Open Access
Fate of a Pseudomonas savastanoi pv. savastanoi type III secretion system mutant in olive plants (Olea europaea L.)
(American Society for Microbiology, 2010) Pérez Martínez, Isabel; Rodríguez Moreno, Luis; Lambertsen, Lotte; Matas Casado, Isabel María; Murillo Martínez, Jesús; Tegli, Stefania; Jiménez, Antonio J.; Ramos, Cayo; Producción Agraria; Nekazaritza Ekoizpena
Pseudomonas savastanoi pv. savastanoi strain NCPPB 3335 is a model bacterial pathogen for studying the molecular basis of disease production in woody hosts. We report the sequencing of the hrpS-to-hrpZ region of NCPPB 3335, which has allowed us to determine the phylogenetic position of this pathogen with respect to previously sequenced Pseudomonas syringae hrp clusters. In addition, we constructed a mutant of NCPPB 3335, termed T3, which carries a deletion from the 3 end of the hrpS gene to the 5 end of the hrpZ operon. Despite its inability to multiply in olive tissues and to induce tumor formation in woody olive plants, P. savastanoi pv. savastanoi T3 can induce knot formation on young micropropagated olive plants. However, the necrosis and formation of internal open cavities previously reported in knots induced by the wild-type strain were not observed in those induced by P. savastanoi pv. savastanoi T3. Tagging of P. savastanoi pv. savastanoi T3 with green fluorescent protein (GFP) allowed real-time monitoring of its behavior on olive plants. In olive plant tissues, the wild-type strain formed aggregates that colonized the intercellular spaces and internal cavities of the hypertrophic knots, while the mutant T3 strain showed a disorganized distribution within the parenchyma of the knot. Ultrastructural analysis of knot sections revealed the release of extensive outer membrane vesicles from the bacterial cell surface of the P. savastanoi pv. savastanoi T3 mutant, while the wild-type strain exhibited very few vesicles. This phenomenon has not been described before for any other bacterial phytopathogen during host infection.
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
Recovery of nonpathogenic mutant bacteria from tumors caused by several Agrobacterium tumefaciens strains: a frequent event?
(American Society for Microbiology, 2009) Llop, Pablo; Murillo Martínez, Jesús; Lastra, Beatriz; López, María Milagros; Producción Agraria; Nekazaritza Ekoizpena
We have evaluated the interaction that bacterial genotypes and plant hosts have with the loss of pathogenicity in tumors, using seven Agrobacterium tumefaciens strains inoculated on 12 herbaceous and woody hosts. We performed a screening of the agrobacteria present inside the tumors, looking for nonpathogenic strains, and found a high variability of those strains in this niche. To verify the origin of the putative nonpathogenic mutant bacteria, we applied an efficient, reproducible, and specific randomly amplified polymorphic DNA analysis method. In contrast with previous studies, we recovered a very small percentage (0.01%) of nonpathogenic strains that can be considered true mutants. Of 5,419 agrobacterial isolates examined, 662 were nonpathogenic in tomato, although only 7 (from pepper and tomato tumors induced by two A. tumefaciens strains) could be considered to derive from the inoculated strain. Six mutants were affected in the transferred DNA (T-DNA) region; one of them contained IS426 inserted into the iaaM gene, whereas the whole T-DNA region was apparently deleted in three other mutants, and the virulence of the remaining two mutants was fully restored with the T-DNA genes as well. The plasmid profile was altered in six of the mutants, with changes in the size of the Ti plasmid or other plasmids and/or the acquisition of new plasmids. Our results also suggest that the frequent occurrence of nonpathogenic clones in the tumors is probably due to the preferential growth of nonpathogenic agrobacteria, of either endophytic or environmental origin, but different from the bacterial strain inducing the tumor.
Open Access
La patología vegetal en los últimos 25 años
(Phytoma España, 2013) Murillo Martínez, Jesús; Ortiz Barredo, Amaia; Ayllón, María Ángeles; López, María Milagros; Producción Agraria; Nekazaritza Ekoizpena
En los últimos 25 años hemos asistido a una verdadera revolución de la Ciencia y la Tecnología que, por supuesto, también ha influido de manera importante sobre el desarrollo de la Patología Vegetal. A petición de la editorial Phytoma, en este artículo hemos recogido 25 acontecimientos (avances, logros, riesgos y amenazas) de especial importancia. Indudablemente, la revolución electrónica ha facilitado la comunicación entre investigadores, la diseminación del conocimiento científico y el desarrollo de nuevas técnicas de análisis, entre otras, que han facilitado la generación y análisis de datos. Además, en estos 25 años se han producido diversos avances y acontecimientos que han impactado de forma particular sobre el desarrollo de la Patología Vegetal en España y en el mundo.
