Open Access
The Pbo cluster from Pseudomonas syringae pv. phaseolicola NPS3121 is thermoregulated and required for phaseolotoxin biosynthesis
(MDPI, 2021) Guardado-Valdivia, Lizeth; Chacón-López, Alejandra; Murillo Martínez, Jesús; Poveda Arias, Jorge; Hernández Flores, José Luis; Xoca-Orozco, Luis; Aguilera, Selene; Institute for Multidisciplinary Research in Applied Biology - IMAB
The bean (Phaseolus vulgaris) pathogen Pseudomonas syringae pv. phaseolicola NPS3121 synthe-sizes phaseolotoxin in a thermoregulated way, with optimum production at 18 °C. Gene PSPPH_4550 was previously shown to be thermoregulated and required for phaseolotoxin bio-synthesis. Here, we established that PSPPH_4550 is part of a cluster of 16 genes, the Pbo cluster, included in a genomic island with a limited distribution in P. syringae and unrelated to the posses-sion of the phaseolotoxin biosynthesis cluster. We identified typical non-ribosomal peptide syn-thetase, and polyketide synthetase domains in several of the pbo deduced products. RT-PCR and the analysis of polar mutants showed that the Pbo cluster is organized in four transcriptional units, including one monocistronic and three polycistronic. Operons pboA and pboO are both es-sential for phaseolotoxin biosynthesis, while pboK and pboJ only influence the amount of toxin produced. The three polycistronic units were transcribed at high levels at 18 °C but not at 28 °C, whereas gene pboJ was constitutively expressed. Together, our data suggest that the Pbo cluster synthesizes secondary metabolite(s), which could participate in the regulation of phaseolotoxin biosynthesis.
Open Access
Host range determinants of Pseudomonas savastanoi pathovars of woody hosts revealed by comparative genomics and cross-pathogenicity tests
(Frontiers Media, 2020) Moreno Pérez, Alba; Pintado, Adrián; Murillo Martínez, Jesús; Caballo Ponce, Eloy; Tegli, Stefania; Moretti, Chiaraluce; Rodríguez Palenzuela, Pablo; Ramos, Cayo; Institute for Multidisciplinary Research in Applied Biology - IMAB
The study of host range determinants within the Pseudomonas syringae complex is gaining renewed attention due to its widespread distribution in non-agricultural environments, evidence of large variability in intra-pathovar host range, and the emergence of new epidemic diseases. This requires the establishment of appropriate model pathosystems facilitating integration of phenotypic, genomic and evolutionary data. Pseudomonas savastanoi pv. savastanoi is a model pathogen of the olive tree, and here we report a closed genome of strain NCPPB 3335, plus draft genome sequences of three strains isolated from oleander (pv. nerii), ash (pv. fraxini) and broom plants (pv. retacarpa). We then conducted a comparative genomic analysis of these four new genomes plus 16 publicly available genomes, representing 20 strains of these four P. savastanoi pathovars of woody hosts. Despite overlapping host ranges, cross-pathogenicity tests using four plant hosts clearly separated these pathovars and lead to pathovar reassignment of two strains. Critically, these functional assays were pivotal to reconcile phylogeny with host range and to define pathovar-specific genes repertoires. We report a pan-genome of 7,953 ortholog gene families and a total of 45 type III secretion system effector genes, including 24 core genes, four genes exclusive of pv. retacarpa and several genes encoding pathovar-specific truncations. Noticeably, the four pathovars corresponded with well-defined genetic lineages, with core genome phylogeny and hierarchical clustering of effector genes closely correlating with pathogenic specialization. Knot-inducing pathovars encode genes absent in the canker-inducing pv. fraxini, such as those related to indole acetic acid, cytokinins, rhizobitoxine, and a bacteriophytochrome. Other pathovar-exclusive genes encode type I, type II, type IV, and type VI secretion system proteins, the phytotoxine phevamine A, a siderophore, c-di-GMP-related proteins, methyl chemotaxis proteins, and a broad collection of transcriptional regulators and transporters of eight different superfamilies. Our combination of pathogenicity analyses and genomics tools allowed us to correctly assign strains to pathovars and to propose a repertoire of host range-related genes in the P. syringae complex.
Open Access
GacA reduces virulence and increases competitiveness in planta in the tumorigenic olive pathogen Pseudomonas savastanoi pv. savastanoi
(Frontiers Media, 2024) Lavado-Benito, Carla; Murillo Martínez, Jesús; Martínez Gil, Marta; Ramos, Cayo; Rodríguez Moreno, Luis; Institute for Multidisciplinary Research in Applied Biology - IMAB
GacS/GacA is a widely distributed two-component system playing an essential role as a key global regulator, although its characterization in phytopathogenic bacteria has been deeply biased, being intensively studied in pathogens of herbaceous plants but barely investigated in pathogens of woody hosts. P. savastanoi pv. savastanoi (Psv) is characterized by inducing tumours in the stem and branches of olive trees. In this work, the model strain Psv NCPPB 3335 and a mutant derivative with a complete deletion of gene gacA were subjected to RNA-Seq analyses in a minimum medium and a medium mimicking in planta conditions, accompanied by RT-qPCR analyses of selected genes and phenotypic assays. These experiments indicated that GacA participates in the regulation of at least 2152 genes in strain NCPPB 3335, representing 37.9 % of the annotated CDSs. GacA also controls the expression of diverse rsm genes, and modulates diverse phenotypes, including motility and resistance to oxidative stresses. As occurs with other P. syringae pathovars of herbaceous plants, GacA regulates the expression of the type III secretion system and cognate effectors. In addition, GacA also regulates the expression of WHOP genes, specifically encoded in P. syringe strains isolated from woody hosts, and genes for the biosynthesis of phytohormones. A gacA mutant of NCPPB 3335 showed increased virulence, producing large immature tumours with high bacterial populations, but showed a significantly reduced competitiveness in planta. Our results further extend the role of the global regulator GacA in the virulence and fitness of a P. syringae pathogen of woody hosts.
