Search

Global population growth makes it necessary to increase agricultural production yields. However, climate change impacts and diseases caused by plant pathogens are challenging modern agriculture. Therefore, it is necessary to look for alternatives to the excessive use of chemical fertilizers and pesticides. The plant microbiota plays an essential role in plant nutrition and health, and offers enormous potential to meet future challenges of agriculture. In this context, here we characterized the antifungal properties of the rhizosphere bacterium Pantoea agglomerans 9Rz4, which is active against a broad spectrum of plant pathogenic fungi. Chemical analyses revealed that strain 9Rz4 produces the antifungal herbicolin A and its biosynthetic gene cluster was identified and characterized. We found that the only acyl-homoserine lactone-based quorum sensing system of 9Rz4 modulates herbicolin A gene cluster expression. No role of plasmid carriage in the production of herbicolin A was observed. Plant assays revealed that herbicolin A biosynthesis does not affect the root colonization ability of P. agglomerans 9Rz4. Current legislative restrictions are aimed at reducing the use of chemical pesticides in agriculture, and the results derived from this study may lay the foundations for the development of novel biopesticides from rhizosphere microorganisms.

Resumen del poster presentado en: I Jornada de Juventud Investigadora Estación Experimental del Zaidín. EEZ Granada; 20 noviembre (2022), The plant microbiome plays a crucial role in plant growth and protection against phytopathogens through multiple mechanisms, for example, improving nutrient acquisition and disease resistance. The bacterium Pantoea agglomerans 9Rz4 was isolated from the rhizosphere of oilseed rape due to its ability to antagonise the growth of different fungal pathogens of agronomic relevance [1]. However, the mechanisms behind these antifungal properties are unknown. By screening a library of random transposon mutants, we have isolated mutants of 9Rz4 deficient in their antifungal properties. The combination of de novo genome sequencing and chemical analysis allowed us to determine that the strain 9Rz4 produces the non-ribosomal peptide antifungal herbicolin A, as well as to identify and characterise the corresponding biosynthetic cluster. The expression of the herbicolin A gene cluster is maximal in stationary phase of growth. A quorum sensing mutant of 9Rz4, defective in the biosynthesis of acyl-homoserine lactones, shows reduced production of herbicolin A and, as a consequence, a lower antifungal activity. In planta assays revealed that 9Rz4 does not negatively impact on plant growth, but also that herbicolin A does not affect rhizosphere colonisation by the 9Rz4. Assessing bacterial potential as biopesticides is fundamental for transitioning to green alternatives to agrochemicals. Pantoea agglomerans 9Rz4 is active against a broad range of fungal phytopathogens and our results lay the foundation for the possible future use of this bacterium as a biocontrol agent in agriculture., This work was supported by a grant from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 (PID2019-103972GA-I00) to Miguel A. Matilla]

Resumen de la comunicación oral presentada en: I Jornada de Juventud Investigadora Estación Experimental del Zaidín. EEZ Granada; 20 noviembre (2022), Plant pathogens contain many more chemoreceptors than the bacterial average. Sequence analysis reveal that many chemoreceptor families are specific for plant associated bacteria. However, there is only very scarce information available on the function or ligands recognized by plant pathogens. To address this question, we use Pectobacterium atrosepticum SCRI1043 as model organism, since it is among the top 10 most relevant bacterial phytopathogens and causes soft rot diseases in many agriculturally relevant crops [1]. It contains 36 chemoreceptors, mostly of unknown function. To identify chemoreceptor function, the individual chemoreceptor sensor domains were overexpressed in Escherichia coli and purified by affinity chromatography. Purified protein was submitted to differential scanning fluorimetry based thermal shift assays to screen different compound arrays for potential ligands. The compounds that increased the midpoint of protein unfolding were tested by isothermal titration calorimetry to derive binding constants. We identify the function of three chemoreceptors encoded in a gene cluster containing four paralogous genes. PacG (Pectobacterium atrosepticum chemoreceptor G) sensor domain bound with negative cooperativity agmatine, feruloylagmatine and p-coumaroylagmatine, whereas the PacH sensor domain recognized the plant hormone salicylic acid, 4-clorobenzoyc acid, 4-methoxybenzoic acid, 4- hydroxibenzoic acid, benzoate, 4-nitrobenzoic acid and protocatechuate. Vanillin and salicylic acid interacted with the PacI sensor domain. Interestingly, a large part of these ligands are plant-derived compounds like salicylic acid, vanillin and the agmatine derivatives. The latter compounds were found to be secreted by plants in response to bacterial infection [2]. Two receptors recognize salicylic acid, a plant hormone that controls many physiologic features. In addition, its cellular accumulation induces the upregulation of pathogenesis-related proteins. It has also been demonstrated that plant infection by fungi triggers the release of vanillin [3]. Subsequent studies will reveal whether and to which degree P. atrosepticum responds to the ligands identified and whether these responses are associated with virulence., This work was supported by Ministerio de Ciencia e Innovación – Agencia Estatal de Investigación, grant number PID2020-12612GB-I00 with predoctoral grant number PRE2021-096885

