Phaseolicola 1448A Research Articles

A novel strategy to control Pseudomonas syringae through inhibition of type III secretion system

Pseudomonas syringae (P. syringae) is a highly prevalent Gram-negative pathogen with over 60 pathogenic variants that cause yield losses of up to 80% in various crops. Traditional control methods mainly involve the application of antibiotics to inactivate pathogenic bacteria, but large-scale application of antibiotics has led to the development of bacterial resistance. Gram-negative pathogens including P. syringae commonly use the type III secretion system (T3SS) as a transport channel to deliver effector proteins into host cells, disrupting host defences and facilitating virulence, providing a novel target for antibacterial drug development. In this study, we constructed a high-throughput screening reporter system based on our previous work to screen for imidazole, oxazole and thiazole compounds. The screening indicated that the three compounds (II-14, II-15 and II-24) significantly inhibited hrpW and hrpL gene promoter activity without influencing the growth of P. syringae, and the inhibitory activity was better than that of the positive control sulforaphane (4-methylsulfinylbutyl isothiocyanate, SFN) at 50 μM. Three compounds suppressed the transcript levels of representative T3SS genes to different degrees, suggesting that the compounds may suppress the expression of T3SS by modulating the HrpR/S-HrpL regulatory pathway. Inoculation experiments indicated that all three compounds suppressed the pathogenicity of Pseudomonas syringae pv. tomato DC3000 in tomato and Pseudomonas syringae pv. phaseolicola 1448A in bean to varying degrees. One representative compound, II-15, significantly inhibited the secretion of the Pst DC3000 AvrPto effector protein. These findings provide a theoretical basis for the development of novel P. syringae T3SS inhibitors for application in disease prevention and control.

Remodeling of Lipid A in Pseudomonas syringae pv. phaseolicola In Vitro.

Pseudomonas species infect a variety of organisms, including mammals and plants. Mammalian pathogens of the Pseudomonas family modify their lipid A during host entry to evade immune responses and to create an effective barrier against different environments, for example by removal of primary acyl chains, addition of phosphoethanolamine (P-EtN) to primary phosphates, and hydroxylation of secondary acyl chains. For Pseudomonas syringae pv. phaseolicola (Pph) 1448A, an economically important pathogen of beans, we observed similar lipid A modifications by mass spectrometric analysis. Therefore, we investigated predicted proteomes of various plant-associated Pseudomonas spp. for putative lipid A-modifying proteins using the well-studied mammalian pathogen Pseudomonas aeruginosa as a reference. We generated isogenic mutant strains of candidate genes and analyzed their lipid A. We show that the function of PagL, LpxO, and EptA is generally conserved in Pph 1448A. PagL-mediated de-acylation occurs at the distal glucosamine, whereas LpxO hydroxylates the secondary acyl chain on the distal glucosamine. The addition of P-EtN catalyzed by EptA occurs at both phosphates of lipid A. Our study characterizes lipid A modifications in vitro and provides a useful set of mutant strains relevant for further functional studies on lipid A modifications in Pph 1448A.

HopH1 effectors of Pseudomonas syringae pv. tomato DC3000 and pv. syringae B728a induce HR cell death in nonhost eggplant Solanum torvum

HopH1 is an effector protein of Pseudomonas syringae pv. tomato DC3000 and P. syringae pv. syringae B728a and is a homolog of the putative Zn-dependent protease effector Rip36 of Ralstonia solanacearum, which induces hypersensitive response (HR) cell death in a nonhost plant, Solanum torvum Sw. cv. Torubamubiga. Although P. syringae pv. phaseolicola (Pph) 1448A neither produces HopH1 nor induces HR cell death, hopH1-introduced Pph 1448A acquired the ability to induce HR. These results indicate that the putative Zn-protease HopH1 effector induces HR cell death in nonhost S. torvum.

Two Homologues of the Global Regulator Csr/Rsm Redundantly Control Phaseolotoxin Biosynthesis and Virulence in the Plant Pathogen Pseudomonas amygdali pv. phaseolicola 1448A.