Open Access
Secreted LysM proteins are required for niche competition and full virulence in Pseudomonas savastanoi during host plant infection
(PLOS, 2025-08-01) Domínguez-Cerván, Hilario; Barrientos-Moreno, Laura; Díaz-Martínez, Luis; Murillo Martínez, Jesús; Pérez-Dorado, Inmaculada; Ramos, Cayo; Rodríguez Moreno, Luis; Institute for Multidisciplinary Research in Applied Biology - IMAB
Phytopathogenic bacteria secrete diverse virulence factors to manipulate host defenses and establish infection. Characterization of the type III secretion system (T3SS)- and HrpL-independent secretome (T3-IS) in Pseudomonas savastanoi pv. savastanoi (Psv), the causal agent of olive knot disease, identified five secreted LysM-containing proteins (LysM1–LysM5) associated with distinct physiological processes critical for infection. Functional predictions from network analyses suggest that LysM1, LysM2, and LysM4 may participate in type IV pilus-related functions, while LysM3 and LysM5 are likely to possess peptidoglycan hydrolase domains critical for cell division. Supporting these predictions, loss of LysM1 function resulted in impaired twitching and swimming motility, highlighting a role in pilus-mediated movement and early host colonization. In contrast, mutants lacking LysM3 or LysM5 exhibited pronounced filamentation and defective bacterial division, underscoring their essential role in septation, a process crucial for both in planta fitness and tumor formation. Structural modeling and protein stability assays demonstrate that LysM3 interacts with peptidoglycan fragments such as tetra-N-acetylglucosamine and meso-diaminopimelic acid, as well as with zinc ions, through conserved LysM and M23 domains. LysM3 also displayed selective bacteriostatic activity against co-inhabiting Gram-negative bacterial competitors, such as Pantoea agglomerans and Erwinia toletana. Our findings highlight the relevance of LysM proteins in maintaining bacterial integrity, motility, and competitive fitness, which are crucial for successful host infection. This study expands the functional repertoire of LysM-containing proteins and reveals their broader impact on bacterial virulence and adaptation to the plant-associated niche.
Open Access
Necesidades de formación especializada en Fitopatología de los profesionales de la Sanidad Vegetal
(Phytoma España, 2012) López, María Milagros; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
La Fitopatología es la ciencia que estudia las enfermedades de las plantas, que como es bien sabido, constituyen un importante factor limitante de la producción agrícola. Esta ciencia, que trata de la naturaleza, etiología y epidemiología de las enfermedades, estudia asimismo métodos de control de virus, viroides, bacterias, mollicutes, hongos, oomicetos, nematodos y plantas parásitas, por lo que presenta una indudable complejidad que es imposible simplificar. Es necesaria, por lo tanto, una formación especializada en Fitopatología que permita a los profesionales asesorar con conocimiento sobre los diferentes temas, cumpliendo los requisitos de la Directiva 2009/128 CE. Ello obliga a reconocer que los planes de estudio actualmente vigentes en España no proporcionan a los estudiantes la formación que será requerida para el desempeño de sus funciones profesionales en dicho campo y que tanto los fitopatólogos como los organismos públicos y privados, debemos poner todos los medios a nuestro alcance para su mejora.
Open Access
Comparative genomics of native plasmids from plant pathogenic Gammaproteobacteria
(Oxford University Press, 2025-04-01) Urriza Leoz, Miriam; Dimaria, Giulio; Oliveira, Luiz Orlando de; Catara, Vittoria; Murillo Martínez, Jesús; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Plasmids are key in the evolution and adaptation of plant pathogenic Gammaproteobacteria (PPG), yet their diversity and functional contributions remain underexplored. Here, comparative genomics revealed extensive variation in plasmid size, replicon types, mobility, and genetic content across PPG. Most plasmids are small (< 200 kb), except in Pantoea, exhibiting high coding densities (76% to 78%). Five ancestral replicon types were identifed across multiple orders, indicating vertical descent yet effcient horizontal transfer across taxa, although with limited genetic conservation. Virulence plasmids are widespread (56% to 68%) but differ in virulence gene content across orders: type III effector (T3E) genes are common in Pseudomonas and Xanthomonas, but rare in Enterobacterales and Xylella, aligning with their smaller effector repertoires. Plasmids frequently carry regulatory genes, highlighting their role in bacterial phenotype modulation. Distinct patterns were observed among orders: Enterobacterales plasmids often harbor thiamin biosynthesis operons and transcriptional regulators but lack post-transcriptional regulators, while most Pseudomonas and Xanthomonas plasmids are mobile, enriched in T3E genes, and exhibit high insertion sequence densities, fostering DNA mobility. Resistance to ultraviolet light is common, but not to antimicrobial compounds. These fndings highlight the dynamic role of plasmids in spreading adaptive traits, shaping virulence, and driving the evolution of plant pathogenic bacteria.
