Open Access
Coping with environmental eukaryotes; identification of Pseudomonas syringae genes during the interaction with alternative hosts or predators
(MDPI, 2018) Dorati, Federico; Barrett, Glyn A.; Sánchez Contreras, María; Murillo Martínez, Jesús; Caballero Murillo, Primitivo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Understanding the molecular mechanisms underpinning the ecological success of plant pathogens is critical to develop strategies for controlling diseases and protecting crops. Recent observations have shown that plant pathogenic bacteria, particularly Pseudomonas, exist in a range of natural environments away from their natural plant host e.g., water courses, soil, non-host plants. This exposes them to a variety of eukaryotic predators such as nematodes, insects and amoebae present in the environment. Nematodes and amoeba in particular are bacterial predators while insect herbivores may act as indirect predators, ingesting bacteria on plant tissue. We therefore postulated that bacteria are probably under selective pressure to avoid or survive predation and have therefore developed appropriate coping mechanisms. We tested the hypothesis that plant pathogenic Pseudomonas syringae are able to cope with predation pressure and found that three pathovars show weak, but significant resistance or toxicity. To identify the gene systems that contribute to resistance or toxicity we applied a heterologous screening technique, called Rapid Virulence Annotation (RVA), for anti-predation and toxicity mechanisms. Three cosmid libraries for P. syringae pv. aesculi, pv. tomato and pv. phaseolicola, of approximately 2000 cosmids each, were screened in the susceptible/non-toxic bacterium Escherichia coli against nematode, amoebae and an insect. A number of potential conserved and unique genes were identified which included genes encoding haemolysins, biofilm formation, motility and adhesion. These data provide the first multi-pathovar comparative insight to how plant pathogens cope with different predation pressures and infection of an insect gut and provide a foundation for further study into the function of selected genes and their role in ecological success.
Open Access
Avances en el conocimiento sobre los organismos fitopatógenos y su repercusión en la Fitopatología en los últimos 35 años
(Phytoma España, 2023) Landa, Blanca B.; Cambra Álvarez, Mariano; Castillo, Pablo; Escobar Lucas, Carolina; García Arenal, Fernando; Jiménez Díaz, Rafael M.; López, María Milagros; Montesinos, Emilio; Murillo Martínez, Jesús; Pallás, Vicente; Palomares Rius, Juan Emilio; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB
Los pasados 35 años han sido testigos de importantes avances en el conocimiento sobre los organismos fitopatógenos, demasiado numerosos y diversos en naturaleza para poder resumirlos en unas páginas. Muchos de dichos avances han estado mediados por el desarrollo de nuevas metodologías, instrumentos y protocolos de estudio, en particular los concernientes a las tecnologías de análisis y secuenciación del ADN que comenzaron con su amplificación mediada por una ADN polimerasa termoestable (PCR) ¿y sus posteriores derivados: PCR cuantitativa, en tiempo real, digital, etc. Estos avances continuaron con el uso de las plataformas de secuenciación masiva para el análisis de los genomas de estos organismos, incluso a partir de la matriz vegetal que infectaban sin necesidad de su aislamiento, así como el uso de diversas tecnologías -ómicas para el análisis masivo de la expresión diferencial de genes (genómica), proteínas (proteómica) y metabolitos (metabolómica). Todo ello ha tenido profundas repercusiones, por ejemplo, sobre la taxonomía y relaciones filogenéticas de estos organismos fitopatógenos, la comprensión de la regulación genética de la patogenicidad y de los factores (efectores) de virulencia, la resistencia a la infección en la planta. Asimismo, las tecnologías de observación microscópica y el uso de genes que codifican proteínas fluorescentes de diferentes propiedades espectrales han propiciado una mejor compresión de los procesos de infección (Deal, 2011). En las siguientes secciones del artículo se presentan algunos avances ilustrativos seleccionados por expertos para cada uno de los grandes grupos de organismos fitopatógenos: hongos, oomicetos, bacterias, virus y nematodos. Cabe decir que muchos de los avances presentados para cada organismo fitopatógeno son aplicables a todos los demás. Si bien, con objeto de no incurrir en reiteraciones, y extendernos en exceso, se ha intentado, en la medida de lo posible, seleccionar avances con aspectos diferenciadores.
Open Access
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
(American Society for Microbiology, 2005) Rivas Mena, Luis; Mansfield, John W.; Tsiamis, George; Jackson, Robert W.; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
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.