The widely conserved Csr/Rsm (carbon storage regulator/repressor of stationary-phase metabolites) post-transcriptional regulatory system controls diverse phenotypes involved in bacterial pathogenicity and virulence. Here we show that Pseudomonas amygdali pv. phaseolicola 1448A contains seven rsm genes, four of which are chromosomal. In RNAseq analyses, only rsmE was thermoregulated, with increased expression at 18 °C, whereas the antagonistic sRNAs rsmX1, rsmX4, rsmX5 and rsmZ showed increased levels at 28 °C. Only double rsmA-rsmE mutants showed significantly altered phenotypes in functional analyses, being impaired for symptom elicitation in bean, including in planta growth, and for induction of the hypersensitive response in tobacco. Double mutants were also non-motile and were compromised for the utilization of different carbon sources. These phenotypes were accompanied by reduced mRNA levels of the type III secretion system regulatory genes hrpL and hrpA, and the flagellin gene, fliC. Biosynthesis of the phytotoxin phaseolotoxin by mutants in rsmA and rsmE was delayed, occurring only in older cultures, indicating that these rsm homologues act as inductors of toxin synthesis. Therefore, genes rsmA and rsmE act redundantly, although with a degree of specialization, to positively regulate diverse phenotypes involved in niche colonization. Additionally, our results suggest the existence of a regulatory molecule different from the Rsm proteins and dependent on the GacS/GacA (global activator of antibiotic and cyanide production) system, which causes the repression of phaseolotoxin biosynthesis at high temperatures.

Two Strategies of Pseudomonas syringae to Avoid Recognition of the HopQ1 Effector in Nicotiana Species.

Pseudomonas syringae employs a battery of type three secretion effectors to subvert plant immune responses. In turn, plants have developed receptors that recognize some of the bacterial effectors. Two strain-specific HopQ1 effector variants (for Hrp outer protein Q) from the pathovars phaseolicola 1448A (Pph) and tomato DC3000 (Pto) showed considerable differences in their ability to evoke disease symptoms in Nicotiana benthamiana. Surprisingly, the variants differ by only six amino acids located mostly in the N-terminal disordered region of HopQ1. We found that the presence of serine 87 and leucine 91 renders PtoHopQ1 susceptible to N-terminal processing by plant proteases. Substitutions at these two positions did not strongly affect PtoHopQ1 virulence properties in a susceptible host but they reduced bacterial growth and accelerated onset of cell death in a resistant host, suggesting that N-terminal mutations rendered PtoHopQ1 susceptible to processing in planta and, thus, represent a mechanism of recognition avoidance. Furthermore, we found that co-expression of HopR1, another effector encoded within the same gene cluster masks HopQ1 recognition in a strain-dependent manner. Together, these data suggest that HopQ1 is under high host-pathogen co-evolutionary selection pressure and P. syringae may have evolved differential effector processing or masking as two independent strategies to evade HopQ1 recognition, thus revealing another level of complexity in plant – microbe interactions.

Spatial and temporal dynamics of primary and secondary metabolism in Phaseolus vulgaris challenged by Pseudomonas syringae.

Many defense mechanisms contribute to the plant immune system against pathogens, involving the regulation of different processes of the primary and secondary metabolism. At the same time, pathogens have evolved mechanisms to hijack the plant defense in order to establish the infection and proliferate. Localization and timing of the host response are essential to understand defense mechanisms and resistance to pathogens (Rico et al. 2011). Imaging techniques, such as fluorescence imaging and thermography, are a very valuable tool providing spatial and temporal information about a series of plant processes. In this study, bean plants challenged with two pathovars of Pseudomonas syringae have been investigated. Pseudomonas syringae pv. phaseolicola 1448A and P. syringae pv. tomato DC3000 elicit a compatible and incompatible interaction in bean, respectively. Both types of host-pathogen interaction triggered different changes in the activity of photosynthesis and the secondary metabolism. We conclude that the combined analysis of leaf temperature, chlorophyll fluorescence and green fluorescence emitted by phenolics allows to discriminate compatible from incompatible P. syringae-Phaseolus vulgaris interactions in very early times of the infection, prior to the development of symptoms. These can constitute disease signatures that would allow an early identification of emerging plagues in crops.

