BUSCADOR RECOLECTA

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., This research was supported by project grants PID2020-115177RB-C21 (to CR and LR-M) and PID2020-115177RB-C22 (to JM) financed by the Ministerio de Ciencia, Innovación y Universidades (MICIU)/ Agencia Estatal de Investigación (AEI)/10.13039/501100011033/ and by the European Regional Development Fund (ERDF) “A way to make Europe”, and by grant 2024ICT153 (to IP-D) from the Consejo Superior de Investigaciones Científicas (CSIC). HD-C was supported by predoctoral grant PRE2021-099113 (MICIU). LB-M was supported by the A4 Program of the Universidad de Málaga, Peer reviewed

Plasmids are key in the evolution and adaptation of plant pathogenic Gammaproteobacteria (PPG), yet their diversity and functional contributions remain underexplored. Here, comparative genomics revealed extensive variation in plasmid size, replicon types, mobility, and genetic content across PPG. Most plasmids are small (< 200 kb), except in Pantoea, exhibiting high coding densities (76% to 78%). Five ancestral replicon types were identifed across multiple orders, indicating vertical descent yet effcient horizontal transfer across taxa, although with limited genetic conservation. Virulence plasmids are widespread (56% to 68%) but differ in virulence gene content across orders: type III effector (T3E) genes are common in Pseudomonas and Xanthomonas, but rare in Enterobacterales and Xylella, aligning with their smaller effector repertoires. Plasmids frequently carry regulatory genes, highlighting their role in bacterial phenotype modulation. Distinct patterns were observed among orders: Enterobacterales plasmids often harbor thiamin biosynthesis operons and transcriptional regulators but lack post-transcriptional regulators, while most Pseudomonas and Xanthomonas plasmids are mobile, enriched in T3E genes, and exhibit high insertion sequence densities, fostering DNA mobility. Resistance to ultraviolet light is common, but not to antimicrobial compounds. These fndings highlight the dynamic role of plasmids in spreading adaptive traits, shaping virulence, and driving the evolution of plant pathogenic bacteria., This research was supported by project grants PQ 314124/2023-3 from Conselho Nacional de Desenvolvimento Científco e Tecnológico (CNPq) and fellowship grant 88887.878703/2023-00 from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), to LOO; by Incentive Plan for Research (PIA.CE.RI.) 2024-2026 line 2 (DIME-SIECO Research Project) of the University of Catania (Italy), to VC; and PID2020-115177RB-C22 fnanced by the Spanish Ministry of Science and Innovation (MCIN)/Agencia Estatal de Investigación (AEI) /10.13039/501100011033/ and by the European Regional Development Fund (ERDF) 'A way to make Europe', to JM. Open access funding provided by Universidad Pública de Navarra.

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., This research was supported by project grants PID2020-115177RB-C21 (to CR and LR-M) and PID2020-115177RB-C22 (to JM) financed by the Ministerio de Ciencia, Innovación y Universidades (MICIU)/ Agencia Estatal de Investigación (AEI)/10.13039/501100011033/ and by the European Regional Development Fund (ERDF) 'A way to make Europe', and by grant 2024ICT153 (to IP-D) from the Consejo Superior de Investigaciones Científicas (CSIC). HD-C was supported by predoctoral grant PRE2021-099113 (MICIU). LB-M was supported by the A4 Program of the Universidad de Málaga.

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 di ering eciencies. We also identified a functional MOBQ
mobilization region in pC, which could only mobilize this plasmid. Plasmid
pB could be eciently 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., This work was funded by the Spanish Plan Nacional
I+D+i grants PID2020-115177RB-C21 and PID2020-
115177RB-C22 financed by the Ministerio de Ciencia
e Innovación and the Agencia Estatal de Investigación,
MCIN/AEI/10.13039/501100011033/, Spain.

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., This research was supported by project grants PID2020-115177RB-C21 and PID2020-115177RB-C22 financed by the Spanish Ministry of Science and Innovation (MCIN)/Agencia Estatal de Investigación (AEI)/10.13039/501100011033/ and by the European Regional Development Fund (ERDF) “A way to make Europe”. CL-B was supported by the FPI2018-084276 predoctoral grant. Open access was partially funded by grant QUAL21 012 IHSM, Consejería de Universidad, Investigación e Innovación, Junta de Andalucía.