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

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

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

Los factores ambientales, luz y temperatura, modifican la formación de la biopelícula en Pseudomonas syringae pv. syringae.

Comunicación a congreso en formato pósterLas biopelículas bacterianas están formadas por agregados celulares embebidos en una matriz extracelular de producción propia, formada principalmente por exopolisacáridos (EPS), proteínas y ADN extracelular. Existen datos recientes que sugieren que entre el 40-80% de las bacterias se encuentran formando biopelículas en la naturaleza, y que la formación de las biopelículas se ve afectada por factores ambientales. Pseudomonas syringae pv. syringae (Pss) es una bacteria fitopatógena causante de la necrosis apical del mango (NAM), enfermedad limitante de la producción de este cultivo en la región mediterránea. En Pss se ha descrito una conexión entre factores ambientales y la incidencia y severidad de la NAM, jugando los EPS celulosa y uno tipo-Psl, un papel relevante en la transición entre un estilo de vida epífito o patogénico. En este trabajo, se ha analizado el papel de algunos factores ambientales relevantes como son la luz y la temperatura en la formación de la biopelícula in vitro en una colección de cepas de Pss aisladas de mango. Los resultados obtenidos sugieren que la temperatura y particularmente la luz pueden influir en la formación de las biopelículas a través de la biosíntesis de los EPS, principalmente celulosa. Además, también se ha observado como niveles más bajos de producción de celulosa podrían estar asociados con una mayor virulencia. Estos resultados indican que los EPS, y principalmente celulosa juegan un papel clave en la ecología de Pss sobre la planta de mango a través de la formación de la biopelícula.Este trabajo ha sido financiado por Proyectos de Excelencia de la Junta de Andalucía (P12- AGR-1473), cofinanciado con ayudas a proyectos I+D+i en el marco del Programa Operativo FEDER Andalucía (UMA20-FEDERJA-060), y la Universidad de Málaga. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The Erwinia chrysanthemi phoP-phoQ operon plays an important role in growth at low pH, virulence and bacterial survival in plant tissue

The Erwinia chrysanthemi phoP-phoQ operon plays an important role in growth at low pH, virulence and bacterial survival in plant tissu

Functional and evolutionary significance of unknown genes from uncultivated taxa

25 Pág.Many of the Earth's microbes remain uncultured and understudied, limiting our understanding of the functional and evolutionary aspects of their genetic material, which remain largely overlooked in most metagenomic studies1. Here we analysed 149,842 environmental genomes from multiple habitats2-6 and compiled a curated catalogue of 404,085 functionally and evolutionarily significant novel (FESNov) gene families exclusive to uncultivated prokaryotic taxa. All FESNov families span multiple species, exhibit strong signals of purifying selection and qualify as new orthologous groups, thus nearly tripling the number of bacterial and archaeal gene families described to date. The FESNov catalogue is enriched in clade-specific traits, including 1,034 novel families that can distinguish entire uncultivated phyla, classes and orders, probably representing synapomorphies that facilitated their evolutionary divergence. Using genomic context analysis and structural alignments we predicted functional associations for 32.4% of FESNov families, including 4,349 high-confidence associations with important biological processes. These predictions provide a valuable hypothesis-driven framework that we used for experimental validatation of a new gene family involved in cell motility and a novel set of antimicrobial peptides. We also demonstrate that the relative abundance profiles of novel families can discriminate between environments and clinical conditions, leading to the discovery of potentially new biomarkers associated with colorectal cancer. We expect this work to enhance future metagenomics studies and expand our knowledge of the genetic repertory of uncultivated organisms.This project has received funding from the National Programme for Fostering Excellence in Scientific and Technical Research (grant no. PGC2018-098073-A-I00 MCIU/AEI/FEDER, UE) and, partially, by MCIN/AEI/10.13039/501100011033/ and FEDER Una manera de hacer Europa (grant no. PID2021-127210NB-I00). A.R.d.R. was supported by a fellowship from la Caixa Foundation (ID 100010434, fellowship code no. LCF/BQ/DI18/11660009), cofunded by the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement no. 713673. C.P.C., S.S.-H. and Z.D. acknowledge support by Severo Ochoa Centres of Excellence Programme from the State Research Agency of Spain (grant nos. SEV-2016-0672 (2017–2021) and CEX2020-000999-S). J.B. acknowledges support by a grant from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (no. 2020-218584). A.H.-P. was supported by Research Technical Support Staff Aid (no. PTA2019-017593-I/AEI/10.13039/501100011033). M.M.-P., J.J.R.-H. and E.L.-S. acknowledge support from Ministerio de Ciencia e Innovación, MCIN/AEI/10.13039/501100011033 (grant no. PID2021-125673OB-I00). S.S. acknowledges support from the Swiss National Science Foundation project (grant no. 205321_184955) and NCCR Microbiomes (no. 51NF40_180575), and thanks the staff at ETH Zurich IT Services and HPC facilities.Peer reviewe