Variable Suites of Non-effector Genes Are Co-regulated in the Type III Secretion Virulence Regulon across the Pseudomonas syringae Phylogeny

Pseudomonas syringae is a phylogenetically diverse species of Gram-negative bacterial plant pathogens responsible for crop diseases around the world. The HrpL sigma factor drives expression of the major P. syringae virulence regulon. HrpL controls expression of the genes encoding the structural and functional components of the type III secretion system (T3SS) and the type three secreted effector proteins (T3E) that are collectively essential for virulence. HrpL also regulates expression of an under-explored suite of non-type III effector genes (non-T3E), including toxin production systems and operons not previously associated with virulence. We implemented and refined genome-wide transcriptional analysis methods using cDNA-derived high-throughput sequencing (RNA-seq) data to characterize the HrpL regulon from six isolates of P. syringae spanning the diversity of the species. Our transcriptomes, mapped onto both complete and draft genomes, significantly extend earlier studies. We confirmed HrpL-regulation for a majority of previously defined T3E genes in these six strains. We identified two new T3E families from P. syringae pv. oryzae 1_6, a strain within the relatively underexplored phylogenetic Multi-Locus Sequence Typing (MLST) group IV. The HrpL regulons varied among strains in gene number and content across both their T3E and non-T3E gene suites. Strains within MLST group II consistently express the lowest number of HrpL-regulated genes. We identified events leading to recruitment into, and loss from, the HrpL regulon. These included gene gain and loss, and loss of HrpL regulation caused by group-specific cis element mutations in otherwise conserved genes. Novel non-T3E HrpL-regulated genes include an operon that we show is required for full virulence of P. syringae pv. phaseolicola 1448A on French bean. We highlight the power of integrating genomic, transcriptomic, and phylogenetic information to drive concise functional experimentation and to derive better insight into the evolution of virulence across an evolutionarily diverse pathogen species.

Comparative analysis of flagellin glycans among pathovars of phytopathogenic Pseudomonas syringae

Flagellin is a principal component of the flagellum filament. Previously, we reported that the flagellin of Pseudomonas syringae pv. tabaci 6605 (Pta6605) was glycosylated by oligosaccharides composed of two or three l-rhamnosyl (l-Rha) residues and a terminal 4,6-dideoxy-4-(3-hydroxybutanamide)-2-O-methylglucopyranosyl residue. In this study, we characterized the chemical structure of flagellin glycans in P. syringae pathovars glycinea race 4 (Pgl4), phaseolicola 1448A (Pph1448A), tomato DC3000 (PtoDC3000), and syringae B728a (PsyB728a). Flagellin glycans were released by hydrazinolysis, labeled on the reducing ends with 2-aminopyridine (PA), and the PA-labeled oligosaccharides were isolated by high-performance liquid chromatography. The purified PA-labeled glycans were analyzed by mass spectrometry and NMR spectroscopy. The results showed that the glycans on flagellin of Pgl4, PtoDC3000, and Pph1448A were identical to those of Pta6605, which were characterized previously. The flagellin of PsyB728a is O-glycosylated with a novel trisaccharide identified as 2-acetamide-2-deoxy-β-d-glucopyranosyl-(1→2)-3-O-methyl-α-l-rhamnopyranosyl-(1→2)-l-rhamnose. Our data indicate that flagellin glycosylation of P. syringae pathovars has universality with little diversity.

Genetic analysis of the individual contribution to virulence of the type III effector inventory of Pseudomonas syringae pv. phaseolicola.

