Host specificity,pathogenicity and the presence of virulence genes in Iranian strains of Pseudomonas syringae pv. syringae from different hosts

Pseudomonas syringae pv. syringae (Pss) strains were isolated from almond, apricot, peach, pear, sweet cheery and wheat in Kohgiluye and Boyer-Ahmad, Kordestan, Fras and Chaharmahal and Bakhtiari provinces of Iran. The strains were examined for host specificity, the presence of virulence genes and pathogenicity on different hosts. After inoculation of isolates, in compatible reactions bacterial populations increased within six days of inoculation and final cell numbers increased several-fold over initial inoculum levels, but in incompatible reactions, bacterial populations declined within four days of inoculation. Almond, sweet cherry and wheat isolates induced progressive necrotic symptoms on almond leaves and stems. Apricot, peach and sweet cherry isolates induced necrotic lesions when inoculated on apricot leaves. On pear leaves and stems, only the pear isolate incited pathogenic reaction and isolates from other hosts did not. The syrB gene was detected in all of the tested isolates. Almond and pear isolates did not have the syrD gene. The sypA gene was detected in the almond, peach, pear and sweet cherry isolates while the sypB gene was detected in the apricot, peach, sweet cherry and wheat isolates. Almond, apricot, pear and wheat isolates gave negative results for the detection of nit gene. The gene Ach, was detected only in the peach isolate and gene hrmA, was detected only in the wheat isolate. This study indicates that host specificity exists among different Pss strains, and genes responsible for syringomycin and syringopeptin production contribute to the virulence of Pss strains.     

Characterization and genetic diversity of Pseudomonas syringae isolates from stone fruits in north‐western Iran

From 33 Iranian fluorescent Pseudomonas isolates originating from symptomatic tissues of peach (Prunus persica), plum (Prunus domestica), sweet (Prunus avium) and sour cherry (Prunus cerasus), 27 were identified as Pseudomonas syringae using LOPAT tests. Further characterization of those isolates by GATTa and L‐lactate utilization tests and the detection of syringomycin and coronatine and yersiniabactin coding genes showed that five of them belonged to race 1 and four to race 2 of P. syringae pv. morsprunorum (Psm) and eighteen other isolates were identified as P. syringae pv. syringae (Pss). Based on the analysis of the fingerprint patterns generated by REP, ERIC and BOX‐PCR, the strains were differentiated into three main groups at the 67% similarity level. Strains of the groups 1, 2 and 3 belong to Psm race 1, Psm race 2 and Pss, respectively. Rep‐PCR analysis showed high intra‐pathovar variation within the Pss isolates, which grouped into four distinct clusters. Using the REP primers, the percentage of polymorphic loci was 74.61%, whereas with BOX and ERIC primers, it was 60.5 and 55.21%, respectively. Finally, this study is the first report of the isolation of P. syringae pv. morsprunorum race 1 and 2 strains from stone fruit trees in Iran.     

Pseudomonas corrugata contains a conserved N-acyl homoserine lactone quorum sensing system; its role in tomato pathogenicity and tobacco hypersensitivity response

Pseudomonas corrugata is a phytopathogenic bacterium, causal agent of tomato pith necrosis, yet it is an ubiquitous bacterium that is part of the microbial community in the soil and in the rhizosphere of different plant species. Although it is a very heterogeneous species, all the strains tested were able to produce short chain acyl homoserine lactone (AHL) quorum sensing signal molecules. The main AHL produced was N-hexanoyl-L-homoserine lactone (C(6)-AHL). An AHL quorum sensing system, designated PcoI/PcoR, was identified and characterized. The role of the quorum sensing system in the expression of a variety of traits was evaluated. Inactivation of pcoI abolished the production of AHLs. The pcoR mutant, but not the pcoI mutant, was impaired in swarming, unable to cause a hypersensitivity response on tobacco and resulted in a reduced tomato pith necrosis phenotype. The pcoI mutant showed a reduced antimicrobial activity against various fungi and bacteria when assayed on King's B medium. These results demonstrate that the AHL quorum sensing in Ps. corrugata regulates traits that contribute to virulence, antimicrobial activity and fitness. This is the first report of genes of Ps. corrugata involved in the disease development and biological control activity.     

