Open Access
The toxic guardians: multiple toxin-antitoxin systems provide stability, avoid deletions and maintain virulence genes of Pseudomonas syringae virulence plasmids
(BMC, 2019) Bardají Goikoetxea, Leire; Añorga García, Maite; Echeverría Ancín, Myriam; Ramos, Cayo; Murillo Martínez, Jesús; Institute for Multidisciplinary Research in Applied Biology - IMAB
Background: Pseudomonas syringae is a y-proteobacterium causing economically relevant diseases in practically all cultivated plants. Most isolates of this pathogen contain native plasmids collectively carrying many pathogenicity and virulence genes. However, P. syringae is generally an opportunistic pathogen primarily inhabiting environmental reservoirs, which could exert a low selective pressure for virulence plasmids. Additionally, these plasmids usually contain a large proportion of repeated sequences, which could compromise plasmid integrity. Therefore, the identification of plasmid stability determinants and mechanisms to preserve virulence genes is essential to understand the evolution of this pathogen and its adaptability to agroecosystems. Results: The three virulence plasmids of P. syringae pv. savastanoi NCPPB 3335 contain from one to seven functional stability determinants, including three highly active toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C. The TA systems reduced loss frequency of pPsv48A by two orders of magnitude, whereas one of the two replicons of pPsv48C likely confers stable inheritance by itself. Notably, inactivation of the TA systems from pPsv48C exposed the plasmid to high-frequency deletions promoted by mobile genetic elements. Thus, recombination between two copies of MITEPsy2 caused the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 x 10-3. Likewise, one-ended transposition of IS801 generated plasmids containing deletions of variable size, with a frequency of 5.5 ± 2.1 x 1 0- 4, of which 80% had lost virulence gene idi. These deletion derivatives were stably maintained in the population by replication mediated by repJ, which is adjacent to IS801. IS801 also promoted deletions in plasmid pPsv48A, either by recombination or one-ended transposition. In all cases, functional TA systems contributed significantly to reduce the occurrence of plasmid deletions in vivo. Conclusions: Virulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Importantly, we showed that multiple plasmid-borne TA systems have a prominent role in preserving plasmid integrity and ensuring the maintenance of virulence genes in free-living conditions. This strategy is likely widespread amongst native plasmids of P. syringae and other bacteria.
Open Access
Pseudomonas savastanoi pv. savastanoi: some like it knot
(Wiley, 2012) Ramos, Cayo; Matas Casado, Isabel María; Bardají Goikoetxea, Leire; Aragón, Isabel M.; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
Pseudomonas savastanoi pv. savastanoi is the causal agent of olive (Olea europaea) knot disease and an unorthodox member of the P. syringae complex, causing aerial tumours instead of the foliar necroses and cankers characteristic of most members of this complex. Olive knot is present wherever olive is grown; although losses are difficult to assess, it is assumed that olive knot is one of the most important diseases of the olive crop. The last century has witnessed a good deal of scientific articles describing the biology, epidemiology and control of this pathogen. However, most P. savastanoi pv. savastanoi strains are highly recalcitrant to genetic manipulation, which has effectively left the pathogen out of the scientific progress in molecular biology that has elevated the foliar pathogens of the P. syringae complex to supermodels. A series of studies in the last years have made significant advances in the biology, ecology and genetics of P. savastanoi pv. savastanoi, paving the way for the molecular dissection of its interaction with other non-pathogenic bacteria and their woody hosts. The selection of a genetically pliable model strain was soon followed by the development of rapid methods for virulence assessment with micropropagated olive plants and the analysis of cellular interactions with the plant host. The generation of a draft genome of strain NCPPB 3335 and the closed sequence of its three native plasmids has allowed for functional and comparative genomic analyses for the identification of its pathogenicity gene complement. This includes 34 putative type III effector genes and genomic regions, shared with other pathogens of woody hosts, that encode metabolic pathways associated with the degradation of lignin-derived compounds. Now, the time is right to explore the molecular basis of the P. savastanoi pv. savastanoi-olive interaction and to get insights into why some pathovars like it necrotic and why some like it knot. Synonyms: Pseudomonas syringae pv. savastanoi Taxonomy: Kingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Family Pseudomonadaceae; Genus Pseudomonas; included in genomospecies 2 together with at least P. amygdali, P. ficuserectae, P. meliae and 16 other pathovars from the P. syringae complex (aesculi, ciccaronei, dendropanacis, eriobotryae, glycinea, hibisci, mellea, mori, myricae, phaseolicola, photiniae, sesami, tabaci, ulmi, and certain strains of lachrymans and morsprunorum); when a formal proposal is made for the unification of these bacteria, the species name P. amygdali would take priority over P. savastanoi. Microbiological properties: Gram-negative rods, 0.4-0.8 by 1.0-3.0 µm, aerobic. Motile by one to four polar flagella, rather slow growing, optimal temperatures for growth of 25–30 °C, oxidase negative, arginine dihydrolase negative, elicits the hypersensitive response on tobacco, most isolates are fluorescent and levan negative although some isolates are non-fluorescent and levan positive. Host range: P. savastanoi pv. savastanoi causes tumours in cultivated and wild olive and ash (Fraxinus excelsior). Although strains from olive were reported to infect oleander (Nerium oleander), this is generally not the case; however, strains of P. savastanoi pv. nerii can infect olive. Pathovars fraxini and nerii differentiate from pv. savastanoi mostly in their host range, and were not formally recognized until 1996. Literature previous to about 1996 generally name strains of the three pathovars as P. syringae subsp. savastanoi or P. savastanoi subsp. savastanoi, contributing to confusion about host range and biological properties. Disease symptoms: Symptoms of infected trees include hyperplastic growths (tumorous galls or knots) on the stems and branches of the host plant and, occasionally, on leaves and fruits. Epidemiology: The pathogen can survive and multiply on aerial plant surfaces, as well as in knots, from where it can be dispersed by rain, wind, insects and human activities, entering the plant through wounds. Populations are very unevenly distributed in the plant, and suffer drastic fluctuations throughout the year, with maximum numbers of bacteria occurring during rainy and warm months. Populations of P. savastanoi pv. savastanoi are normally associated to non-pathogenic bacteria, both epiphytically and endophytically, and were demonstrated to form mutualistic consortia with Erwinia toletana and Pantoea agglomerans that could result in increased bacterial populations and disease symptoms. Disease control: Based on preventive measures, mostly sanitary and cultural practices. Integrated control programs benefit from regular applications of copper formulations, which should be maintained at least a few years for maximum benefit. Olive cultivars vary in their susceptibility to olive knot, but there are no known cultivars with full resistance to the pathogen. Useful websites: http://www.pseudomonas-syringae.org/; http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl; ASAP access to the P. savastanoi pv. savastanoi NCPPB 3335 genome sequence https://asap.ahabs.wisc.edu/asap/logon.php.

Bardají Goikoetxea, Leire

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