Bacterial pathogens of woody plants represent a pervasive yet underexplored threat to global agriculture and forestry, causing devastating diseases in fruit trees, shrubs, forest trees and ornamental species. Unlike pathogens of herbaceous plants, the molecular strategies enabling bacteria to colonise and damage woody tissues remain poorly understood, owing to the experimental challenges associated with woody host systems and the distinctive biology of these plants. Moreover, persistence within long-lived woody tissues requires prolonged modulation of host immune signalling, enabling pathogens to evade or attenuate defence responses across growing seasons. This review synthesises current knowledge of the virulence mechanisms underlying bacterial infection of woody hosts, highlighting host-specific strategies and conserved pathways critical for disease establishment. Key mechanisms include manipulation of plant immune responses through type III secretion system effectors and other secreted proteins, catabolism of host-derived metabolites, degradation of structural barriers, and the activity of bacterial toxins and extracellular polysaccharides. By integrating these molecular insights, we examine how bacterial pathogens have evolved adaptive strategies to overcome the unique challenges posed by woody tissues and emphasise the implications for disease management in economically important crops. Addressing knowledge gaps in woody-host infections is essential for developing innovative, targeted strategies to mitigate the impact of these pathogens and safeguard global woody crops and forestry systems.

Phytopathogenic bacteria secrete diverse virulence factors to manipulate host defenses and establish infection. Characterization of the type III secretion system (T3SS)- and HrpL-independent secretome (T3-IS) in Pseudomonas savastanoi pv. savastanoi (Psv), the causal agent of olive knot disease, identified five secreted LysM-containing proteins (LysM1-LysM5) associated with distinct physiological processes critical for infection. Functional predictions from network analyses suggest that LysM1, LysM2, and LysM4 may participate in type IV pilus-related functions, while LysM3 and LysM5 are likely to possess peptidoglycan hydrolase domains critical for cell division. Supporting these predictions, loss of LysM1 function resulted in impaired twitching and swimming motility, highlighting a role in pilus-mediated movement and early host colonization. In contrast, mutants lacking LysM3 or LysM5 exhibited pronounced filamentation and defective bacterial division, underscoring their essential role in septation, a process crucial for both in planta fitness and tumor formation. Structural modeling and protein stability assays demonstrate that LysM3 interacts with peptidoglycan fragments such as tetra-N-acetylglucosamine and meso-diaminopimelic acid, as well as with zinc ions, through conserved LysM and M23 domains. LysM3 also displayed selective bacteriostatic activity against co-inhabiting Gram-negative bacterial competitors, such as Pantoea agglomerans and Erwinia toletana. Our findings highlight the relevance of LysM proteins in maintaining bacterial integrity, motility, and competitive fitness, which are crucial for successful host infection. This study expands the functional repertoire of LysM-containing proteins and reveals their broader impact on bacterial virulence and adaptation to the plant-associated niche.

The type III secretion system in Pseudomonas syringae complex pathogens delivers type III effectors (T3Es) into plant cells to manipulate host processes, enhance survival, and promote disease. While substantial research has focused on herbaceous pathogens, T3Es in strains infecting woody hosts are less understood. This study investigates the HopBL family of effectors in Pseudomonas savastanoi, a pathogen of woody plants. HopBL1 and HopBL2, core effectors in P. savastanoi, are restricted to phylogroup 3 strains of the P. syringae complex, all isolated from woody hosts. Phylogenetic analysis suggests recent horizontal acquisition of these effectors across multiple P. syringae pathovars, integrated into genomic islands flanked by mobile genetic elements. Structural analysis shows that both HopBL effectors contain SUMO protease and DNA-binding domains, with HopBL1 also possessing an ethylene-responsive motif, all characteristic of XopD from Xanthomonas spp. Despite low sequence identity, HopBL effectors exhibit structural similarity to XopD, with HopBL1 showing greater resemblance, particularly in the arrangement of these domains. Functional assays in olive and oleander revealed strain-specific contributions of HopBL1 and HopBL2 to virulence. In oleander, the natural host of P. savastanoi pv. nerii, mutation of either effector gene resulted in reduced symptom development. We show that HopBL2 localised predominantly to subnuclear foci and associated with plasmodesmata, with partial overlap observed along microtubules, suggesting a potential role in cytoskeleton manipulation. These findings underscore the importance of T3Es unique to P. syringae strains infecting woody hosts and their adaptation to modulate host cellular structures to promote disease.