The c‐di‐GMP phosphodiesterase BifA is involved in the virulence of bacteria from the Pseudomonas syringae complex

In a recent screen for novel virulence factors involved in the interaction between Pseudomonas savastanoi pv. savastanoi and the olive tree, a mutant was selected that contained a transposon insertion in a putative cyclic diguanylate (c‐di‐GMP) phosphodiesterase‐encoding gene. This gene displayed high similarity to bifA of Pseudomonas aeruginosa and Pseudomonas putida. Here, we examined the role of BifA in free‐living and virulence‐related phenotypes of two bacterial plant pathogens in the Pseudomonas syringae complex, the tumour‐inducing pathogen of woody hosts, P. savastanoi pv. savastanoi NCPPB 3335, and the pathogen of tomato and Arabidopsis, P. syringae pv. tomato DC3000. We showed that deletion of the bifA gene resulted in decreased swimming motility of both bacteria and inhibited swarming motility of DC3000. In contrast, overexpression of BifA in P. savastanoi pv. savastanoi had a positive impact on swimming motility and negatively affected biofilm formation. Deletion of bifA in NCPPB 3335 and DC3000 resulted in reduced fitness and virulence of the microbes in olive (NCPPB 3335) and tomato (DC3000) plants. In addition, real‐time monitoring of olive plants infected with green fluorescent protein (GFP)‐tagged P. savastanoi cells displayed an altered spatial distribution of mutant ΔbifA cells inside olive knots compared with the wild‐type strain. All free‐living phenotypes that were altered in both ΔbifA mutants, as well as the virulence of the NCPPB 3335 ΔbifA mutant in olive plants, were fully rescued by complementation with P. aeruginosa BifA, whose phosphodiesterase activity has been demonstrated. Thus, these results suggest that P. syringae and P. savastanoi BifA are also active phosphodiesterases. This first demonstration of the involvement of a putative phosphodiesterase in the virulence of the P. syringae complex provides confirmation of the role of c‐di‐GMP signalling in the virulence of this group of plant pathogens.     

Recombineering and stable integration of the Pseudomonas syringae pv. syringae 61 hrp/hrc cluster into the genome of the soil bacterium Pseudomonas fluorescens Pf0‐1

Many Gram‐negative bacteria use a type III secretion system (T3SS) to establish associations with their hosts. The T3SS is a conduit for direct injection of type‐III effector proteins into host cells, where they manipulate the host for the benefit of the infecting bacterium. For plant‐associated pathogens, the variations in number and amino acid sequences of type‐III effectors, as well as their functional redundancy, make studying type‐III effectors challenging. To mitigate this challenge, we developed a stable delivery system for individual or defined sets of type‐III effectors into plant cells. We used recombineering and Tn5‐mediated transposition to clone and stably integrate, respectively, the complete hrp/hrc region from Pseudomonas syringae pv. syringae 61 into the genome of the soil bacterium Pseudomonas fluorescens Pf0‐1. We describe our development of Effector‐to‐Host Analyzer (EtHAn), and demonstrate its utility for studying effectors for their in planta functions.     