Open Access
Miniature transposable sequences are frequently mobilized in the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola
(Public Library of Science, 2011) Bardají Goikoetxea, Leire; Añorga García, Maite; Jackson, Robert W.; Martínez Bilbao, Alejandro; Yanguas Casas, Natalia; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
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 x 10(-5) and 1.1 x 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.
Open Access
Multiple relaxases contribute to the horizontal transfer of the virulence plasmids from the tumorigenic bacterium Pseudomonas syringae pv. savastanoi NCPPB 3335
(Frontiers Media, 2022) Añorga García, Maite; Urriza Leoz, Miriam; Ramos, Cayo; Murillo Martínez, Jesús; Institute for Multidisciplinary Research in Applied Biology - IMAB
Pseudomonas syringae pv. savastanoi NCPPB 3335 is the causal agent of olive knot disease and contains three virulence plasmids: pPsv48A (pA), 80 kb; pPsv48B (pB), 45 kb, and pPsv48C (pC), 42 kb. Here we show that pB contains a complete MPFT (previously type IVA secretion system) and a functional origin of conjugational transfer adjacent to a relaxase of the MOBP family; pC also contains a functional oriT-MOBP array, whereas pA contains an incomplete MPFI (previously type IVB secretion system), but not a recognizable oriT. Plasmid transfer occurred on solid and in liquid media, and on leaf surfaces of a non-host plant (Phaseolus vulgaris) with high (pB) or moderate frequency (pC); pA was transferred only occasionally after cointegration with pB. We found three plasmid-borne and three chromosomal relaxase genes, although the chromosomal relaxases did not contribute to plasmid dissemination. The MOBP relaxase genes of pB and pC were functionally interchangeable, although with di ering eciencies. We also identified a functional MOBQ mobilization region in pC, which could only mobilize this plasmid. Plasmid pB could be eciently transferred to strains of six phylogroups of P. syringae sensu lato, whereas pC could only be mobilized to two strains of phylogroup 3 (genomospecies 2). In two of the recipient strains, pB was stably maintained after 21 subcultures in liquid medium. The carriage of several relaxases by the native plasmids of P. syringae impacts their transfer frequency and, by providing functional diversity and redundancy, adds robustness to the conjugation system.
Open Access
Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences
(Blackwell Publishing, 1994) Murillo Martínez, Jesús; Keen, Noel T.; Producción Agraria; Nekazaritza Ekoizpena
Strain PT23 of Pseudomonas syringae pv, tomato contains four native plasmids, designated A, B, C, and D. By DNA hybridization of genomic and plasmid DNA digests from the wild type and a plasmid-cured strain, we determined that c. 61 kb (c. 74%) of pPT23B is repeated in pPT23A and only c. 17 kb (c. 21%) is in single copy in strain PT23. pPT23B also contains DNA repeated in the chromosome that occurs in three DNA fragments of 0.6, 4.6, and 9.6 kb that might be transposable elements. Additionally, the 9.6 kb fragment also shares sequences with the three other plasmids of strain PT23. By DNA hybridization with the origin of replication from a native plasmid of P. syringae pv. syringae and in vivo replication tests, we identified the origins of replication of plasmids A, B, and D and showed that they cross-hybridize. The putative par region from pPT23 A has also been identified and is not conserved in the other three native plasmids from strain PT23. By using the defined minimal origin of replication from pPT23 A as a probe, we showed that it is highly conserved in 14 strains belonging to nine different pathovars of P. syringae and that as many as five different native plasmids with closely related origins of replication coexist in the same cell. The duplication and reorganization of plasmids might therefore occur at high frequency and could be responsible for the existence of large numbers of native plasmids in P. syringae strains.
