Stability of plasmid profile of highly virulent races of Xanthomonas campestris pv. malvacearum during storage and subculturing

Four races of Xanthomonas campestris pv. mal-vacearum (Xcm) viz. races 23, 27 and 32 (isolated from Gossypium hirsutum) and race 23b (from Gossypium barbadense) were studied. The plasmid profile of the natural isolates showed four plasmids in races 23 and 23b (ca. 60, 40, 23, 8.2 kb), five in race 27 (ca. 60, 40, 23, 8.2 and 3.7 kb) and six in race 32 (ca. 60, 40, 23, 8.2, 3.7 and 1.6 kb). Continuously sub-cultured laboratory isolates of the Xcm races resulted in the loss of all but two plasmids, ca. 60 and 40 kb in size. When the laboratory isolates were passed through cotton (Gossypium hirsutum), they regained certain plasmids so that four plasmids were found in race 23 and 23b (ca. 60, 40, 23 and 8.2 kb), five in race 27 (ca. 60, 40, 23, 8.2 and 3.7 kb) and six in race 32 (ca. 60, 40, 23, 8.2, 3.7 and 1.6 kb), which was more or less similar to the original isolates. The isolates recovered from cotton maintained their plasmid profile (except for minor changes in the miniplasmids) after storage for six months at -70degreesC in 50% glycerol. It is suggested that plasmid profiles among highly virulent races of Xcm are unstable during repeated sub-culturing at room temperature, resulting in rapid loss of some plasmids. However, when the cultures were sub-cultured and stored at -70degreesC the plasmid profile was fairly stable except for the miniplasmids (ca. 3.7 and 1.6 kb).

Stability in miniplasmids of Xanthomonas campestris pv. malvacearum during subculturing and storage - Abstract 7

Abstract 7

Can the dynamical impact of the stratosphere on the troposphere be described by large-scale adjustment to the stratospheric PV distribution?

Recent numerical experiments have demonstrated that the state of the stratosphere has a dynamical impact on the state of the troposphere. To account for such an effect, a number of mechanisms have been proposed in the literature, all of which amount to a large-scale adjustment of the troposphere to potential vorticity (PV) anomalies in the stratosphere. This paper analyses whether a simple PV adjustment suffices to explain the actual dynamical response of the troposphere to the state of the stratosphere, the actual response being determined by ensembles of numerical experiments run with an atmospheric general-circulation model. For this purpose, a new PV inverter is developed. It is shown that the simple PV adjustment hypothesis is inadequate. PV anomalies in the stratosphere induce, by inversion, flow anomalies in the troposphere that do not coincide spatially with the tropospheric changes determined by the numerical experiments. Moreover, the tropospheric anomalies induced by PV inversion are on a larger scale than the changes found in the numerical experiments, which are linked to the Atlantic and Pacific storm-tracks. These findings imply that the impact of the stratospheric state on the troposphere is manifested through the impact on individual synoptic-scale systems and their self-organization in the storm-tracks. Changes in these weather systems in the troposphere are not merely synoptic-scale noise on a larger scale tropospheric response, but an integral part of the mechanism by which the state of the stratosphere impacts that of the troposphere.

An interpretation of baroclinic initial value problems: results for simple basic states with nonzero interior PV gradients

In the Eady model, where the meridional potential vorticity (PV) gradient is zero, perturbation energy growth can be partitioned cleanly into three mechanisms: (i) shear instability, (ii) resonance, and (iii) the Orr mechanism. Shear instability involves two-way interaction between Rossby edge waves on the ground and lid, resonance occurs as interior PV anomalies excite the edge waves, and the Orr mechanism involves only interior PV anomalies. These mechanisms have distinct implications for the structural and temporal linear evolution of perturbations. Here, a new framework is developed in which the same mechanisms can be distinguished for growth on basic states with nonzero interior PV gradients. It is further shown that the evolution from quite general initial conditions can be accurately described (peak error in perturbation total energy typically less than 10%) by a reduced system that involves only three Rossby wave components. Two of these are counterpropagating Rossby waves—that is, generalizations of the Rossby edge waves when the interior PV gradient is nonzero—whereas the other component depends on the structure of the initial condition and its PV is advected passively with the shear flow. In the cases considered, the three-component model outperforms approximate solutions based on truncating a modal or singular vector basis.

Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum.

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.

Confocal imaging of pseudomonas syringae pv. phaseolicola colony development in bean reveals reduced multiplication of strains containing the genomic island PPHGI-1.