LOV‐domain photoreceptor,encoded in a genomic island,attenuates the virulence of Pseudomonas syringae in light‐exposed Arabidopsis leaves

In Arabidopsis thaliana, light signals modulate the defences against bacteria. Here we show that light perceived by the LOV domain‐regulated two‐component system (Pst–Lov) of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) modulates virulence against A. thaliana. Bioinformatic analysis and the existence of an episomal circular intermediate indicate that the locus encoding Pst–Lov is present in an active genomic island acquired by horizontal transfer. Strains mutated at Pst–Lov showed enhanced growth on minimal medium and in leaves of A. thaliana exposed to light, but not in leaves incubated in darkness or buried in the soil. Pst–Lov repressed the expression of principal and alternative sigma factor genes and their downstream targets linked to bacterial growth, virulence and quorum sensing, in a strictly light‐dependent manner. We propose that the function of Pst–Lov is to distinguish between soil (dark) and leaf (light) environments, attenuating the damage caused to host tissues while releasing growth out of the host. Therefore, in addition to its direct actions via photosynthesis and plant sensory receptors, light may affect plants indirectly via the sensory receptors of bacterial pathogens.     

The cloning and characterization of phage promoters, directing high expression of luciferase in Pseudomonas syringae pv. phaseolicola, allowing single cell and microcolony detection

Regions of DNA containing promoter sequences from a Pseudomonas syringae pv. phaseolicola -specific phage (φ11P) were identified by shotgun cloning into a broad-host-range promoter-probe vector (pQF70). When used in conjunction with the luciferase reporter genes, one of these DNA fragments, 19H, directed gene expression at a level which enabled the subsequent light output (bioluminescence) of single cells of P. syringae pv. phaseolicola to be detected and visualized using a charge-coupled device (CCD). The P. syringae pv. phaseolicola φ11P, 19H and P. aeruginosa φPLS27, HcM promoters gave a 50-fold increase in bioluminescence (maximum relative light output) compared to similar constructs containing other well-characterized promoters, for example, tetracycline. Similar bioluminescent characteristics of the transformed bacterium, were observed during growth with and without antibiotic-selection. When lux ⁺ bacteria were inoculated onto French bean leaf ( Phaseolus vulgaris L.), the resultant secondary halo blight lesions were bioluminescent and during phylloplane colonization by the lux ⁺ bacterium, bioluminescence on leaf surfaces was detected and imaged by the CCD. Use of these newly identified promoters, combined with the greatly increased sensitivity of bioluminescence detection by the CCD, thus provided a new dimension for the study of natural ecological populations during the bacterial colonization of plants.     

IDL6‐HAE/HSL2 impacts pectin degradation and resistance to Pseudomonas syringae pv tomato DC3000 in Arabidopsis leaves

Plant cell walls undergo dynamic structural and chemical changes during plant development and growth. Floral organ abscission and lateral root emergence are both accompanied by cell‐wall remodeling, which involves the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)‐derived peptide and its receptors, HAESA (HAE) and HAESA‐LIKE2 (HSL2). Plant cell walls also act as barriers against pathogenic invaders. Thus, the cell‐wall remodeling during plant development could have an influence on plant resistance to phytopathogens. Here, we identified IDA‐like 6 (IDL6), a gene that is prominently expressed in Arabidopsis leaves. IDL6 expression in Arabidopsis leaves is significantly upregulated when the plant is suffering from attacks of the bacterial Pseudomonas syringae pv. tomato (Pst) DC3000. IDL6 overexpression and knockdown lines respectively decrease and increase the Arabidopsis resistance to Pst DC3000, indicating that the gene promotes the Arabidopsis susceptibility to Pst DC3000. Moreover, IDL6 promotes the expression of a polygalacturonase (PG) gene, ADPG2, and increases PG activity in Arabidopsis leaves, which in turn reduces leaf pectin content and leaf robustness. ADPG2 overexpression restrains Arabidopsis resistance to Pst DC3000, whereas ADPG2 loss‐of‐function mutants increase the resistance to the bacterium. Pst DC3000 infection elevates the ADPG2 expression partially through HAE and HSL2. Taken together, our results suggest that IDL6‐HAE/HSL2 facilitates the ingress of Pst DC3000 by promoting pectin degradation in Arabidopsis leaves, and Pst DC3000 might enhance its infection by manipulating the IDL6‐HAE/HSL2‐ADPG2 signaling pathway.