Comunicación oral presentada en: Gordon Research Conference 2022 on Molecular Basis and Applications of Microbial Signal Detection, Transduction and Response. Ventura, California EEUU, 25-30 sep (2022), Bacteria adapt to environmental changes through multiple signal transduction pathways that sense a broad diversity of signals. Some of these signals can serve as nutrient and energy sources, whereas others provide information about the environmental niche. Indole-3-acetic acid (IAA) is the main auxin phytohormone and modulates plant growth and development. However, IAA is synthesized by species that belong to all domains of life and growing evidence supports that acts as a signaling molecule regulating a variety of bacterial physiological processes. Here, we investigate the role of IAA in the regulation of antibiotic production and chemotaxis in two plant-associated bacteria. We showed that IAA inhibits the synthesis of the antibiotic andrimid though its binding to the pathwayspecific regulator AdmX. This binding causes conformational changes in AdmX that result in the inhibition of transcription of the andrimid biosynthetic cluster. We also found that IAA serves as a chemoattractant in a IAA degrading bacterium. This chemotactic behaviour was independent of IAA metabolism. Comparative genomics, mutagenesis and phenotypic screening allowed us to identify the chemoreceptor responsible for auxin chemotaxis. Our results contribute to highlighting the role of IAA as an intra- and interkingdom signal molecule, as well as to deciphering the corresponding molecular mechanisms.

Resumen del poster presentado en: XIII Reunión del Grupo de Microbiología Molecular de La SEM. Granada, 7-9 septiembre (2022), This work was supported by grants PID2019-103972GA-I00 (to M.A.M.), PID2020-116261GBI00 (to J.A.G.) and PID2020-112612GB-I00 (to T.K.) from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033, grant P18-FR-1621 (to T.K.) from the Junta de Andalucía.

Resumen del poster presentado en: XIII Reunión del Grupo de Microbiología Molecular de La SEM. Granada, 7-9 septiembre (2022), This work was supported by grants PID2019-103972GA-I00 (to M.A.M.) and PID2020-112612GBI00 (to T.K.) from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 and grant P18-FR-1621(to T.K.) from the Junta de Andalucía.

Resumen del poster presentado en: XIII Reunión del Grupo de Microbiología Molecular de La SEM. Granada, 7-9 septiembre (2022), This work was supported by grants PID2019-103972GA-I00 (to M.A.M.) and PID2020-112612GBI00 (to T.K.) from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 and grant P18-FR-1621(to T.K.) from the Junta de Andalucía. A.R. was supported by the Ramon y Cajal R&D&i Programme (RYC2019-026481-I) from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigaci _on/10.13039/501100011033 y FSE ‘El FSE invierte en tu futuro

Resumen del poster presentado en: XIII Reunión del Grupo de Microbiología Molecular de La SEM. Granada, 7-9 septiembre (2022)

Resumen del poster presentado en: XIII Reunión del Grupo de Microbiología Molecular de La SEM. Granada, 7-9 septiembre (2022)

Conferencia invitada on line presentada en: Receptor Zoomfest. 13-14 Julio (2022), Acetylcholine is a central biological signal molecule present in all kingdoms of life. In humans, acetylcholine is the primary neurotransmitter of the peripheral nervous system; it mediates signal transmission at neuromuscular junctions. Here, we show that the opportunistic human pathogen Pseudomonas aeruginosa exhibits chemoattraction toward acetylcholine over a concentration range of 1 mM to 100 mM. The maximal magnitude of the response was superior to that of many other P. aeruginosa chemoeffectors. We demonstrate that this chemoattraction is mediated by the PctD (PA4633) chemoreceptor. Using microcalorimetry, we show that the PctD ligand-binding domain (LBD) binds acetylcholine with a equilibrium dissociation constant (KD) of 23 mM. It also binds choline and with lower affinity betaine. Highly sensitive responses to acetylcholine and choline, and less sensitive responses to betaine and L-carnitine, were observed in Escherichia coli expressing a chimeric receptor comprising the PctD-LBD fused to the Tar chemoreceptor signaling domain. We also identified the PacA (ECA_RS10935) chemoreceptor of the phytopathogen Pectobacterium atrosepticum, which binds choline and betaine but fails to recognize acetylcholine. To identify the molecular determinants for acetylcholine recognition, we report high-resolution structures of PctD-LBD (with bound acetylcholine and choline) and PacA-LBD (with bound betaine). We identified an amino acid motif in PctD-LBD that interacts with the acetylcholine tail. This motif is absent in PacA-LBD. Significant acetylcholine chemotaxis was also detected in the plant pathogens Agrobacterium tumefaciens and Dickeya solani. To the best of our knowledge, this is the first report of acetylcholine chemotaxis and extends the range of host signals perceived by bacterial chemoreceptors.