Identification and Characterization of a Well-Defined Series of Coronatine Biosynthetic Mutants of Pseudomonas syringae pv. tomato DC3000

To identify Pseudomonas syringae pv. tomato genes involved in pathogenesis, we carried out a screen for Tn5 mutants of P. syringae pv. tomato DC3000 with reduced virulence on Arabidopsis thaliana. Several mutants defining both known and novel virulence loci were identified. Six mutants contained insertions in biosynthetic genes for the phytotoxin coronatine (COR). The P. syringae pv. tomato DC3000 COR genes are chromosomally encoded and are arranged in two separate clusters, which encode enzymes responsible for the synthesis of coronafacic acid (CFA) or coronamic acid (CMA), the two defined intermediates in COR biosynthesis. High-performance liquid chromatography fractionation and exogenous feeding studies confirmed that Tn5 insertions in the cfa and cma genes disrupt CFA and CMA biosynthesis, respectively. All six COR biosynthetic mutants were significantly impaired in their ability to multiply to high levels and to elicit disease symptoms on A. thaliana plants. To assess the relative contributions of CFA, CMA, and COR in virulence, we constructed and characterized cfa6 cmaA double mutant strains. These exhibited virulence phenotypes on A. thalliana identical to those observed for the cmaA or cfa6 single mutants, suggesting that reduced virulence of these mutants on A. thaliana is caused by the absence of the intact COR toxin. This is the first study to use biochemically and genetically defined COR mutants to address the role of COR in pathogenesis.

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Pseudomonas syringae pv. tomato DC3000 CmaL (PSPTO4723), a DUF1330 Family Member, Is Needed To Produce l-allo-Isoleucine, a Precursor for the Phytotoxin Coronatine

Journal of Bacteriology ◽

10.1128/jb.01352-12 ◽

2012 ◽

Vol 195 (2) ◽

pp. 287-296 ◽

Cited By ~ 16

Author(s):

Jay N. Worley ◽

Alistair B. Russell ◽

Aaron G. Wexler ◽

Philip A. Bronstein ◽

Brian H. Kvitko ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Genomic Region ◽

Tomato Dc3000 ◽

Content Type ◽

Type Iii Effectors ◽

Type Iii ◽

Coronafacic Acid ◽

Feeding Experiments ◽

Leaf Chlorosis

ABSTRACTPseudomonas syringaepv. tomato DC3000 produces the phytotoxin coronatine, a major determinant of the leaf chlorosis associated with DC3000 pathogenesis. The DC3000 PSPTO4723 (cmaL) gene is located in a genomic region encoding type III effectors; however, it promotes chlorosis in the model plantNicotiana benthamianain a manner independent of type III secretion. Coronatine is produced by the ligation of two moieties, coronafacic acid (CFA) and coronamic acid (CMA), which are produced by biosynthetic pathways encoded in separate operons. Cross-feeding experiments, performed inN. benthamianawithcfa,cma, andcmaLmutants, implicate CmaL in CMA production. Furthermore, analysis of bacterial supernatants under coronatine-inducing conditions revealed that mutants lacking either thecmaoperon orcmaLaccumulate CFA rather than coronatine, supporting a role for CmaL in the regulation or biosynthesis of CMA. CmaL does not appear to regulate CMA production, since the expression of proteins with known roles in CMA production is unaltered incmaLmutants. Rather, CmaL is needed for the first step in CMA synthesis, as evidenced by the fact that wild-type levels of coronatine production are restored to a ΔcmaLmutant when it is supplemented with 50 μg/mll-allo-isoleucine, the starting unit for CMA production.cmaLis found in all other sequencedP. syringaestrains with coronatine biosynthesis genes. This characterization of CmaL identifies a critical missing factor in coronatine production and provides a foundation for further investigation of a member of the widespread DUF1330 protein family.