Several reports have recently contributed to determine the effector inventory of the sequenced strain Pseudomonas syringae pv. phaseolicola (Pph) 1448a. However, the contribution to virulence of most of these effectors remains to be established. Genetic analysis of the contribution to virulence of individual P. syringae effectors has been traditionally hindered by the lack of phenotypes of the corresponding knockout mutants, largely attributed to a high degree of functional redundancy within their effector inventories. In support of this notion, effectors from Pseudomonas syringae pv. tomato (Pto) DC3000 have been classified into redundant effector groups (REGs), analysing virulence of polymutants in the model plant Nicotiana benthamiana. However, using competitive index (CI) as a virulence assay, we were able to establish the individual contribution of AvrPto1Pto DC3000 to Pto DC3000 virulence in tomato, its natural host, even though typically, contribution to virulence of AvrPto1 is only shown in strains also lacking AvrPtoB (also called HopAB2), a member of its REG. This report raised the possibility that even effectors targeting the same defence signalling pathway may have an individual contribution to virulence, and pointed out to CI assays as the means to establish such a contribution for individual effectors. In this work, we have analysed the individual contribution to virulence of the majority of previously uncharacterised Pph 1448a effectors, by monitoring the development of disease symptoms and determining the CI of single knockout mutants at different stages of growth within bean, its natural host. Despite their potential functional redundancy, we have found individual contributions to virulence for six out of the fifteen effectors analysed. In addition, we have analysed the functional relationships between effectors displaying individual contribution to virulence, highlighting the diversity that these relationships may present, and the interest of analysing their functions within the context of the infection.

Characterisation of the mgo operon in Pseudomonas syringae pv. syringae UMAF0158 that is required for mangotoxin production.

BackgroundMangotoxin is an antimetabolite toxin that is produced by strains of Pseudomonas syringae pv. syringae; mangotoxin-producing strains are primarily isolated from mango tissues with symptoms of bacterial apical necrosis. The toxin is an oligopeptide that inhibits ornithine N-acetyl transferase (OAT), a key enzyme in the biosynthetic pathway of the essential amino acids ornithine and arginine. The involvement of a putative nonribosomal peptide synthetase gene (mgoA) in mangotoxin production and virulence has been reported.ResultsIn the present study, we performed a RT-PCR analysis, insertional inactivation mutagenesis, a promoter expression analysis and terminator localisation to study the gene cluster containing the mgoA gene. Additionally, we evaluated the importance of mgoC, mgoA and mgoD in mangotoxin production. A sequence analysis revealed an operon-like organisation. A promoter sequence was located upstream of the mgoB gene and was found to drive lacZ transcription. Two terminators were located downstream of the mgoD gene. RT-PCR experiments indicated that the four genes (mgoBCAD) constitute a transcriptional unit. This operon is similar in genetic organisation to those in the three other P. syringae pathovars for which complete genomes are available (P. syringae pv. syringae B728a, P. syringae pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A). Interestingly, none of these three reference strains is capable of producing mangotoxin. Additionally, extract complementation resulted in a recovery of mangotoxin production when the defective mutant was complemented with wild-type extracts.ConclusionsThe results of this study confirm that mgoB, mgoC, mgoA and mgoD function as a transcriptional unit and operon. While this operon is composed of four genes, only the last three are directly involved in mangotoxin production.

Use of Low-Coverage, Large-Insert, Short-Read Data for Rapid and Accurate Generation of Enhanced-Quality Draft Pseudomonas Genome Sequences

Next-generation genomic technology has both greatly accelerated the pace of genome research as well as increased our reliance on draft genome sequences. While groups such as the Genomics Standards Consortium have made strong efforts to promote genome standards there is a still a general lack of uniformity among published draft genomes, leading to challenges for downstream comparative analyses. This lack of uniformity is a particular problem when using standard draft genomes that frequently have large numbers of low-quality sequencing tracts. Here we present a proposal for an “enhanced-quality draft” genome that identifies at least 95% of the coding sequences, thereby effectively providing a full accounting of the genic component of the genome. Enhanced-quality draft genomes are easily attainable through a combination of small- and large-insert next-generation, paired-end sequencing. We illustrate the generation of an enhanced-quality draft genome by re-sequencing the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola 1448A (Pph 1448A), which has a published, closed genome sequence of 5.93 Mbp. We use a combination of Illumina paired-end and mate-pair sequencing, and surprisingly find that de novo assemblies with 100x paired-end coverage and mate-pair sequencing with as low as low as 2–5x coverage are substantially better than assemblies based on higher coverage. The rapid and low-cost generation of large numbers of enhanced-quality draft genome sequences will be of particular value for microbial diagnostics and biosecurity, which rely on precise discrimination of potentially dangerous clones from closely related benign strains.