The Pseudomonas syringae complex WHOP genomic island underpins virulence in woody hosts by mediating the catabolism of aromatic compounds. However, the biochemical functions of the ipoABC and dhoAB operons and the regulatory gene whpR remain unknown. Comparative genomics revealed WHOP-like clusters beyond P. syringae, found in diverse plant-associated, environmental and clinical bacteria, including indole degraders. We propose that ipoABC and dhoAB mediate indole degradation via anthranilate, linking indole detoxification to central metabolism through the β-ketoadipate pathway. In the olive pathogen P. savastanoi pv. savastanoi, ipoABC promotes indole degradation, indigo production, cell aggregation and biofilm formation. WhpR, an AraC-family regulator structurally related to CuxR and ToxT, defines a regulon comprising repression of most WHOP operons along with genes outside this region, including trpAB, reflecting integrated regulation of indole catabolism and tryptophan biosynthesis. In line with the observed transcriptional repression of WHOP genes, deletion of whpR led to hypervirulence and significantly altered bacterial fitness in woody olive plants. These findings define the WHOP region as a regulatory hub linking indole detoxification, multicellular behaviour and virulence, emerging as a target for novel control strategies against woody plant diseases.

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.

Bacteria have an extensive adaptive ability to live in close association with eukaryotic hosts, exhibiting detrimental, neutral or beneficial effects on host growth and health. However, the genes involved in niche adaptation are mostly unknown and their functions poorly characterized. Here, we present bacLIFE ( https://github.com/Carrion-lab/bacLIFE ) a streamlined computational workflow for genome annotation, large-scale comparative genomics, and prediction of lifestyle-associated genes (LAGs). As a proof of concept, we analyzed 16,846 genomes from the Burkholderia / Paraburkholderia and Pseudomonas genera, which led to the identification of hundreds of genes potentially associated with a plant pathogenic lifestyle. Site-directed mutagenesis of 14 of these predicted LAGs of unknown function, followed by plant bioassays, showed that 6 predicted LAGs are indeed involved in the phytopathogenic lifestyle of Burkholderia plantarii and Pseudomonas syringae pv. phaseolicola. These 6 LAGs encompassed a glycosyltransferase, extracellular binding proteins, homoserine dehydrogenases and hypothetical proteins. Collectively, our results highlight bacLIFE as an effective computational tool for prediction of LAGs and the generation of hypotheses for a better understanding of bacteria-host interactions.

Indole-3-acetic acid (IAA) production is a pathogenicity/virulence factor in the Pseudomonas syringae complex, including Pseudomonas savastanoi. P. savastanoi pathovars (pvs.) genomes contain the iaaL gene, encoding an enzyme that catalyzes the biosynthesis of the less biologically active compound 3-indole-acetyl-ϵ-L–lysine (IAA–Lys). Previous studies have reported the identification of IAA–Lys in culture filtrates of P. savastanoi strains isolated from oleander (pv. nerii), but the conversion of IAA into a conjugate was not detectable in olive strains (pv. savastanoi). In this paper, we show the distribution of iaaL alleles in all available P. savastanoi genomes of strains isolated from woody hosts. Most strains encode two different paralogs, except for those isolated from broom (pv. retacarpa), which contain a single allele. In addition to the three previously reported iaaL alleles (iaaLPsv, iaaLPsn and iaaLPto), we identified iaaLPsf, an exclusive allele of strains isolated from ash (pv. fraxini). We also found that the production of IAA–Lys in P. savastanoi pv. savastanoi and pv. nerii depends on a functional iaaLPsn allele, whereas in pv. fraxini depends on iaaLPsf. The production of IAA–Lys was detected in cultures of an olive strain heterologously expressing IaaLPsn-1, IaaLPsf-1 and IaaLPsf-3, but not when expressing IaaLPsv-1. In addition, Arabidopsis seedlings treated with the strains overproducing the conjugate, and thus reducing the free IAA content, alleviated the root elongation inhibitory effect of IAA. IAA–Lys synthase activity assays with purified allozymes confirmed the functionality and specificity of lysine as a substrate of IaaLPsn-1 and IaaLPsf-3, with IaaLPsf-3 showing the highest catalytic efficiency for both substrates. The IAA–Lys synthase activity of IaaLPsn-1 was abolished by the insertion of two additional tyrosine residues encoded in the inactive allozyme IaaLPsv-1. These results highlight the relevance of allelic variation in a phytohormone-related gene for the modulation of auxin production in a bacterial phytopathogen.