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Characterization of Regulatory Pathways in Xylella fastidiosa: Genes and Phenotypes Controlled by gacA

Applied and Environmental Microbiology ◽

10.1128/aem.01964-08 ◽

2009 ◽

Vol 75 (8) ◽

pp. 2275-2283 ◽

Cited By ~ 26

Author(s):

Xiang Yang Shi ◽

C. Korsi Dumenyo ◽

Rufina Hernandez-Martinez ◽

Hamid Azad ◽

Donald A. Cooksey

Keyword(s):

Pseudomonas Syringae ◽

Biofilm Formation ◽

Xylella Fastidiosa ◽

Cell Aggregation ◽

Toxin Gene ◽

Tomato Dc3000 ◽

Disease Symptoms ◽

Regulatory Pathways ◽

Physiological Processes

ABSTRACT The xylem-limited, insect-transmitted bacterium Xylella fastidiosa causes Pierce's disease in grapes through cell aggregation and vascular clogging. GacA controls various physiological processes and pathogenicity factors in many gram-negative bacteria, including biofilm formation in Pseudomonas syringae pv. tomato DC3000. Cloned gacA of X. fastidiosa was found to restore the hypersensitive response and pathogenicity in gacA mutants of P. syringae pv. tomato DC3000 and Erwinia amylovora. A gacA mutant of X. fastidiosa (DAC1984) had significantly reduced abilities to adhere to a glass surface, form biofilm, and incite disease symptoms on grapevines, compared with the parent (A05). cDNA microarray analysis identified 7 genes that were positively regulated by GacA, including xadA and hsf, predicted to encode outer membrane adhesion proteins, and 20 negatively regulated genes, including gumC and an antibacterial polypeptide toxin gene, cvaC. These results suggest that GacA of X. fastidiosa regulates many factors, which contribute to attachment and biofilm formation, as well as some physiological processes that may enhance the adaptation and tolerance of X. fastidiosa to environmental stresses and the competition within the host xylem.

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Percepción de los entrenadores de alto rendimiento de atletismo: caracterización de su perfil polifacético (Perception of high performance track and field coaches: characterization of their multifaceted profile)

Retos ◽

10.47197/retos.v0i29.36511 ◽

2015 ◽

pp. 42-47

Author(s):

Angeles Filgueira Perez

Keyword(s):

High Performance ◽

Target Population ◽

Track And Field ◽

Personal Qualities ◽

High Level ◽

Almost All ◽

Methodological Aspects ◽

The Ideal

En el presente estudio se ha tratado de obtener una aproximación al perfil ideal del entrenador de alto rendimiento en atletismo. En el mundo del deporte de alta competición, el entrenador debe desarrollar funciones de maestro, técnico y líder, lo cual hace difícil delimitar sus competencias (conocimientos, habilidades y cualidades personales). Por tanto, la principal motivación de este trabajo es establecer el papel que debe desempeñar el entrenador para la preparación física, técnica, táctica, psicológica y moral del deportista de alto nivel. Para ello, se ha considerado como población objeto de estudio al conjunto de entrenadores de alto rendimiento que en el momento de la investigación estuviesen activo, ya que interesaba realizar este estudio desde su propia perspectiva. Los datos recogidos forman parte de una investigación más amplia, llevada a cabo mediante encuesta, para la que se diseñó un cuestionario de 78 preguntas en el que se abarcaban tres temas: el perfil del entrenador y del deportista, así como la figura del formador de entrenadores en el Practicum. En este trabajo nos centramos en el primer tema y el análisis de la información obtenida nos permite concluir que la que práctica totalidad de los entrenadores de atletismo consideran necesario dominar con precisión los aspectos técnico-metodológicos. Además, consideran que su ética profesional debe estar regida por los principios de autonomía y beneficencia, de modo que valores como la honestidad y la justicia deben primar en el desarrollo de sus funciones.Abstract: In the current study, we have tried to get an approximation of the ideal profile of high performance coaches in athletics. In the high-level sports world, the coach must develop the role of teacher, technician and leader, which makes it difficult to delimit his/her competencies (knowledge, skills and personal qualities). Therefore, the main motivation of this work is to define the role that the coach plays in relation to the physical, technical, tactical, psychological and moral preparation of elite athletes. Keeping this aim as an objective, we have considered as the target population the high performance coaches who are active at the time of the investigation, since we were interested in knowing their own perspective. The data that was collected is a part of a wider investigation, conducted by a survey, for which we designed a questionnaire of 78 questions divided on three topics: the profile of the coach and the athlete, as well as the figure of the coach educator in the Practicum. In this work, we focus on the first topic and the analysis of the information that we obtained allows us to conclude that almost all the track and field coaches find it necessary to dominate the technical and methodological aspects. They also consider that professional ethics must be governed by the principles of autonomy and care that values like honesty and justice must prevail in the performance of their work.

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