Análisis genómico y funcional de los efectores de las familias HopAF y HopAO del sistema de secreción tipo III de Pseudomonas savastanoi pv. savastanoi NCPPB 3335

Pseudomonas savastanoi pv. savastanoi (Psv) es el agente causal de la tuberculosis del olivo. El análisis bioinformático del borrador del genoma de Psv NCPPB 3335 permitió identificar 33 posibles efectores (T3E) del sistema de secreción tipo III (T3SS). Además, la secuenciación de los tres plásmidos de esta cepa reveló que los genes codificantes de los T3E HopAF1 y HopAO1 se localizan en los plásmidos pPsv48A y pPsv48B, respectivamente, codificándose en el cromosoma de esta cepa un homólogo de HopAF1 (HopAF1-2). Análisis posteriores revelaron que Psv NCPPB 3335 también codifica en el cromosoma un T3E (HopAO2) que contiene un dominio enzimático tirosina fosfatasa (PTP), similar al que posee HopAO1. El análisis filogenético de las familias HopAF y HopAO permitió identificar que ambas se encuentran ampliamente distribuidas dentro del complejo P. syringae. Análisis de translocación y transcripcionales validaron a los T3E HopAF1, HopAF1-2, HopAO1 y HopAO2 como nuevos T3E del secretoma del T3SS de Psv NCPPB 3335. La expresión heteróloga de estos 4 T3E tiene como consecuencia la interferencia con la respuesta de defensa primaria (PTI) de Nicotiana tabacum, lo que implica una reducción de la deposición de calosa y de la formación de especies reactivas de oxígeno (ROS). Asimismo, los T3E HopAF1-2, HopAO1 y HopAO2 también inhiben la inmunidad mediada por efectores (ETI) en este mismo hospedador. Por otro lado, y utilizando fusiones traduccionales a la proteína verde fluorescente (GFP), se localizaron los T3E HopAF1, HopAF1-2, HopAO1 y HopAO2 próximos a la membrana plasmática de las células de Nicotiana benthamiana. Además, HopAO2 también se localizó en vesículas del aparato de Golgi. La deleción del gen hopAF1 del plásmido pPsv48A en Psv NCPPB 3335 tuvo como consecuencia una ligera reducción en el tamaño de los tumores inducidos por este patógeno en plantas de olivo lignificadas, mientras que la deleción del gen hopAO1 conllevó una clara disminución de la virulencia del mismo.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Comparative Analysis of the Type III Secretion System Effector Repertoires of Pseudomonas savastanoi Pathovars Pathogenic on Woody Hosts

Comunicación de tipo pósterThe species Pseudomonas savastanoi, a member of the Pseudomonas syringae complex, includes four pathovars causing knots or excrescences in woody hosts: P. savastanoi pv. savastanoi (Psv), pv. fraxini (Psf), pv. nerii (Psn) and pv. retacarpa (Psr), comprising isolates from olive, ash, oleander and broom plants, respectively. Pathogenicity of P. savastanoi is dependent, among other factors, on the type III secretion system (T3SS) and its effector (T3E) repertoire. Furthermore, a putative role in the interaction with woody hosts has been suggested for several of these T3E. The recent availability of the genome sequences of several P. savastanoi strains isolated from different hosts has facilitated bioinformatics predictions of their T3SS genes and T3E pools, the study of their distribution in other strains of the P. syringae complex isolated from woody hosts and the functional analysis of several of these secreted proteins. As previously reported for Psv, Psn and Psf, here we show that pathogenicity of Psr ICMP16945, is also dependent on the T3SS. Psv strains NCPPB 3335, ICMP4352 and PseNe107 share a core set of at least 22 T3E, 18 of which are also encoded in Psn ICMP16943, Psf ICMP7711 and Psr ICMP16945. However, these three strains encode truncated versions of 1-2 of these 18 T3E and, Psr ICMP16945 contains three pathovarspecific T3E. Our results also show that several T3E, including HopAO1, are phylogenetically clustered across the P. syringae complex according to the woody/herbaceous nature of their host of isolation, suggesting host specialization of these effectors in this complex.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Features associated to woody hosts in the bacterial pathogen of olive plants Pseudomonas savastanoi pv. savastanoi

