scholarly journals Re-evaluation of a Tn5::gacAmutant ofPseudomonas syringaepv.tomatoDC3000 uncovers roles foruvrCandanmKin promoting virulence

2019 ◽  
Author(s):  
Megan R. O’Malley ◽  
Alexandra J. Weisberg ◽  
Jeff H. Chang ◽  
Jeffrey C. Anderson
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Insertional Mutagenesis ◽  
Leaf Tissue ◽  
Wild Type ◽  
Two Component System ◽  
Gene Encoding ◽  
Interior Space ◽  
Damage Repair

ABSTRACTPseudomonas syringaeis a taxon of plant pathogenic bacteria that can colonize and proliferate within the interior space of leaf tissue. This process requiresP. syringaeto rapidly upregulate the production of virulence factors including a type III secretion system (T3SS) that suppress host defenses. GacS/A is a two-component system that regulates virulence of many plant and animal pathogenic bacteria includingP. syringae. We recently investigated the virulence defect of strain AC811, a Tn5::gacAmutant ofP. syringae pv.tomatoDC3000 that is less virulent on Arabidopsis. We discovered that decreased virulence of AC811 is not caused by loss of GacA function. Here, we report the molecular basis of the virulence defect of AC811. We show that AC811 possesses a nonsense mutation inanmK, a gene predicted to encode a 1,6-anhydromuramic acid kinase involved in cell wall recycling. Expression of a wild-type allele ofanmKpartially increased growth of AC811 in Arabidopsis leaves. In addition to the defectiveanmKallele, we also show that the Tn5insertion ingacAexerts a polar effect onuvrC, a downstream gene encoding a regulator of DNA damage repair. Expression of the wild-typeanmKallele together with increased expression ofuvrCfully restored the virulence of AC811 during infection of Arabidopsis. These results demonstrate that defects inanmKanduvrCare together sufficient to account for the decreased virulence of AC811, and suggest caution is warranted in assigning phenotypes to GacA function based on insertional mutagenesis of thegacA-uvrClocus.

scholarly journals Identification of Novel Type III Secretion Effectors in Xanthomonas oryzae pv. oryzae

2009 ◽  
Vol 22 (1) ◽  
pp. 96-106 ◽  
Author(s):  
Ayako Furutani ◽  
Minako Takaoka ◽  
Harumi Sanada ◽  
Yukari Noguchi ◽  
Takashi Oku ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Regulatory Protein ◽  
Effector Proteins ◽  
Xanthomonas Oryzae ◽  
Dependent Manner ◽  
Plant Pathogenic Bacteria ◽  
Type Iii ◽  
Genes Encoding

Many gram-negative bacteria secrete so-called effector proteins via a type III secretion (T3S) system. Through genome screening for genes encoding potential T3S effectors, 60 candidates were selected from rice pathogen Xanthomonas oryzae pv. oryzae MAFF311018 using these criteria: i) homologs of known T3S effectors in plant-pathogenic bacteria, ii) genes with expression regulated by hrp regulatory protein HrpX, or iii) proteins with N-terminal amino acid patterns associated with T3S substrates of Pseudomonas syringae. Of effector candidates tested with the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter for translocation into plant cells, 16 proteins were translocated in a T3S system-dependent manner. Of these 16 proteins, nine were homologs of known effectors in other plant-pathogenic bacteria and seven were not. Most of the effectors were widely conserved in Xanthomonas spp.; however, some were specific to X. oryzae. Interestingly, all these effectors were expressed in an HrpX-dependent manner, suggesting coregulation of effectors and the T3S system. In X. campestris pv. vesicatoria, HpaB and HpaC (HpaP in X. oryzae pv. oryzae) have a central role in recruiting T3S substrates to the secretion apparatus. Secretion of all but one effector was reduced in both HpaB– and HpaP– mutant strains, indicating that HpaB and HpaP are widely involved in efficient secretion of the effectors.

scholarly journals The Chlamydomonas IDA7 Locus Encodes a 140-kDa Dynein Intermediate Chain Required to Assemble the I1 Inner Arm Complex