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 differing efficiencies. We also identified a functional MOBQ mobilization region in pC, which could only mobilize this plasmid. Plasmid pB could be efficiently 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.

HomeMolecular Plant-Microbe Interactions®Vol. 35, No. 12The Genomic Landscape Resource of Pseudomonas syringae pv. syringae Strains Isolated from Mango Trees PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseThe Genomic Landscape Resource of Pseudomonas syringae pv. syringae Strains Isolated from Mango TreesJosé A. Gutiérrez-Barranquero, Zaira Heredia-Ponce, Lorena Aguilera-Cobos, Adrián Pintado, M. Gonzalo Claros, Cayo Ramos, Francisco M. Cazorla, and Antonio de VicenteJosé A. Gutiérrez-Barranquero†Corresponding author: J. A. Gutiérrez-Barranquero; E-mail Address: jagutierrez@uma.eshttps://orcid.org/0000-0003-1810-699XInstituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC)Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, Málaga, SpainSearch for more papers by this author, Zaira Heredia-PonceInstituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC)Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, Málaga, SpainSearch for more papers by this author, Lorena Aguilera-CobosPlataforma Andaluza de Bioinformática-SCBI, Universidad de Málaga, Málaga, SpainSearch for more papers by this author, Adrián PintadoInstituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC)Departamento de Biología Celular, Genética y Fisiología, Área de Genética, Facultad de Ciencias, Universidad de Málaga, Málaga, SpainSearch for more papers by this author, M. Gonzalo Claroshttps://orcid.org/0000-0002-0112-3550Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC)Plataforma Andaluza de Bioinformática-SCBI, Universidad de Málaga, Málaga, SpainDepartamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Málaga, SpainSearch for more papers by this author, Cayo RamosInstituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC)Departamento de Biología Celular, Genética y Fisiología, Área de Genética, Facultad de Ciencias, Universidad de Málaga, Málaga, SpainSearch for more papers by this author, Francisco M. CazorlaInstituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC)Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, Málaga, SpainSearch for more papers by this author, and Antonio de Vicentehttps://orcid.org/0000-0003-2716-9861Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC)Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, Málaga, SpainSearch for more papers by this authorAffiliationsAuthors and Affiliations José A. Gutiérrez-Barranquero1 2 † Zaira Heredia-Ponce1 2 Lorena Aguilera-Cobos3 Adrián Pintado1 4 M. Gonzalo Claros1 3 5 Cayo Ramos1 4 Francisco M. Cazorla1 2 Antonio de Vicente1 2 1Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC) 2Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain 3Plataforma Andaluza de Bioinformática-SCBI, Universidad de Málaga, Málaga, Spain 4Departamento de Biología Celular, Genética y Fisiología, Área de Genética, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain 5Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain Published Online:12 Dec 2022https://doi.org/10.1094/MPMI-05-22-0107-AAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleGenome Resource AnnouncementPseudomonas syringae pv. syringae is the causal agent of bacterial apical necrosis (BAN) disease of mango trees (Cazorla et al. 1998). BAN disease is the most limiting factor of mango trees in the Mediterranean region because it severely affects mango yield in seasons when weather conditions are favorable to develop the infection by P. syringae pv. syringae (Gutiérrez-Barranquero et al. 2012). P. syringae pv. syringae isolates from mango possess a vast arsenal of virulence factors highlighting the production of the antimetabolite toxin mangotoxin (Arrebola et al. 2003). Other important adaptation traits were also present is P. syringae pv. syringae isolates from mango, such as the production of the exopolysaccharide (EPS) cellulose. The presence of different variants of copper resistance genes have been detected in plasmids belonging to the pPT23A family of plasmids, a family that appears to be indigenous to P. syringae (Sesma et al. 1998). These adaptation traits enhance epiphytic fitness on mango tree surfaces (Gutiérrez-Barranquero et al. 2013a, 2017, 2019; Heredia-Ponce et al. 2020). P. syringae pv. syringae strains from mango form a specific phylotype within the pathovar syringae, strongly associated with the mango host and with mangotoxin production (Gutiérrez-Barranquero et al. 2013b, 2019). To date, genomic information of P. syringae pv. syringae belonging to this specific phylotype remain scarce, with only three genome sequences available in GenBank (Aprile et al. 2021; Martinez-García et al. 2015). Based on a previous phylogenetic distribution study performed within this specific phylotype, including strains from two groups isolated at different times (Aprile et al. 2021), the presence of various phylogenetic subgroups (PSGs) was observed.From these important PSGs, different strains were selected for genome sequencing, to obtain a comprehensive overview of the genomic landscape of the P. syringae pv. syringae mango phylotype. The main characteristics of the P. syringae pv. syringae strains that were selected are summarized in Supplementary Table S1. P. syringae pv. syringae strains were streaked onto King's B agar plates from −80°C frozen stocks and were grown for 48 h at 25°C. Single colonies from these plates were sampled using sterile toothpicks to inoculate 5 ml lysogenic broth (LB) tubes that were grown for 18 h at 25°C with shaking at 150 rpm. Total DNA extractions were performed using the DNAeasy UltraClean microbial kit (Qiagen) following manufacturer instructions. DNA libraries were prepared using the Nextera XT DNA library preparation kit for small genomes (Illumina, Inc.). Whole draft genome sequencing was performed by the Supercomputing and Bioinnovation Center of the University of Málaga, using the