridization-based comparative genomic analyses with nine sequenced phytopathogenic pseudomonads identified dozens of specific islands (SIs), and revealed that the genomes of Psg strains are more similar to those belonging to the same genomospecies such as Pph 1448A than to other phytopathogenic pseudomonads. The number of highly conserved genes (core genome) among them decreased dramatically when more genomes were included in the subtraction, suggesting the diversification of pseudomonads, and further indicating the genome heterogeneity among pseudomonads. However, the number of specific genes did not change significantly, suggesting these genes are indeed specific in Psg genomes. These results reinforce the idea of a species complex of P. syringae and support the reclassification of P. syringae into different species