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
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
Phylogenetic analysis of the pPT23A plasmid family of Pseudomonas syringae
(American Society for Microbiology, 2006) Ma, Zhonghua; Smith, James J.; Zhao, Youfu; Jackson, Robert W.; Arnold, Dawn L.; Murillo Martínez, Jesús; Sundin, George W.; Producción Agraria; Nekazaritza Ekoizpena
The pPT23A plasmid family of Pseudomonas syringae contains members that contribute to the ecological and pathogenic fitness of their P. syringae hosts. In an effort to understand the evolution of these plasmids and their hosts, we undertook a comparative analysis of the phylogeny of plasmid genes and that of conserved chromosomal genes from P. syringae. In total, comparative sequence and phylogenetic analyses were done utilizing 47 pPT23A family plasmids (PFPs) from 16 pathovars belonging to six genomospecies. Our results showed that the plasmid replication gene (repA), the only gene currently known to be distributed among all the PFPs, had a phylogeny that was distinct from that of the P. syringae hosts of these plasmids and from those of other individual genes on PFPs. The phylogenies of two housekeeping chromosomal genes, those for DNA gyrase B subunit (gyrB) and primary sigma factor (rpoD), however, were strongly associated with genomospecies of P. syringae. Based on the results from this study, we conclude that the pPT23A plasmid family represents a dynamic genome that is mobile among P. syringae pathovars.
Open Access
Characterisation of the mgo operon in Pseudomonas syringae pv. syringae UMAF0158 that is required for mangotoxin production
(BioMed Central, 2012) Arrebola, Eva; Carrión, Víctor J.; Cazorla, Francisco M.; Pérez García, Alejandro; Murillo Martínez, Jesús; Vicente, Antonio de; Producción Agraria; Nekazaritza Ekoizpena
Background: Mangotoxin is an antimetabolite toxin that is produced by strains of Pseudomonas syringae pv. syringae; mangotoxin-producing strains are primarily isolated from mango tissues with symptoms of bacterial apical necrosis. The toxin is an oligopeptide that inhibits ornithine N-acetyl transferase (OAT), a key enzyme in the biosynthetic pathway of the essential amino acids ornithine and arginine. The involvement of a putative nonribosomal peptide synthetase gene (mgoA) in mangotoxin production and virulence has been reported. Results: In the present study, we performed a RT-PCR analysis, insertional inactivation mutagenesis, a promoter expression analysis and terminator localisation to study the gene cluster containing the mgoA gene. Additionally, we evaluated the importance of mgoC, mgoA and mgoD in mangotoxin production. A sequence analysis revealed an operon-like organisation. A promoter sequence was located upstream of the mgoB gene and was found to drive lacZ transcription. Two terminators were located downstream of the mgoD gene. RT-PCR experiments indicated that the four genes (mgoBCAD) constitute a transcriptional unit. This operon is similar in genetic organisation to those in the three other P. syringae pathovars for which complete genomes are available (P. syringae pv. syringae B728a, P. syringae pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A). Interestingly, none of these three reference strains is capable of producing mangotoxin. Additionally, extract complementation resulted in a recovery of mangotoxin production when the defective mutant was complemented with wild-type extracts. Conclusions: The results of this study confirm that mgoB, mgoC, mgoA and mgoD function as a transcriptional unit and operon. While this operon is composed of four genes, only the last three are directly involved in mangotoxin production.
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.
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
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
Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola
(National Academy of Sciences, 1999) Jackson, Robert W.; Athanassopoulos, Evangelos; Tsiamis, George; Mansfield, John W.; Sesma, Ane; Arnold, Dawn L.; Gibbon, Marjorie J.; Murillo Martínez, Jesús; Taylor, John D.; Vivian, Alan; Producción Agraria; Nekazaritza Ekoizpena
The 154-kb plasmid was cured from race 7 strain 1449B of the phytopathogen Pseudomonas syringae pv. phaseolicola (Pph). Cured strains lost virulence toward bean, causing the hypersensitive reaction in previously susceptible cultivars. Restoration of virulence was achieved by complementation with cosmid clones spanning a 30-kb region of the plasmid that contained previously identified avirulence (avr) genes avrD, avrPphC, and avrPphF. Single transposon insertions at multiple sites (including one located in avrPphF) abolished restoration of virulence by genomic clones. Sequencing 11 kb of the complementing region identified three potential virulence (vir) genes that were predicted to encode hydrophilic proteins and shared the hrp-box promoter motif indicating regulation by HrpL. One gene achieved partial restoration of virulence when cloned on its own and therefore was designated virPphA as the first (A) gene from Pph to be identified for virulence function. In soybean, virPphA acted as an avr gene controlling expression of a rapid cultivar-specific hypersensitive reaction. Sequencing also revealed the presence of homologs of the insertion sequence IS100 from Yersinia and transposase Tn501 from P. aeruginosa. The proximity of several avr and vir genes together with mobile elements, as well as G1C content significantly lower than that expected for P. syringae, indicates that we have located a plasmidborne pathogenicity island equivalent to those found in mammalian pathogens.
Open Access
Molecular characterization of Tunisian strains of Erwinia amylovora
(Italian Phytopathological Society, 2017) Dardouri, Sana; Chehimi, Sonia; Murillo Martínez, Jesús; Hajlaoui, Mohamed Rabeh; Producción Agraria; Nekazaritza Ekoizpena
The present study focused on the molecular characterization of a collection of Erwinia amylovora isolates recovered from different outbreaks in Tunisia between 2012 and 2014. Analysis of 54 isolates, including the reference type strain CFBP 1430, revealed that all Tunisian isolates produced the expected amplicons with diverse primer pairs routinely used for molecular diagnostics of E. amylovora. We also evaluated the genetic variability of these isolates by PCR fingerprinting, using specific primers for clustered regularly interspaced short palindromic repeats (CRISPRs) and for variable number of tandem repeats (VNTR) sequences. For the first method, our results revealed that all the primers used, except those for CRISPR3, which produced an identical amplicon for all isolates, showed some variability among Tunisian isolates. For the second method, forty-nine isolates showed the same fingerprint patterns as the reference type strain CFBP 1430 with all the primers used, whereas four of the isolates showed very divergent patterns. These results suggest that there has been a main introduction of European-type isolates in Tunisia, and possibly a few mutations or other independent introductions of the pathogen. Additionally, these results indicate that PCR fingerprinting using VNTR markers is a most useful tool for discriminating among E. amylovora strains and for their identification in epidemiological studies.
Open Access
Venturia inaequalis resistance in local Spanish cider apple germplasm under controlled and ﬁeld conditions
(Springer, 2012) Martínez Bilbao, Alejandro; Ortiz Barredo, Amaia; Montesinos, Emilio; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua: PA123
Host resistance is a key method for the integrated management of apple scab caused by Venturia inaequalis, which is one of the most important diseases of apple. Artificial inoculation of 92 cider apple cultivars with a mixed inoculum of V. inaequalis identified 19 weakly resistant and 19 resistant cultivars. Twelve of these resistant cultivars were previously classified as having low susceptibility to fire blight, and four of them showed complete or weak resistance to races (1), (1, 6) and (6, 7, 13) of V. inaequalis. The analysis of a selection of 72 cultivars for 6 years under field conditions identified 14 cultivars that were classified as resistant to apple scab under high disease pressure involving one to six Mills periods of severe risk of infection each year. Eight out of these 14 cultivars previously showed high levels of resistance to fire blight, which would allow the incorporation of genetic resistance in the integrated production of cider apples in Spain through their use in breeding programs.