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Trabajo presentado a la XVI Reunión de Biología Molecular de Plantas-Meeting of Plant Molecular Biology (RBMP) celebrada en Sevilla entre el 14 y el 16 de diciembre de 2022., Plants possess a battery of dedicated intracellular immune receptors known as nucleotide-binding leucine-rich repeat proteins (NLRs) involved in recognition of pathogen derived efectors and activation of efector-triggered immunity (ETI). In a recent work, we report a two-tiered regulatory network in Arabidopsis mediated by a microRNA (miR825-5p) and a wave of secondary NLRderived phased small RNAs (phasi-NLRs) involved in silencing dozens of NLR genes encoding Toll/interleukin-1 NLRs (TNLs). In our model, targeting of MIST1 (microRNA-silenced TNL1) transcripts by miR825-5p, triggers the generation of phasi-NLRs that subsequently silence a wide network of TNL encoding genes and reinforce the silencing of MIST1 (López-Márquez et al., 2021). Although miRNA and phasi-NLR mediated regulation of NLRs has been characterized to some extent, the current knowledge regarding how these miRNA-target-phasiRNA networks are modulated and their contribution to the immune response during diferent developmental stages is still scarce (Deng et al., 2018; Cui et al., 2019). Thus, through generation of GUS-based A. thaliana reporter lines we have characterized the expression pattern of both genes (MIR825 and MIST1) as well as the domain activity of miR825-5p. Moreover, a comprehensive analysis of RNA and small RNA-seq datasets, as well as the use of diferent orthogonal approaches, unravelled a hitherto unknow connection between miR825-5p/phasi-NLR mediated regulation of NLRs and plant development. Further analysis also supports the notion that the action of this regulatory mechanism is restricted to control NLR expression in an organ specifc way, mainly operating in leaves of the model plant. At present, we are working to understand how the changes on miRNA/phasi-NLR levels during developmental stages modulate the strength of the immune response and the biological impact of this developmental-dependent regulation on plant health. In summary, our results point to a developmentally regulated sRNA-based control of NLR expression in Arabidopsis and shed light on how levels of these regulatory molecules (miRNAs and phasi-NLRs) are modulated during development or in an organ specifc manner to fne-tune NLR expression., This work was supported by Project Grant RTI2018-095069-B-I00 fnanced by MCIN/AEI/10.13039/501100011033/ and FEDER, and by Project Grant PY18-2398 fnanced by Junta de Andalucía. ADE was funded by a FPU Grant (Predoctoral Fellowship from the Spanish Ministerio de Ciencia, Innovación y Universidades; FPU17/03520).

Additional file 1. Supplementary methods, figures and table.

Pseudomonas syringae es un patógeno bacteriano modelo capaz de infectar y desarrollar la enfermedad en una gran variedad de plantas. Para ello, P. syringae utiliza un sistema de secreción de tipo III para translocar proteínas efectoras dentro del citosol de la célula vegetal, muchas de las cuales suprimen la defensa basal PTI (PAMP-Triggered Immunity), disparada por el reconocimiento de patrones moleculares asociados a patógenos (PAMPs). Algunos de esos efectores suprimen la ETI (Effector-Triggered Immunity), respuesta de defensa más específica e intensa desencadenada por el reconocimiento de efectores y cuyo resultado final es la muerte celular programada llamada HR (Hypersensitive Response). Además de las respuestas locales, PTI y ETI, la planta puede activar una respuesta sistémica de defensa SAR (Systemic Acquire Resistance) que la protege contra ataques posteriores del patógeno. El efector HopZ1a es una acetiltransferasa perteneciente a la superfamilia de efectores YopJ, capaz de suprimir las respuestas de defensa PTI, ETI y SAR en Arabidopsis. De entre todas las diana descritas para este efector, ZED1 es una pseudokinasa cuya única función conocida es ser acetilada por HopZ1a, activando así a una proteína R llamada ZAR1, y desencadenando ETI. Esta función de decoy de ZED1, junto a la existencia de interacciones entre otros efectores de la familia YopJ con kinasas de animales, nos lleva a pensar que una kinasa pueda ser diana de HopZ1a. Nuestro trabajo identifica a la kinasa ZIP1, regulador positivo de defensa, como diana de HopZ1a. Nosotros caracterizamos su interacción y demostramos que HopZ1a acetila a ZIP1 en una lisina esencial para su función kinasa para suprimir las defensas de la planta. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Durante un estrés biótico, las plantas modulan la expresión de una batería de genes involucrados en la respuesta de defensa, proceso donde recientemente se ha determinado el papel esencial que desempeña el silenciamiento génico. El silenciamiento génico es un mecanismo de regulación de la expresión génica, donde destacan como principales moléculas efectoras los pequeños RNAs (sRNAs). En plantas, estos sRNAs, son clasificados en pequeños RNAs interferentes (siRNAs) o microRNAs (miRNAs), presentando tamaños similares (20-24 nt) pero difiriendo en su biogénesis y modo de acción. Los miRNAs son pequeños RNAs de cadena sencilla que actúan regulando negativamente la expresión de genes, mediante su unión al complejo RISC (Rna Induced Silencing Complex) y en una forma dependiente de secuencia. En nuestro laboratorio, mediante el análisis de datos transcriptómicos, y el uso de herramientas bioinformáticas, identificamos un miRNA* de 22 nt como potencial regulador de la expresión de genes de resistencia (“R”) del tipo TIR-NBS-LRR. Posteriormente hemos validado dicha regulación y caracterizado los patrones de expresión tanto del Pri-miRNA como de un gen “R” regulado por este, en diferentes tejidos y estadios del desarrollo, así como durante la interacción con P. syringae. Por otro lado, hemos generado plantas transgénicas que presentan niveles alterados del miRNA* (incremento y reducción) y hemos observado que muestran fenotipos alterados de PTI y una mayor/menor colonización de P. syringae. Finalmente hemos identificado la producción de sRNAs (phasiRNAs) a partir del gen de resistencia, en una forma dependiente de miRNA*-RDR6-DCL4, pudiendo estos sRNAs secundarios regular otros transcritos de la misma familia de genes de resistencia. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