The causal agent of olive knot disease, Pseudomonas savastanoi pv. savastanoi, belongs to the Pseudomonas syringae complex, a bacterial group causing diseases in a broad variety of both woody and herbaceous plant species. Here we summarize our results regarding a set of P. savastanoi pv. savastanoi features exclusively found in the genomes of bacteria from the P. syringae complex isolated from woody hosts. Comparative genomics and evolutionary studies allowed us to identify a 15 kb genomic island (WHOP, from woody host and Pseudomonas), carrying a set of genes involved in degradation of phenolic compounds and exclusively found in bacterial pathogens of woody hosts. Deletion of several WHOP-encoded genes in Pseudomonas savastanoi pv. savastanoi NCPPB 3335 revealed that they play a role in the virulence of the strain in woody olive plants but not in in vitro-grown (nonwoody) plants. In addition, several type III secretion system effectors belonging to the HopAF, HopAO and HopBL families were shown to be clustered across the P. syringae complex according to the woody/herbaceous nature of their host of isolation. Further functional analyses of these virulence factors are needed to facilitate the design of novel strategies directed to control bacterial pathogens of woody hosts.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Translocation and Functional Analysis of Pseudomonas savastanoi pv. savastanoi NCPPB 3335 Type III Secretion System Effectors Reveals Two Novel Effector Families of the Pseudomonas syringae Complex

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 causes olive knot disease and is a model pathogen for exploring bacterial infection of woody hosts. The type III secretion system (T3SS) effector repertoire of this strain includes 31 effector candidates plus two novel candidates identified in this study which have not been reported to translocate into plant cells. In this work, we demonstrate the delivery of seven NCPPB 3335 effectors into Nicotiana tabacum leaves, including three proteins from two novel families of the P. syringae complex effector super-repertoire (HopBK and HopBL), one of which comprises two proteins (HopBL1 and HopBL2) that harbor a SUMO protease domain. When delivered by P. fluorescens heterologously expressing a P. syringae T3SS, all seven effectors were found to suppress the production of defense-associated reactive oxygen species. Moreover, six of these effectors, including the truncated versions of HopAA1 and HopAZ1 encoded by NCPPB 3335, suppressed callose deposition. The expression of HopAZ1 and HopBL1 by functionally effectorless P. syringae pv. tomato DC3000D28E inhibited the hypersensitive response in tobacco and, additionally, expression of HopBL2 by this strain significantly increased its competitiveness in N. benthamiana. DNA sequences encoding HopBL1 and HopBL2 were uniquely detected in a collection of 31 P. savastanoi pv. savastanoi strains and other P. syringae strains isolated from woody hosts, suggesting a relevant role of these two effectors in bacterial interactions with olive and other woody plants

Differential modulation of plant immune responses by diverse 2 members of the Pseudomonas savastanoi pv. savastanoi HopAF 3 type III effector family

The Pseudomonas savastanoi pv. savastanoi NCPPB 3335 type III secretion system (T3SS) effector repertoire includes 33 candidates, seven of which translocate into host cells and interfere with plant defences. The present study was performed to investigate the co-existence of both plasmid- and chromosomalencoded members of the HopAF effector family, HopAF1-1 and HopAF1-2, respectively, in the genome of NCPPB 3335. Here, we show that the HopAF1 paralogues are widely distributed in the Pseudomonas syringae complex, where HopAF1-1 is most similar to the homologues encoded by other P. syringae pathovars infecting woody hosts that belong to phylogroups 1 and 3. We show that the expression of both HopAF1-1 and HopAF-2 is transcriptionally dependent on HrpL and demonstrate their delivery into Nicotiana tabacum leaves. Although the heterologous delivery of either HopAF1-1 or HopAF1-2 significantly suppressed the production of defence-associated reactive oxygen species levels, only HopAF1-2 reduced the levels of callose deposition. Moreover, the expression of HopAF1-2 by functionally effectorless P. syringae pv. tomato DC3000D28E completely inhibited the hypersensitive response in tobacco and significantly increased the competitiveness of the strain in Nicotiana benthamiana. Despite their functional differences, subcellular localization studies reveal that green fluorescent protein (GFP) fusions to either HopAF1-1 or HopAF1-2 are targeted to the plasma membrane when they are expressed in plant cells, a process that is completely dependent on the integrity of their N-myristoylation motif. Our results further support the notion that highly similar T3SS effectors might differentially interact with diverse plant targets, even when they co-localize in the same cell compartment