Miniature Transposable Sequences Are Frequently Mobilized in the Bacterial Plant Pathogen Pseudomonas syringae pv. phaseolicola

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.6×10−5 and 1.1×10−6, depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria.

Characterization of pyoverdine and achromobactin in Pseudomonas syringae pv. phaseolicola 1448a.

BackgroundPseudomonas syringae pv. phaseolicola 1448a (P. syringae 1448a), the causative agent of bean halo blight, is a bacterium capable of occupying diverse biological niches. Under conditions of iron starvation P. syringae 1448a secretes siderophores for active uptake of iron. The primary siderophore of P. syringae 1448a is pyoverdine, a fluorescent molecule that is assembled from amino acid precursors by non-ribosomal peptide synthetase (NRPS) enzymes. Whereas other species of Pseudomonas often exhibit structural variations in the pyoverdine produced by different strains, all P. syringae pathovars previously tested have been found to make an identical pyoverdine molecule. P. syringae 1448a also appears to have the genetic potential to make two secondary siderophores, achromobactin and yersiniabactin, each of which has previously been detected in different P. syringae pathovars.ResultsFive putative pyoverdine NRPS genes in P. syringae 1448a were characterized in-silico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Pyoverdine was purified from P. syringae 1448a and analyzed by MALDI-TOF and MS/MS spectroscopy. Peaks were detected corresponding to the expected sizes for the pyoverdine structure previously found in other P. syringae pathovars, but surprisingly P. syringae 1448a appears to also produce a variant pyoverdine species that has an additional 71 Da monomer incorporated into the peptide side chain. Creation of pyoverdine null mutants of P. syringae 1448a revealed that this strain also produces achromobactin as a temperature-regulated secondary siderophore, but does not appear to make yersiniabactin. Pyoverdine and achromobactin null mutants were characterized in regard to siderophore production, iron uptake, virulence and growth in iron limited conditions.ConclusionsThis study provides the first evidence of a P. syringae pathovar producing a side chain variant form of pyoverdine. We also describe novel IC50 and liquid CAS assays to quantify the contribution of different siderophores across a range of iron starvation conditions, and show that although achromobactin has potential to contribute to fitness its contribution is masked by the presence of pyoverdine, which is a significantly more effective siderophore. Neither pyoverdine nor achromobactin appear to be required for P. syringae 1448a to cause bean halo blight, indicating that these siderophores are not promising targets for crop protection strategies.

MOLECULAR AND PHENOTYPIC VARIABILITY AMONG PSEUDOMONAS AVELLANAE, P. SYRINGAE pv. ACTINIDIAE AND P. SYRINGAE pv. THEAE: THE GENOMOSPECIES 8 SENSU GARDAN et al. (1999)