sequencing platform NextSeq 550 with paired-end reads, with a read length of 2 × 150 bp. To obtain high-quality reads for assembly, raw reads were pre-processed using SeqTrimBB, a modified software of SeqTrim (Falgueras et al. 2010), to eliminate low-quality reads and contaminants. The assembly of high-quality filtered reads was conducted using the A5-MiSeq pipeline with default parameters (Coil et al. 2015).Genome sequence annotation and gene identifications were obtained using two different approaches: i) the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP), using default parameters, and ii) Prokka (version 1.14.6), a command line software, to obtain a rapid prokaryotic genome annotation (Seemann 2014) with default parameters. The main features of the draft genome sequences obtained in this work, including their accession numbers, are listed in Table 1. The whole draft genome sizes ranged from 5.83 to 6.02 Mb, with a total number of coding DNA sequences ranging from 5,030 to 5,206 and G + C content from 59.1 to 59.3%, values that are typical for strains belonging to P. syringae species. The sequencing mean coverage of each genome sequence ranged from 605 to 1,118× and the content of transfer RNAs ranged from 57 to 63, based on PGAP results.Table 1. Accession numbers and genome assembly features of Pseudomonas syringae pv. syringae strains used in this studyaStrainsPSGGenBank accession no.Genome size (Mb)No. of scaffoldsN50 (bp)Coverage (×)No. of CDSNo. of tRNAsG + C content (%)UMAF2044PSG-IJAJPOB0000000005.8738809,1291,1185,0815959.3UMAF0049PSG-IIJAJPON0000000005.9524493,8859505,1395959.2UMAF0081PSG-IIJAJPOM0000000005.8724411,1701,0525,0305859.3UMAF0271PSG-IIJAJPOF0000000005.9520753,3529595,1336159.2UMAF7000PSG-IIIJAJPNY0000000005.9222669,5709635,0716159.2UMAF0176PSG-IVJAJPOK0000000005.8715907,0061,0415,0385959.2UMAF0297PSG-IVJAJPOD0000000005.9418819,9678705,1015659.2UMAF1013PSG-IVJAJPOC0000000005.9929663,1696815,2065959.2UMAF1029PSG-IVJAJPOI0000000006.0120820,0421,0355,1865859.2UMAF2801PSG-IVJAJPOH0000000005.9415564,1991,0175,0935759.1UMAF2815PSG-IVJAJPNZ0000000005.8912810,1341,0635,0976359.1EPS17APSG-VIJAJPOO0000000005.9125556,0857025,1115759.2UMAF1003PSG-VIJAJPOJ0000000006.0228551,3716315,2055859.2UMAF0170PSG-VIIJAJPOL0000000005.9265473,4116055,1105759.1UMAF0273PSG-VIIJAJPOE0000000005.83101,076,7719765,0725959.3UMAF2016PSG-VIIJAJPOG0000000005.8929414,3596285,1015759.2UMAF2600PSG-VIIJAJPOA0000000005.9227660,2828845,1636059.2DAR77789PSG-VIIJAJPOP0000000005.8722468,6186815,1025759.2aPSG = phylogenetic subgroup, CDS = coding DNA sequences, and tRNA = transfer RNA.Table 1. Accession numbers and genome assembly features of Pseudomonas syringae pv. syringae strains used in this studyaView as image HTML The Roary pipeline (Page et al. 2015) and the Bacterial Pan Genome Analysis Pipeline (BPGA) (Chaudhari et al. 2016) were both used to obtain the pangenome and core genomes. Based on Roary, the pangenome and core genomes (including the P. syringae pv. syringae B728a strain in our analysis) numbered 8,673 and 4,106 gene clusters, respectively, and using BPGA, the pangenome and core genomes were 8,202 and 3,963 protein-coding sequences, respectively. BGPA also uses curve fitting with Heaps’ law to estimate whether a pangenome is open or closed. Contrary to what was observed previously for the P. syringae complex (Dillon et al. 2019a), the pangenome of P. syringae pv. syringae isolates from mango was nearly closed, with a fitting parameter (γ) of 0.14, excluding the output P. syringae pv. syringae B728a strain (pangenome and core genomes by BPGA excluding P. syringae pv. syringae B728a were 7,590 and 4,273, respectively) (Supplementary Fig. S1A). A nearly closed pangenome was expected, as the 21 genomes of P. syringae pv. syringae strains isolated from mango would be suitable to capture most of the diversity of this genetically homogeneous group, in comparison to genomes of the entire P. syringae complex. The randomized axelerated maximum likelihood (RAxML, version 7.7.6) (Stamatakis 2014) method was used to analyze the phylogenetic distribution of the P. syringae pv. syringae strains based on the concatenated nucleotide sequences of genes belonging to the core genome obtained by the Roary pipeline. The RAxML phylogenetic tree was constructed using the GRT + gamma model with 1,000 fast bootstrap runs. The core genome phylogeny generated a rearrangement of certain P. syringae pv. syringae strains in comparison with the previous results, obtained using partial sequences of gyrB and rpoD genes (Aprile et al. 2021) (Fig. 1A). This result was considered more robust because of the inclusion of P. syringae pv. syringae strains isolated from pear in the same PSG. There is a correlation in the distribution of the strains, based on the area of isolation in some PSGs, in which old and new P. syringae pv. syringae strains isolated from related areas are part of the same PSGs (PSG-VIIa, PSG-I, PSG-II, and PSG-VI). In addition, there is a strong correlation based on their resistance or sensitivity to copper. Furthermore, the BLAST Atlas tool from the Gview server was used to perform a graphical genomic comparison, using the genome of P. syringae pv. syringae B728a as reference (Fig. 1B).Fig. 1. Core genome phylogeny and graphical comparative genomic analysis of Pseudomonas syringae pv. syringae strains isolated from mango. A, Phylogenetic analysis based on the multialigment of the concatenated nucleotide sequences belonging to the core genome of each P. syringae pv. syringae strain sequenced in this study (4,106 genes), including three other previously sequenced P. syringae pv. syringae strains isolated from mango (UMAF0158, UMAF0291, and UMAF3028). Strain P. syringae pv. syringae B728a was used as outgroup. Bootstrap values are included in the node of each branch. In addition, the year and location of isolation have been included. B, The BLAST Atlas tool was used for graphical comparative genomic analysis of P. syringae pv. syringae mango isolates, using the genome of P. syringae pv. syringae B728a as a reference. The different ring colors