1998 ◽  
Vol 9 (12) ◽  
pp. 3351-3365 ◽  
Author(s):  
Catherine A. Perrone ◽  
Pinfen Yang ◽  
Eileen O’Toole ◽  
Winfield S. Sale ◽  
Mary E. Porter
Keyword(s):  
Insertional Mutagenesis ◽  
Gene Transformation ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Radial Spoke ◽  
Important Target ◽  
Dynein Intermediate Chain ◽  
Biochemical Analyses ◽  
Intermediate Chain

To identify new loci that are involved in the assembly and targeting of dynein complexes, we have screened a collection of motility mutants that were generated by insertional mutagenesis. One such mutant, 5B10, lacks the inner arm isoform known as the I1 complex. This isoform is located proximal to the first radial spoke in each 96-nm axoneme repeat and is an important target for the regulation of flagellar motility. Complementation tests reveal that 5B10 represents a new I1 locus, IDA7. Biochemical analyses confirm thatida7 axonemes lack at least five I1 complex subunits. Southern blots probed with a clone containing the gene encoding the 140-kDa intermediate chain (IC) indicate that theida7 mutation is the result of plasmid insertion into the IC140 gene. Transformation with a wild-type copy of the IC140 gene completely rescues the mutant defects. Surprisingly, transformation with a construct of the IC140 gene lacking the first four exons of the coding sequence also rescues the mutant phenotype. These studies indicate that IC140 is essential for assembly of the I1 complex, but unlike other dynein ICs, the N-terminal region is not critical for its activity.

The 73-kb pIAA plasmid increases competitive fitness of Pseudomonas syringae subspecies savastanoi in oleander

1993 ◽  
Vol 39 (7) ◽  
pp. 659-664 ◽  
Author(s):  
Sara E. Silverstone ◽  
David G. Gilchrist ◽  
Richard M. Bostock ◽  
Tsune Kosuge
Keyword(s):  
Population Density ◽  
Pseudomonas Syringae ◽  
Indoleacetic Acid ◽  
Leaf Tissue ◽  
Insertion Mutant ◽  
Wild Type ◽  
In Planta ◽  
Bacterial Strains ◽  
Growth And Survival ◽  
Iaa Production

Pseudomonas syringae subsp. savastanoi causes tumors on olive and oleander by producing the plant growth regulators indoleacetic acid (IAA) and cytokinins following infection of the plant. The contribution of IAA production to the ability of P. syringae subsp. savastanoi to grow and survive in oleander leaf tissue was studied. Bacterial strains differing only with respect to IAA production were characterized. Growth and survival of wild-type and two mutant strains of P. syringae subsp. savastanoi in oleander leaf tissue were monitored by weekly colony counts and IAA plate assays. Growth rate of the three strains in culture and in planta did not differ significantly. However, the wild-type strain reached a higher population density and maintained its maximum density at least 9 weeks longer than either mutant population. An insertion mutant containing the IAA plasmid (pIAA), but incapable of IAA production, did not maintain a higher population density than a strain cured of the IAA plasmid. The pIAA-cured strain maintained a higher population density when coinoculated with an IAA-producing strain than when inoculated alone. These results suggest that IAA production may contribute to the fitness of P. syringae subsp. savastanoi in oleander tissue and that the iaa operon alone may be responsible for the competitive advantage of cells harboring pIAA.Key words: indoleacetic acid, bacterial ecology.

scholarly journals Components of the Pseudomonas syringae Type III Secretion System Can Suppress and May Elicit Plant Innate Immunity

2010 ◽  
Vol 23 (6) ◽  
pp. 727-739 ◽  
Author(s):  
Hye-Sook Oh ◽  
Duck Hwan Park ◽  
Alan Collmer
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Leaf Tissue ◽  
Secretion System ◽  
Colony Development ◽  
Plant Responses ◽  
In Planta ◽  
Callose Deposition ◽  
Type Iii