SUMMARY Genomospecie 8, sensu Gardan et al. (1999), includes Pseudomonas avellanae, P. syringae pv. theae and P. s . pv. actinidiae. To further characterize this genomospecies, 14 P. avellanae, three P. s . pv. theae and 18 P. s. pv. actinidiae strains were analysed by multilocus sequence typing (MLST) using gapA, gltA, gyrB and rpoD gene fragments. These strains were also checked for the presence/absence of 38 effector protein genes based on the corresponding sequences of P. syringae. pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A. Nutritional tests and a comparison of the 16S rDNA gene sequences deposited at NCBI database were also done to detect possible differences. MLST analysis, based on 2.5 kb sequences, revealed that P. s. pv. theae and P. s. pv. actinidiae are more closely-related to one another than to P. avellanae. This technique clearly revealed that the P. s . pv. actinidiae strains causing the current severe epidemics in Italy are different from those of past outbreaks in Japan and central Italy. Nine effector protein genes were displayed by all strains of genomospecies 8. However, each pathogen of this genomospecies displays some distinctive effector protein genes. HopA1 and hopH1 are unique to P. s. pv. actinidiae strains of the recent epidemics of bacterial canker on Actinidia chinensis and A. deliciosa in Italy. A triplet, in position 461-463 of the 16S rDNA gene, is different in P. s. pv. actinidiae, namely GAT, and in P. avellanae and P. s. pv. theae, namely ATC. Contrarily to P. s. pv. theae and P. s. pv. actinidiae, P. avellanae did not utilize sorbitol.

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.

In silico analysis reveals multiple putative type VI secretion systems and effector proteins in Pseudomonas syringae pathovars

Type VI secretion systems (T6SS) of Gram-negative bacteria form injectisomes that have the potential to translocate effector proteins into eukaryotic host cells. In silico analysis of the genomes in six Pseudomonas syringae pathovars revealed that P. syringae pv. tomato DC3000, pv. tabaci ATCC 11528, pv. tomato T1 and pv. oryzae 1-6 each carry two putative T6SS gene clusters (HSI-I and HSI-II; HSI: Hcp secretion island), whereas pv. phaseolicola 1448A and pv. syringae B728 each carry one. The pv. tomato DC3000 HSI-I and pv. tomato T1 HSI-II possess a highly similar organization and nucleotide sequence, whereas the pv. tomato DC3000, pv. oryzae 1-6 and pv. tabaci 11528 HSI-II are more divergent. Putative effector orthologues vary in number among the strains examined. The Clp-ATPases and IcmF orthologues form distinct phylogenetic groups: the proteins from pv. tomato DC3000, pv. tomato T1, pv. oryzae and pv. tabaci 11528 from HSI-II group together with most orthologues from other fluorescent pseudomonads, whereas those from pv. phaseolicola, pv. syringae, pv. tabaci, pv. tomato T1 and pv. oryzae from HSI-I group closer to the Ralstonia solanacearum and Xanthomonas orthologues. Our analysis suggests multiple independent acquisitions and possible gene attrition/loss of putative T6SS genes by members of P. syringae.

Analysis of the Role of the Type III Effector Inventory ofPseudomonas syringaepv. phaseolicola 1448a in Interaction with the Plant

In Pseudomonas syringae, the type III secretion system (T3SS) is essential for disease in compatible hosts and for eliciting the hypersensitive response in incompatible hosts. P. syringae pathovars secrete a variable number of type III effectors that form their secretomes. The secretome of Pseudomonas syringae pv. phaseolicola 1448a (Pph1448a) currently includes 22 experimentally validated effectors, one HrpL-regulated candidate for which translocation results have been inconsistent, two translocated candidates for which in planta expression has not been established, one bioinformatically identified candidate, and six candidates that have been experimentally discarded. We analyzed the translocation and/or expression of these and other candidates to complete the Pph1448a effector inventory, bringing this inventory to 27 bona fide effectors, including a new one that does not belong to any of the previously described effector families. We developed a simple process for rapidly making single and double knockout mutants and apply it to the generation of an effector mutant collection that includes single knockouts for the majority of the Pph1448a effector inventory. We also generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the single and double mutant strains as well as strains expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to virulence in bean plants, thus validating this approach for dissecting the contribution of the Pph1448a type III effector inventory to virulence. We also analyzed the effect that the expression of these four effectors from Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) has during its interaction with Arabidopsis thaliana, establishing that AvrB4-1, but not the others, determines a restriction of bacterial growth that takes place mostly independently of the salicylic acid (SA)-signaling pathway.