correspond to each P. syringae pv. syringae mango isolate belonging to different phylogenetic subgroups, with the same color code represented in A.Download as PowerPointGenome mining using BLAST searches revealed the presence of relevant genes associated with virulence or adaptation features essential for P. syringae pv. syringae lifestyle on mango tree surfaces. Regarding virulence factors, the mangotoxin generating operon (mgo operon) involved in the production of mangotoxin (Arrebola et al. 2007), an antimetabolite toxin very specific to P. syringae pv. syringae strains isolated from mango (Arrebola et al. 2003; Gutiérrez-Barranquero et al. 2013b), was present in the genome of all P. syringae pv. syringae strains isolated from mango. This operon has been recently described to synthesize the signaling molecule leudizen, a volatile molecule that controls mangotoxin production (Sieber et al. 2021). In addition, the mangotoxin biosynthetic operon (mbo operon) (Carrión et al. 2012) was also found in the genome of all the P. syringae pv. syringae strains. Interestingly, two different type III secretion systems were also found in all P. syringae pv. syringae mango genomes, as were previously identified in the complete genome sequence of P. syringae pv. syringae UMAF0158 (Martínez-García et al. 2015). The pool of predicted type III effectors was analyzed using the BastionHUb platform (Wang et al. 2021), a webserver that integrates and analyzes substrates secreted by gram-negative bacteria. The output from this database was revised to include the new reassignment of several type III effector families reported by Dillon et al. (2019b). The pan and core type III effectors numbered 21 and 16, respectively (Supplementary Fig. S1B).Regarding adaptation mechanisms, all P. syringae pv. syringae strains from mango harbored the biosynthetic genes of cellulose (wss operon), Psl-like, and alginate EPS. Cellulose and Psl-like EPS have been described to be crucial for adhesion and biofilm formation and to function as switches between epiphytic and pathogenic lifestyles (Arrebola et al. 2015; Heredia-Ponce et al. 2020). Interestingly, and similar to what was observed previously for mangotoxin, the wss operon is a very specific feature of the P. syringae pv. syringae mango phylotype. Another important adaption mechanism is the presence of different variants of copper resistance genes, mainly associated with 62-kb pPT23A plasmids. From the 18 P. syringae pv. syringae draft genomes sequenced in this study, 11 P. syringae pv. syringae strains harbored different variants of copper resistance genes (Supplementary Table S1), which could improve their survival against copper treatments (Aprile et al. 2021; Cazorla et al. 2002; Gutiérrez-Barranquero et al. 2019). Finally, to analyze the genetic diversity of our P. syringae pv. syringae mango genomes, the FastANI method (Jain et al. 2018) was performed using the 21 P. syringae pv. syringae strains from mango, nine P. syringae pv. syringae strains isolated from others hosts, and strain P. syringae pv. tomato DC3000 (Feil et al. 2005) (Supplementary Fig. S2). The highest and average ANI values exclusively for P. syringae pv. syringae mango genomes were 99.98 and 99.09%, respectively. The highest and average ANI values for all P. syringae pv. syringae genomes included in this analysis were 99.98% (between mango P. syringae pv. syringae strains UMAF0158 and UMAF0273) and 96.90%, respectively. P. syringae pv. syringae HS191 showed the highest ANI value in comparison with all the P. syringae pv. syringae strains, reaching values in all cases higher than 95%.The increase of genomic data of P. syringae pv. syringae strains from mango provides a comprehensive overview of the genomic landscape of this specific phylotype, providing important clues to understanding the evolution of molecular mechanisms, especially with respect to their specific virulence and adaptation features. Understanding in depth this genomic information will provide an advantage in the fight against bacterial phytopathogens, improving disease management strategies.Data AvailabilityThe draft genome sequences are deposited in the NCBI GenBank database under BioProject PRJNA786963. The GenBank accession numbers of each draft genome sequences are included in Table 1.AcknowledgmentsWe would like to thank F. Aprile for her technical support during the initial stage of this work. The authors also thank J. Gómez from Supercomputing and Bioinnovation Center of the University of Málaga for the excellent technical support provided in the sequencing processes.Author-Recommended Internet ResourcesBastionHUb platform: http://bastionhub.erc.monash.eduGview server: https://server.gview.caThe author(s) declare no conflict of interest.Literature CitedAprile, F., Heredia-Ponce, Z., Cazorla, F. M., de Vicente, A., and Gutiérrez-Barranquero, J. A. 2021. A large Tn7-like transposon confers hyper-resistance to copper in Pseudomonas syringae pv. syringae. Appl. Environ. Microbiol. 87:e02528-20. https://doi.org/10.1128/AEM.02528-20 Crossref, ISI, Google ScholarArrebola, E., Carrión, V. J., Gutiérrez-Barranquero, J. A., Pérez-García, A., Rodríguez-Palenzuela, P., Cazorla, F. 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This is an open access article distributed under the CC BY-NC-ND 4.0 International license.DetailsFiguresLiterature CitedRelated Vol. 35, No. 12 December 2022ISSN:0894-0282e-ISSN:1943-7706 Download Metrics Article History Issue Date: 19 Dec 2022Published: 12 Dec 2022Accepted: 30 Aug 2022 Pages: 1109-1114 InformationCopyright © 2022 The Author(s).This is an open access article distributed under the CC BY-NC-ND 4.0 International license.Funding CICE-Junta de Andalucía, Proyecto de ExcelenciaGrant/Award Number: P12-AGR-1473 Junta de Andalucía, Proyecto Spanish Plan Nacional I+D+IGrant/Award Number: AGL2017-83368-C2-1-R Junta de Andalucía, ProyectoGrant/Award Number: UMA18-FEDERJA-046 Keywordsexopolysaccharidesgenomicsmango treesPseudomonas syringae pv. syringaevirulence associated genesThe author(s) declare no conflict of interest.PDF download