Open Access
The mangotoxin biosynthetic operon (mbo) is specifically distributed within Pseudomonas syringae genomospecies 1 and was acquired only once during evolution
(American Society for Microbiology, 2013) Carrión, Víctor J.; Gutiérrez Barranquero, José Antonio; Arrebola, Eva; Bardají Goikoetxea, Leire; Codina, Juan Carlos; Vicente, Antonio de; Cazorla, Francisco M.; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Mangotoxin production was ﬁrst described in Pseudomonas syringae pv. syringae strains. A phenotypic characterization of 94 P. syringae strains was carried out to determine the genetic evolution of the mangotoxin biosynthetic operon (mbo). We designed a PCR primer pair speciﬁc for the mbo operon to examine its distribution within the P. syringae complex. These primers ampliﬁed a 692-bp DNA fragment from 52 mangotoxin-producing strains and from 7 non-mangotoxin-producing strains that harbor the mbo operon, whereas 35 non-mangotoxin-producing strains did not yield any ampliﬁcation. This, together with the analysis of draft genomes, allowed the identiﬁcation of the mbo operon in ﬁve pathovars (pathovars aptata, avellanae, japonica, pisi, and syringae), all of which belong to genomospecies 1, suggesting a limited distribution of the mbo genes in the P. syringae complex. Phylogenetic analyses using partial sequences from housekeeping genes differentiated three groups within genomospecies 1. All of the strains containing the mbo operon clustered in groups I and II, whereas those lacking the operon clustered in group III; however, the relative branching order of these three groups is dependent on the genes used to construct the phylogeny. The mbo operon maintains synteny and is inserted in the same genomic location, with high sequence conservation around the insertion point, for all the strains in groups I and II. These data support the idea that the mbo operon was acquired horizontally and only once by the ancestor of groups I and II from genomospecies 1 within the P. syringae complex.
Open Access
Expression of the gene for resistance to phaseolotoxin (argK) depends on the activity of genes phtABC in Pseudomonas syringae pv. Phaseolicola
(Public Library of Science, 2012) Aguilera, Selene; Torre Zavala, Susana de la; Hernández Flores, José Luis; Murillo Martínez, Jesús; Bravo, Jaime; Álvarez Morales, Ariel; Producción Agraria; Nekazaritza Ekoizpena
The bacterium Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally produced between 18 degrees C and 20 degrees C, while no detectable amounts are present above 28 degrees C. Phaseolotoxin is an effective inhibitor of ornithine carbamoyltransferase (OCTase) activity from plant, mammalian and bacterial sources and causes a phenotypic requirement for arginine. To protect the cell from its own toxin, P. syringae pv. phaseolicola synthesizes a phaseolotoxin-resistant OCTase (ROCT). The ROCT is the product of the argK gene and is synthesized only under conditions leading to phaseolotoxin synthesis. The argK gene is included in a chromosomal fragment named Pht cluster, which contains genes involved in the synthesis of phaseolotoxin. The aim of the present work was to investigate the possible involvement of other genes included in the Pht cluster in the regulation of gene argK. We conducted transcriptional analyses of argK in several mutants unable to produce phaseolotoxin, transcriptional fusions and electrophoretic mobility shift assays, which allowed us to determine that genes phtABC, located within the Pht cluster, participate in the transcriptional repression of gene argK at temperatures not permissive for phaseolotoxin biosynthesis. This repression is mediated by a protein present in both toxigenic and nontoxigenic strains of P. syringae and in E. coli, and requires the coordinated participation of phtA, phtB and phtC products in order to carry out an efficient argK repression.
Open Access
Global genomic analysis of Pseudomonas savastanoi pv. savastanoi plasmids
(American Society for Microbiology, 2007) Pérez Martínez, Isabel; Zhao, Youfu; Murillo Martínez, Jesús; Sundin, George W.; Ramos, Cayo; Producción Agraria; Nekazaritza Ekoizpena
Pseudomonas savastanoi pv. savastanoi strains harbor native plasmids belonging to the pPT23A plasmid family (PFPs) which are detected in all pathovars of the related species Pseudomonas syringae examined and contribute to the ecological and pathogenic fitness of their host. However, there is a general lack of information about the gene content of P. savastanoi pv. savastanoi plasmids and their role in the interaction of this pathogen with olive plants. We designed a DNA macroarray containing 135 plasmid-borne P. syringae genes to conduct a global genetic analysis of 32 plasmids obtained from 10 P. savastanoi pv. savastanoi strains. Hybridization results revealed that the number of PFPs per strain varied from one to four. Additionally, most strains contained at least one plasmid (designated non-PFP) that did not hybridize to the repA gene of pPT23A. Only three PFPs contained genes involved in the biosynthesis of the virulence factor indole-3-acetic acid (iaaM, iaaH, and iaaL). In contrast, ptz, a gene involved in the biosynthesis of cytokinins, was found in five PFPs and one non-PFP. Genes encoding a type IV secretion system (T4SS), type IVA, were found in both PFPs and non-PFPs; however, type IVB genes were found only on PFPs. Nine plasmids encoded both T4SSs, whereas seven other plasmids carried none of these genes. Most PFPs and non-PFPs hybridized to at least one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors and one or more insertion sequence transposase genes. These results indicate that non-PFPs may contribute to the virulence and fitness of the P. savastanoi pv. savastanoi host. The overall gene content of P. savastanoi pv. savastanoi plasmids, with their repeated information, mosaic arrangement, and insertion sequences, suggests a possible role in adaptation to a changing environment.