The type III secretion system (T3SS) of Pseudomonas syringae translocates into plant cells multiple effectors that suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). P. syringae pv. tomato DC3000 no longer delivers the T3SS translocation reporter AvrPto-Cya in Nicotiana benthamiana leaf tissue in which PTI was induced by prior inoculation with P. fluorescens(pLN18). Cosmid pLN18 expresses the T3SS system of P. syringae pv. syringae 61 but lacks the hopA1Psy61 effector gene. P. fluorescens(pLN18) expressing HrpHPtoDC3000 or HopP1PtoDC3000, two T3SS-associated putative lytic transglycosylases, suppresses PTI, based on multiple assays involving DC3000 challenge inoculum (AvrPto-Cya translocation, hypersensitive response elicitation, and colony development in planta) or on plant responses (vascular dye uptake or callose deposition). Analysis of additional mutations in pHIR11 derivatives revealed that the pLN18-encoded T3SS elicits a higher level of reactive oxygen species (ROS) than does P. fluorescens without a T3SS, that enhanced ROS production is dependent on the HrpK1 translocator, and that HopA1Psy61 suppresses ROS elicitation attributable to both the P. fluorescens PAMPs and the presence of a functional T3SS.

scholarly journals Plant Small RNA Species Direct Gene Silencing in Pathogenic Bacteria as well as Disease Protection

2019 ◽  
Author(s):  
Meenu Singla-Rastogi ◽  
Magali Charvin ◽  
Odon Thiébeauld ◽  
Alvaro L Perez-Quintero ◽  
Antinéa Ravet ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Pseudomonas Syringae ◽  
Small Rnas ◽  
Pathogenic Bacteria ◽  
Wild Type ◽  
Rnai Machinery ◽  
Phytopathogenic Bacterium ◽  
Associated Bacteria ◽  
Small Rna Species ◽  
Plant Small Rna

AbstractPlant small RNAs (sRNAs) and/or double-stranded RNAs (dsRNAs) trigger RNA interference (RNAi) in interacting eukaryotic pathogens or parasites. However, it is unknown whether this phenomenon could operate in bacterial phytopathogens, which lack a eukaryotic-like RNAi machinery. Here, we first show that Arabidopsis-encoded inverted repeat transgenes trigger silencing of Pseudomonas syringae heterologous reporter and endogenous virulence-associated genes during infection. Antibacterial Gene Silencing (AGS) of the latter was associated with a reduced pathogenesis, which was also observed upon application of corresponding plant-derived RNAs onto wild-type plants prior to infection. We additionally demonstrate that sRNAs directed against virulence factor transcripts were causal for silencing and pathogenesis reduction, while cognate long dsRNAs were inactive. Overall, this study provides the first evidence that plant sRNAs can directly reprogram gene expression in a phytopathogenic bacterium and may have wider implications in the understanding of how plants regulate transcriptome, community composition and genome evolution of associated bacteria.

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

2010 ◽  
Vol 23 (5) ◽  
pp. 682-701 ◽  
Author(s):  
Inmaculada Ortiz-Martín ◽  
Richard Thwaites ◽  
John W. Mansfield ◽  
Carmen R. Beuzón
Keyword(s):  
Gene Expression ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
In Planta ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Bacterial Fitness

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.

scholarly journals XopC and XopJ, Two Novel Type III Effector Proteinsfrom Xanthomonas campestris pv.vesicatoria

2003 ◽  
Vol 185 (24) ◽  
pp. 7092-7102 ◽  
Author(s):  
Laurent Noël ◽  
Frank Thieme ◽  
Jana Gäbler ◽  
Daniela Büttner ◽  
Ulla Bonas
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Cell ◽  
Type Iii Secretion ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Effector Proteins ◽  
Type Iii ◽  
Genome Wide ◽  
A Genome ◽  
Xanthomonas Campestris Pv.Vesicatoria