Pseudomonas syringae pv. phaseolicola is the seed borne causative agent of halo blight in the common bean Phaseolus vulgaris. Pseudomonas syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene hopAR1 (located on a 106-kb genomic island, PPHGI-1, and earlier named avrPphB), which matches resistance gene R3 in P. vulgaris cultivar Tendergreen (TG) and causes a rapid hypersensitive reaction (HR). Here, we have fluorescently labeled selected Pseudomonas syringae pv. phaseolicola 1302A and 1448A strains (with and without PPHGI-1) to enable confocal imaging of in-planta colony formation within the apoplast of resistant (TG) and susceptible (Canadian Wonder [CW]) P. vulgaris leaves. Temporal quantification of fluorescent Pseudomonas syringae pv. phaseolicola colony development correlated with in-planta bacterial multiplication (measured as CFU/ml) and is, therefore, an effective means of monitoring Pseudomonas syringae pv. phaseolicola endophytic colonization and survival in P. vulgaris. We present advances in the application of confocal microscopy for in-planta visualization of Pseudomonas syringae pv. phaseolicola colony development in the leaf mesophyll to show how the HR defense response greatly affects colony morphology and bacterial survival. Unexpectedly, the presence of PPHGI-1 was found to cause a reduction of colony development in susceptible P. vulgaris CW leaf tissue. We discuss the evolutionary consequences that the acquisition and retention of PPHGI-1 brings to Pseudomonas syringae pv. phaseolicola in planta.

Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae

The EfeM protein is a component of the putative EfeUOBM iron-transporter of Pseudomonas syringae pathovar syringae and is thought to act as a periplasmic, ferrous-iron binding protein. It contains a signal peptide of 34 amino acid residues and a C-terminal 'Peptidase_M75' domain of 251 residues. The C-terminal domain contains a highly conserved 'HXXE' motif thought to act as part of a divalent cation-binding site. In this work, the gene (efeM or 'Psyr_3370') encoding EfeM was cloned and over-expressed in Escherichia coli, and the mature protein was purified from the periplasm. Mass spectrometry confirmed the identity of the protein (M(W) 27,772Da). Circular dichroism spectroscopy of EfeM indicated a mainly alpha-helical structure, consistent with bioinformatic predictions. Purified EfeM was crystallised by hanging-drop vapor diffusion to give needle-shaped crystals that diffracted to a resolution of 1.6A. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role.

In planta conditions induce genomic changes in Pseudomonas syringae pv. phaseolicola

The co-evolution of bacterial plant pathogens and their hosts is a complex and dynamic process. Plant resistance can impose stress on invading pathogens that can lead to, and select for, beneficial changes in the bacterial genome. The Pseudomonas syringae pv. phaseolicola (Pph) genomic island PPHGI-1 carries an effector gene, avrPphB (hopAR1), which triggers the hypersensitive reaction in bean plants carrying the R3 resistance gene. Interaction between avrPphB and R3 generates an antimicrobial environment within the plant, resulting in the excision of PPHGI-1 and its loss from the genome. The loss of PPHGI-1 leads to the generation of a Pph strain able to cause disease in the plant. In this study, we observed that lower bacterial densities inoculated into resistant bean (Phaseolus vulgaris) plants resulted in quicker PPHGI-1 loss from the population, and that loss of the island was strongly influenced by the type of plant resistance encountered by the bacteria. In addition, we found that a number of changes occurred in the bacterial genome during growth in the plant, whether or not PPHGI-1 was lost. We also present evidence that the circular PPHGI-1 episome is able to replicate autonomously when excised from the genome. These results shed more light onto the plasticity of the bacterial genome as it is influenced by in planta conditions.

Changes in race-specific virulence in pseudomonas syringae pv. phaseolicola are Associated with a chimeric transposable element and rare deletion events in a plasmid-borne pathogenicity island

Virulence for bean and soybean is determined by effector genes in a plasmid-borne pathogenicity island (PAI) in race 7 strain 1449B of Pseudomonas syringae pv. phaseolicola. One of the effector genes, avrPphF, confers either pathogenicity, virulence, or avirulence depending on the plant host and is absent from races 2, 3, 4, 6, and 8 of this pathogen. Analysis of cosmid clones and comparison of DNA sequences showed that the absence of avrPphF from strain 1448A is due to deletion of a continuous 9.5-kb fragment. The remainder of the PAI is well conserved in strains 1448A and 1449B. The left junction of the deleted region consists of a chimeric transposable element generated from the fusion of homologs of IS1492 from Pseudomonas putida and IS1090 from Ralstonia eutropha. The borders of the deletion were conserved in 66 P. syringae pv. phaseolicola strains isolated in different countries and representing the five races lacking avrPphF. However, six strains isolated in Spain had a 10.5-kb deletion that extended 1 kb further from the right junction. The perfect conservation of the 28-nucleotide right repeat of the IS1090 homolog in the two deletion types and in the other 47 insertions of the IS1090 homolog in the 1448A genome strongly suggests that the avrPphF deletions were mediated by the activity of the chimeric mobile element. Our data strongly support a clonal origin for the races of P. syringae pv. phaseolicola lacking avrPphF.