Open Access
Sequence diversity of rulA among natural isolates of Pseudomonas syringae and effect on function of rulAB-mediated UV radiation tolerance
(American Society for Microbiology, 2000) Sundin, George W.; Jacobs, Janette L.; Murillo Martínez, Jesús; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The rulAB locus confers tolerance to UV radiation and is borne on plasmids of the pPT23A family in Pseudomonas syringae. We sequenced 14 rulA alleles from P. syringae strains representing seven pathovars and found sequence differences of 1 to 12% within pathovar syringae, and up to 15% differences between pathovars. Since the sequence variation within rulA was similar to that of P. syringae chromosomal alleles, we hypothesized that rulAB has evolved over a long time period in P. syringae. A phylogenetic analysis of the deduced amino acid sequences of rulA resulted in seven clusters. Strains from the same plant host grouped together in three cases; however, strains from different pathovars grouped together in two cases. In particular, the rulA alleles from P. syringae pv. lachrymans and P. syringae pv. pisi were grouped but were clearly distinct from the other sequenced alleles, suggesting the possibility of a recent interpathovar transfer. We constructed chimeric rulAB expression clones and found that the observed sequence differences resulted in significant differences in UV (wavelength) radiation sensitivity. Our results suggest that specific amino acid changes in RulA could alter UV radiation tolerance and the competitiveness of the P. syringae host in the phyllosphere.
Open Access
Two homologues of the global regulator Csr/Rsm redundantly control phaseolotoxin biosynthesis and virulence in the plant pathogen Pseudomonas amygdali pv. phaseolicola 1448A
(MDPI, 2020) Ramírez Zapata, Diana; Ramos, Cayo; Aguilera, Selene; Bardají Goikoetxea, Leire; Martínez Gil, Marta; Murillo Martínez, Jesús; Institute for Multidisciplinary Research in Applied Biology - IMAB
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.
Open Access
Detection by multiplex PCR and characterization of nontoxigenic strains of Pseudomonas syringae pv. phaseolicola from different places in Spain. Short communication
(Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), 2006) Rico, A.; Erdozáin García, María; Ortiz Barredo, Amaia; Ruiz de Galarreta, José Ignacio; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
El control eficiente de la grasa de la judía causada por Pseudomonas syringae pv. phaseolicola se basa principalmente en la utilización de semilla libre del patógeno. La detección del patógeno en semilla se efectúa mediante métodos altamente sensibles basados en la detección por PCR de los genes responsables de la biosíntesis de la faseolotoxina, la cual, hasta ahora, se consideraba que era sintetizada por todas las cepas del patógeno con importancia epidemiológica. Sin embargo, en la Comunidad de Castilla y León, España, las epidemias de grasa de la judía en campo se asocian frecuentemente con cepas no toxigénicas de P. syringae pv. phaseolicola, que no pueden ser detectadas con los métodos moleculares y serológicos actuales. Los resultados presentados en este trabajo demuestran la existencia de aislados no toxigénicos de P. syringae pv. phaseolicola en zonas distintas de Castilla y León, lo que implica la necesidad de establecer una metodología fiable para la certificación de semillas de judía. Con este propósito, se presenta un sencillo protocolo en dos fases que permite la identificación de los dos tipos de aislados, y que se basa en una PCR multiplex con enriquecimiento a partir de extractos de semilla y en ensayos de patogenicidad.
Open Access
The mbo operon is specific and essential for biosynthesis of mangotoxin in Pseudomonas syringae
(Public Library of Science, 2012) Carrión, Víctor J.; Arrebola, Eva; Cazorla, Francisco M.; Murillo Martínez, Jesús; Vicente, Antonio de; Universidad Pública de Navarra. Departamento de Producción Agraria; Nafarroako Unibertsitate Publikoa. Nekazaritza Ekoizpena Saila
Mangotoxin is an antimetabolite toxin produced by certain Pseudomonas syringae pv. syringae strains. This toxin is an oligopeptide that inhibits ornithine N-acetyl transferase, a key enzyme in the biosynthesis of ornithine and arginine. Previous studies have reported the involvement of the putative nonribosomal peptide synthetase MgoA in virulence and mangotoxin production. In this study, we analyse a new chromosomal region of P. syringae pv. syringae UMAF0158, which contains six coding sequences arranged as an operon (mbo operon). The mbo operon was detected in only mangotoxin-producing strains, and it was shown to be essential for the biosynthesis of this toxin. Mutants in each of the six ORFs of the mbo operon were partially or completely impaired in the production of the toxin. In addition, Pseudomonas spp. mangotoxin non-producer strains transformed with the mbo operon gained the ability to produce mangotoxin, indicating that this operon contains all the genetic information necessary for mangotoxin biosynthesis. The generation of a single transcript for the mbo operon was confirmed and supported by the allocation of a unique promoter and Rho-independent terminator. The phylogenetic analysis of the P. syringae strains harbouring the mbo operon revealed that these strains clustered together.