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 catalog 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.

Chemosensory pathways are among the most abundant prokaryotic signal transduction systems, allowing bacteria to sense and respond to environmental stimuli. Signaling is typically initiated by the binding of specific molecules to the ligand binding domain (LBD) of chemoreceptor proteins (CRs). Although CRs play a central role in plant-microbiome interactions such as colonization and infection, little is known about their phylogenetic and ecological specificity. Here, we analyzed 82,277 CR sequences from 11,806 representative microbial species covering the whole prokaryotic phylogeny, and we classified them according to their LBD type using a de novo homology clustering method. Through phylogenomic analysis, we identified hundreds of LBDs that are found predominantly in plant-associated bacteria, including several LBDs specific to phytopathogens and plant symbionts. Functional annotation of our catalogue showed that many of the LBD clusters identified might constitute unknown types of LBDs. Moreover, we found that the taxonomic distribution of most LBD types that are specific to plant-associated bacteria is only partially explained by phylogeny, suggesting that lifestyle and niche adaptation are important factors in their selection. Finally, our results show that the profile of LBD types in a given genome is related to the lifestyle specialization, with plant symbionts and phytopathogens showing the highest number of niche-specific LBDs. The LBD catalogue and information on how to profile novel genomes are available at https://github.com/compgenomicslab/CRs. IMPORTANCE Considering the enormous variety of LBDs at sensor proteins, an important question resides in establishing the forces that have driven their evolution and selection. We present here the first clear demonstration that environmental factors play an important role in the selection and evolution of LBDs. We were able to demonstrate the existence of LBD families that are highly enriched in plant-associated bacteria but show a wide phylogenetic spread. These findings offer a number of research opportunities in the field of single transduction, such as the exploration of similar relationships in chemoreceptors of bacteria with a different lifestyle, like those inhabiting or infecting the human intestine. Similarly, our results raise the question whether similar LBD types might be shared by members of different sensor protein families. Lastly, we provide a comprehensive catalogue of CRs classified by their LBD region that includes a large number of putative new LBD types.