The role of secretion systems and small molecules in soft-rot enterobacteriaceae pathogenicity

Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity

Suppression of Plant Immune Responses by the Pseudomonas savastanoi pv. savastanoi NCPPB 3335 Type III Effector Tyrosine Phosphatases HopAO1 and HopAO2

The effector repertoire of the olive pathogen P. savastanoi pv. savastanoi NCPPB 3335 includes two members of the HopAO effector family, one of the most diverse T3E families of the P. syringae complex. The study described here explores the phylogeny of these dissimilar members, HopAO1 and HopAO2, among the complex and reveals their activities as immune defense suppressors. Although HopAO1 is predominantly encoded by phylogroup 3 strains isolated from woody organs of woody hosts, both HopAO1 and HopAO2 are phylogenetically clustered according to the woody/herbaceous nature of their host of isolation, suggesting host specialization of the HopAO family across the P. syringae complex. HopAO1 and HopAO2 translocate into plant cells and show hrpL-dependent expression, which allows their classification as actively deployed type III effectors. Our data also show that HopAO1 and HopAO2 possess phosphatase activity, a hallmark of the members of this family. Both of them exert an inhibitory effect on early plant defense responses, such as ROS production and callose deposition, and are able to suppress ETI responses induced by the effectorless polymutant of P. syringae pv. tomato DC3000 (DC3000D28E) in Nicotiana. Moreover, we demonstrate that a ΔhopAO1 mutant of P. savastanoi NCPBB 3335 exhibits a reduced fitness and virulence in olive plants, which supports the relevance of this effector during the interaction of this strain with its host plants. This work contributes to the field with the first report regarding functional analysis of HopAO homologs encoded by P. syringae or P. savastanoi strains isolated from woody hosts.This research was supported by the Spanish PlanNacional ICDCi grants, AGL2015-63851-R and AGL2014-53242-C2-1-R from the Ministerio de Economía y Competitividad (MINECO), cofinanced by Fondo Europeo de Desarrollo Regional (FEDER). MC-O was supported by a FPI fellowship (Ministerio de Ciencia e Innovación/Ministerio de Economiìa y Competitividad, Spain).Peer reviewedPeer Reviewe

The ybiT Gene of Erwinia chrysanthemi Codes for a Putative ABC Transporter and Is Involved in Competitiveness against Endophytic Bacteria during Infection

We investigated the role in bacterial infection of a putative ABC transporter, designated ybiT, of Erwinia chrysanthemi AC4150. The deduced sequence of this gene showed amino acid sequence similarity with other putative ABC transporters of gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, as well as structural similarity with proteins of Streptomyces spp. involved in resistance to macrolide antibiotics. The gene contiguous to ybiT, designated as pab (putative antibiotic biosynthesis) showed sequence similarity with Pseudomonas and Streptomyces genes involved in the biosynthesis of antibiotics. A ybiT mutant (BT117) was constructed by marker exchange. It retained full virulence in potato tubers and chicory leaves, but it showed reduced ability to compete in planta against the wild-type strain or against selected saprophytic bacteria. These results indicate that the ybiT gene plays a role in the in planta fitness of the bacteria