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
Genes ptz and idi, coding for cytokinin biosynthesis enzymes, are essential for tumorigenesis and in planta growth by P. syringae pv. savastanoi NCPPB 3335
(Frontiers Media, 2020) Añorga García, Maite; Pintado, Adrián; Ramos, Cayo; Diego, Nuria de; Ugena, Lydia; Novák, Ondrej; Murillo Martínez, Jesús; Institute for Multidisciplinary Research in Applied Biology - IMAB
The phytopathogenic bacterium Pseudomonas syringae pv. savastanoi elicits aerial tumors on olive plants and is also able to synthesize large amounts of auxins and cytokinins. The auxin indoleacetic acid was shown to be required for tumorigenesis, but there is only correlational evidence suggesting a role for cytokinins. The model strain NCPPB 3335 contains two plasmid-borne genes coding for cytokinin biosynthesis enzymes: ptz, for an isopentenyl transferase and idi, for an isopentenyl-diphosphate delta-isomerase. Phylogenetic analyses showed that carriage of ptz and idi is not strictly associated with tumorigenic bacteria, that both genes were linked when first acquired by P. syringae, and that a different allele of ptz has been independently acquired by P. syringae pv. savastanoi and closely related bacteria. We generated mutant derivatives of NCPPB 3335 cured of virulence plasmids or with site-specific deletions of genes ptz and/or idi and evaluated their virulence in lignified and micropropagated olive plants. Strains lacking ptz, idi, or both produced tumors with average volumes up to 29 times smaller and reached populations up to two orders of magnitude lower than those induced by strain NCPPB 3335; these phenotypes reverted by complementation with the cloned genes. Trans-zeatin was the most abundant cytokinin in culture filtrates of NCPPB 3335. Deletion of gene ptz abolished biosynthesis of trans-zeatin and dihydrozeatin, whereas a reduced but significant amount of isopentenyladenine was still detected in the medium, suggesting the existence of other genes contributing to cytokinin biosynthesis in P. syringae. Conversely, extracts from strains lacking gene idi contained significantly higher amounts of trans-zeatin than extracts from the wild-type strain but similar amounts of the other cytokinins. This suggests that Idi might promote tumorigenesis by ensuring the biosynthesis of the most active cytokinin forms, their correct balance in planta, or by regulating the expression of other virulence genes. Therefore, gene ptz, but not gene idi, is essential for the biosynthesis of high amounts of cytokinins in culture; however, both ptz and idi are individually essential for the adequate development of tumors on olive plants by Psv NCPPB 3335.
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
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
Pseudomonas savastanoi pv. mandevillae pv. nov., a clonal pathogen causing an emerging, devastating disease of the ornamental plant Mandevilla spp.
(APS Publications, 2021) Caballo Ponce, Eloy; Pintado, Adrián; Moreno Pérez, Alba; Murillo Martínez, Jesús; Smalla, Kornelia; Ramos, Cayo; Institute for Multidisciplinary Research in Applied Biology - IMAB
Commercial production of the ornamental plant dipladenia (Mandevilla spp.) is threatened by dipladenia leaf and stem spot disease, caused by the bacterium Pseudomonas savastanoi. P. savastanoi includes four pathovars of woody hosts differentiated by a characteristic host range in olive, oleander, ash and broom plants. However, isolates from dipladenia have not been ascribed to any particular lineage or P. savastanoi pathovar. Here we report that isolates from dipladenia represent a distinct, clonal lineage. First, dipladenia isolates display very similar plasmid profiles, including a plasmid encoding the iaaM gene for biosynthesis of indole-3-acetic acid. Second, multilocus sequence analysis and core-genome single-nucleotide-polymorphisms phylogenies showed a monophyletic origin for dipladenia isolates, which cluster with isolates from oleander (pathovar nerii) in a distinct clade well separated from other P. savastanoi strains. Metabolic profiling and cross-pathogenicity tests in olive, oleander, ash, broom and dipladenia clearly distinguished dipladenia isolates from the four P. savastanoi pathovars. Comparative genomics of the draft genome sequence of the dipladenia strain Ph3 with the other four pathovars showed that Ph3 encodes very few strain-specific genes, and a similar set of virulence genes to pv. nerii, including its repertoire of type III secretion system effectors. However, hierarchical clustering based on the catalogue of effectors and their allelic variants clearly separated Ph3 from pv. nerii strains. Based on their distinctive pathogenicity profile, we propose a de novo pathovar for P. savastanoi isolates from dipladenia, P. savastanoi pv. mandevillae pv. nov., for which strain Ph3 (CFBP 8832PT) has been designated as the pathotype strain.