The Pseudomonas savastanoi species comprises a group of phytopathogenic bacteria that cause symptoms of disease in woody hosts. This is mediated by the rapid activation of a pool of virulence factors that suppress host defences and hijack the host's metabolism to the pathogen's benefit. The hrpL gene encodes an essential transcriptional regulator of virulence functions, including the type III secretion system (T3SS), in pathogenic bacteria. Here, we analyzed the contribution of HrpL to the virulence of four pathovars (pv.) of P. savastanoi isolated from different woody hosts (oleander, ash, broom, and dipladenia) and characterized the HrpL regulon of P. savastanoi pv. savastanoi NCPPB 3335 using two approaches: whole transcriptome sequencing (RNA-seq) and the bioinformatic prediction of candidate genes containing an hrp-box. Pathogenicity tests carried out for the P. savastanoi pvs. showed that HrpL was essential for symptom development in both non-host and host plants. The RNA-seq analysis of the HrpL regulon in P. savastanoi revealed a total of 53 deregulated genes, 49 of which were downregulated in the ΔhrpL mutant. Bioinformatic prediction resulted in the identification of 50 putative genes containing an hrp-box, 16 of which were shared with genes previously identified by RNA-seq. Although most of the genes regulated by HrpL belonged to the T3SS, we also identified some genes regulated by HrpL that could encode potential virulence factors in P. savastanoi.