ABSTRACT Pathogenicity of the gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria depends on a type III secretion (TTS) system which translocates bacterial effector proteins into the plant cell. Previous transcriptome analysis identified a genome-wide regulon of putative virulence genes that are coexpressed with the TTS system. In this study, we characterized two of these genes, xopC and xopJ. Both genes encode Xanthomonas outer proteins (Xops) that were shown to be secreted by the TTS system. In addition, type III-dependent translocation of both proteins into the plant cell was demonstrated using the AvrBs3 effector domain as a reporter. XopJ belongs to the AvrRxv/YopJ family of effector proteins from plant and animal pathogenic bacteria. By contrast, XopC does not share significant homology to proteins in the database. Sequence analysis revealed that the xopC locus contains several features that are reminiscent of pathogenicity islands. Interestingly, the xopC region is flanked by 62-bp inverted repeats that are also associated with members of the Xanthomonas avrBs3 effector family. Besides xopC, a second gene of the locus, designated hpaJ, was shown to be coexpressed with the TTS system. hpaJ encodes a protein with similarity to transglycosylases and to the Pseudomonas syringae pv. maculicola protein HopPmaG. HpaJ secretion and translocation by the X. campestris pv. vesicatoria TTS system was not detectable, which is consistent with its predicted Sec signal and a putative function as transglycosylase in the bacterial periplasm.

scholarly journals The HopPtoF Locus of Pseudomonas syringae pv. tomato DC3000 Encodes a Type III Chaperone and a Cognate Effector

2004 ◽  
Vol 17 (5) ◽  
pp. 447-455 ◽  
Author(s):  
Libo Shan ◽  
Hye-sook Oh ◽  
Jianfu Chen ◽  
Ming Guo ◽  
Jianmin Zhou ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Host Cells ◽  
Whole Genome Sequence ◽  
Avirulence Gene ◽  
Bacterial Cells ◽  
Tomato Dc3000 ◽  
Virulence Proteins ◽  
Type Iii

Type III secretion systes are highly conserved among gram-negative plant and animal pathogenic bacteria. Through the type III secretion system, bacteria inject a number of virulence proteins into the host cells. Analysis of the whole genome sequence of Pseudomonas syringae pv. tomato DC3000 strain identified a locus, named HopPtoF, that is homologous to the avirulence gene locus avrPphF in P. syringae pv. phaseolicola. The HopPtoF locus harbors two genes, ShcFPto and HopFPto, that are preceded by a single hrp box promoter. We present evidence here to show that ShcFPto and HopFPto encode a type III chaperone and a cognate effector, respectively. ShcFPto interacts with and stabilizes the HopFPto protein in the bacterial cell. Translation of HopFPto starts at a rare initiation codon ATA that limits the synthesis of the HopFPto protein to a low level in bacterial cells.

scholarly journals A quick and robust method for quantification of the hypersensitive response in plants

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e1469 ◽  
Author(s):  
Oskar N. Johansson ◽  
Anders K. Nilsson ◽  
Mikael B. Gustavsson ◽  
Thomas Backhaus ◽  
Mats X. Andersson ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Hypersensitive Response ◽  
Pathogenic Bacteria ◽  
Leaf Tissue ◽  
Defense Responses ◽  
Vacuum Infiltration ◽  
Microbial Pathogens ◽  
Bacterial Titer ◽  
Electrolyte Loss ◽  
Dose Dependent

One of the most studied defense reactions of plants against microbial pathogens is the hypersensitive response (HR). The HR is a complex multicellular process that involves programmed cell death at the site of infection. A standard method to quantify plant defense and the HR is to measure the release of cellular electrolytes into water after infiltration with pathogenic bacteria. In this type of experiment, the bacteria are typically delivered into the plant tissue through syringe infiltration. Here we report the development of a vacuum infiltration protocol that allows multiple plant lines to be infiltrated simultaneously and assayed for defense responses. Vacuum infiltration did not induce more wounding response in Arabidopsis leaf tissue than syringe inoculation, whereas throughput and reproducibility were improved. The method was used to study HR-induced electrolyte loss after treatment with the bacteriumPseudomonas syringaepv.tomatoDC3000 harboring the effector AvrRpm1, AvrRpt2 or AvrRps4. Specifically, the influence of bacterial titer on AvrRpm1-induced HR was investigated. Not only the amplitude, but also the timing of the maximum rate of the HR reaction was found to be dose-dependent. Finally, using vacuum infiltration, we were able quantify induction of phospholipase D activity after AvrRpm1 recognition in leaves labeled with33PO4.

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