Annotation and overview of the Pseudomonas savastanoi pv. savastanoi NCPPB 3335 draft genome reveals the virulence gene complement of a tumour‐inducing pathogen of woody hosts

Pseudomonas savastanoi pv. savastanoi is a tumour-inducing pathogen of Olea europaea L. causing olive knot disease. Bioinformatic analysis of the draft genome sequence of strain NCPPB 3335, which encodes 5232 predicted coding genes on a total length of 5856 998 bp and a 57.12% G + C, revealed a large degree of conservation with Pseudomonas syringae pv. phaseolicola 1448A and P. syringae pv. tabaci 11528. However, NCPPB 3335 contains twelve variable genomic regions, which are absent in all previously sequenced P. syringae strains. Various features that could contribute to the ability of this strain to survive in a woody host were identified, including broad catabolic and transport capabilities for degrading plant-derived aromatic compounds, the duplication of sequences related to the biosynthesis of the phytohormone indoleacetic acid (iaaM, iaaH) and its amino acid conjugate indoleacetic acid-lysine (iaaL gene), and the repertoire of strain-specific putative type III secretion system effectors. Access to this seventh genome sequence belonging to the 'P. syringae complex' allowed us to identify 73 predicted coding genes that are NCPPB 3335-specific. Results shown here provide the basis for detailed functional analysis of a tumour-inducing pathogen of woody hosts and for the study of specific adaptations of a P. savastanoi pathovar.

Negative Regulation of the Hrp Type III Secretion System in Pseudomonas syringae pv. phaseolicola

Many plant-pathogenic bacteria require type III secretion systems (T3SS) to cause disease in compatible hosts and to induce the hypersensitive response in resistant plants. T3SS gene expression is induced within the plant and responds to host and environmental factors. In Pseudomonas syringae, expression is downregulated by the Lon protease in rich medium and by HrpV under inducing conditions. HrpV acts as an anti-activator by binding HrpS. HrpG, which can also bind HrpV, has been reported to act as an anti-anti-activator. Previous studies have used mostly in vitro inducing conditions, different pathovars, and methodology. We have used single and double lon and hrpV mutants of P. syringae pv. phaseolicola 1448a, as well as strains ectopically expressing the regulators, to examine their role in coordinating expression of the T3SS. We applied real-time polymerase chain reaction to analyze gene expression both in vitro and in planta, and assessed bacterial fitness using competitive indices. Our results indicate that i) Lon downregulates expression of the hrp/hrc genes in all conditions, probably by constitutively degrading naturally unstable HrpR; ii) HrpV and HrpT downregulate expression of the hrp/hrc genes in all conditions; and iii) HrpG has an additional, HrpV-independent role, regulating expression of the hrpC operon.

Sensitive and specific detection of phaseolotoxigenic and nontoxigenic strains of Pseudomonas syringae pv. phaseolicola by TaqMan real-time PCR using site-specific recombinase gene sequences

Pseudomonas syringae pv. phaseolicola, the causative agent of halo blight, is the most important bacterial pathogen of bean. Both nontoxigenic (Tox(-)) and toxigenic (Tox+) strains of this pathogen cause halo blight in beans. However, nontoxigenic strains cannot be detected by currently available molecular and serological tools. In this study, a TaqMan probe and primer set were designed based on the phage integrase family site-specific recombinase of P. s. pv. phaseolicola 1448A because it is known that most site-specific recombinases are structurally and functionally diverse. The specificity of the probe and primers was evaluated using purified DNA from 29 isolates of 3 different pathovars of P. syringae. The probe and primer set were able to detect Tox(-) and Tox+ isolates of P. s. pv. Phaseolicola, but no other phytopathogenic bacteria. The assay was also able to detect at least 5 genome equivalents of cloned amplified target DNA, using purified DNA, or 7 colony forming unit (CFU) per reaction when using calibrated cell suspensions. Thus, the TaqMan real-time PCR-based method can be used for the rapid detection of both types of P. s. pv. Phaseolicola, and will potentially simplify and facilitate the diagnosis and monitoring of this pathogen, and guide plant disease management.