AbstractThe endophytic bacterium Pantoea agglomerans DAPP‐PG 734 was previously isolated from olive knots caused by infection with Pseudomonas savastanoi pv. savastanoi DAPP‐PG 722. Whole‐genome analysis of this P. agglomerans strain revealed the presence of a Hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To assess the role of the P. agglomerans T3SS in the interaction with P. savastanoi pv. savastanoi, we generated independent knockout mutants in three Hrp genes of the P. agglomerans DAPP‐PG 734 T3SS (hrpJ, hrpN, and hrpY). In contrast to the wildtype control, all three mutants failed to cause a hypersensitive response when infiltrated in tobacco leaves, suggesting that P. agglomerans T3SS is functional and injects effector proteins in plant cells. In contrast to P. savastanoi pv. savastanoi DAPP‐PG 722, the wildtype strain P. agglomerans DAPP‐PG 734 and its Hrp T3SS mutants did not cause olive knot disease in 1‐year‐old olive plants. Coinoculation of P. savastanoi pv. savastanoi with P. agglomerans wildtype strains did not significantly change the knot size, while the DAPP‐PG 734 hrpY mutant induced a significant decrease in knot size, which could be complemented by providing hrpY on a plasmid. By epifluorescence microscopy and confocal laser scanning microscopy, we found that the localization patterns in knots were nonoverlapping for P. savastanoi pv. savastanoi and P. agglomerans when coinoculated. Our results suggest that suppression of olive plant defences mediated by the Hrp T3SS of P. agglomerans DAPP‐PG 734 positively impacts the virulence of P. savastanoi pv. savastanoi DAPP‐PG 722.

The rhizobacterium Pseudomonas alcaligenes AVO110 exhibits antagonism toward the phytopathogenic fungus Rosellinia necatrix. This strain efficiently colonizes R. necatrix hyphae and is able to feed on their exudates. Here, we report the complete genome sequence of P. alcaligenes AVO110. The phylogeny of all available P. alcaligenes genomes separates environmental isolates, including AVO110, from those obtained from infected human blood and oyster tissues, which cluster together with Pseudomonas otitidis. Core and pan-genome analyses showed that P. alcaligenes strains encode highly heterogenic gene pools, with the AVO110 genome encoding the largest and most exclusive variable region (~1.6 Mb, 1795 genes). The AVO110 singletons include a wide repertoire of genes related to biofilm formation, several of which are transcriptionally modulated by R. necatrix exudates. One of these genes (cmpA) encodes a GGDEF/EAL domain protein specific to Pseudomonas spp. strains isolated primarily from the rhizosphere of diverse plants, but also from soil and water samples. We also show that CmpA has a role in biofilm formation and that the integrity of its EAL domain is involved in this function. This study contributes to a better understanding of the niche-specific adaptations and lifestyles of P. alcaligenes, including the mycophagous behavior of strain AVO110.

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.

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.

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.

SummaryIn a number of compatible plant‐bacterium interactions, a rise in apoplastic Ca2+ levels is observed, suggesting that Ca2+ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP‐PG 722 is mediated by a Na+/Ca2+ exchanger critical for virulence. Using the fluorescent Ca2+ probe Fura 2‐AM, we demonstrate that Ca2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca2+ entry is a Na+/Ca2+ exchanger. In silico analysis of the Psav DAPP‐PG 722 genome revealed the presence of a single gene coding for a Na+/Ca2+ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav‐cneA mutant. Complementation of the Psav‐cneA mutation restored both Ca2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca2+ entry acts as a ‘host signal’ that allows and promotes Psav pathogenicity on olive plants.