17 páginas.-- 6 figuras.-- 5 tablas.-- 89 referencias.-- Additional Supporting Information may be found in the online version of this article at the publisher’s web-site, The adaptation capacity of olive trees to different environments is well recognized. However, the presence of microorganisms in the soil is also a key factor in the response of these trees to drought. The objective of the present study was to elucidate the effects of different arbuscular mycorrhizal (AM) fungi coming from diverse soils on olive plant growth and water relations. Olive plants were inoculated with native AM fungal populations from two contrasting environments, that is, semi-arid – Freila (FL) and humid – Grazalema (GZ) regions, and subjected to drought stress. Results showed that plants grew better on GZ soil inoculated with GZ fungi, indicating a preference of AM fungi for their corresponding soil. Furthermore, under these conditions, the highest AM fungal diversity was found. However, the highest root hydraulic conductivity (Lp) value was achieved by plants inoculated with GZ fungi and growing in FL soil under drought conditions. So, this AM inoculum also functioned in soils from different origins. Nine novel aquaporin genes were also cloned from olive roots. Diverse correlation and association values were found among different aquaporin expressions and abundances and Lp, indicating how the interaction of different aquaporins may render diverse Lp values., The study was supported by the Ministry of Economy and Competitiveness of Spain (Juan de la Cierva Program) and Junta de Andalucía (P10-CVI-5920 project) for research funding.

Bacterial microcolonies with heterogeneous sizes are formed during colonization of Phaseolus vulgaris by Pseudomonas syringae. Heterogeneous expression of structural and regulatory components of the P. syringae type III secretion system (T3SS), essential for colonization of the host apoplast and disease development, is likewise detected within the plant apoplast. T3SS expression is bistable in the homogeneous environment of nutrient-limited T3SS-inducing medium, suggesting that subpopulation formation is not a response to different environmental cues. T3SS bistability is reversible, indicating a non-genetic origin, and the T3SSHIGH and T3SSLOW subpopulations show differences in virulence. T3SS bistability requires the transcriptional activator HrpL, the double negative regulatory loop established by HrpV and HrpG, and may be enhanced through a positive feedback loop involving HrpA, the main component of the T3SS pilus. To our knowledge, this is the first example of phenotypic heterogeneity in the expression of virulence determinants during colonization of a non-mammalian host. Ministerio de Ciencia e Innovación BIO2009-11516, AGL2010-22287-C02-2 Ministerio de Economía y Competitividad BIO2012-35641, BIO2015-64391- R

Trabajo presentado en el Rhizosphere 4: Stretching the interface of life, celebrado en Maastricht del 21 al 25 de junio de 2015., Plant disease epidemics result from interactions of a susceptible host, a virulent pathogen and conducive environment. Shifts in any component can change disease expression to still unknown directions. Verticillium wilt (VW) of olive caused by the soilborne fungus Verticillium dahliae (Vd) is of major concern for olive industry in the Mediterranean basin. We carried out experiments using olive cvs. Picual and Arbequina, which grew in soil infested by the defoliating (D) or non-defoliating (ND) Vd pathotypes at 20, 24 and 28ºC and CO2 levels of 386, 550 and 750 ppm, representative of current and future SRES-IPCC A2 and B2 scenarios for southern Spain. Surface response models quantified the combined effects of temperature and CO2 on VW intensity and plant growth, demonstrating a differential effect of biotic and abiotic factors. Optimum VW development occurred at 20-24°C and current CO2, being faster and severe in 'Picual'/D. Raising CO2 delayed VW and Vd infection. The transcriptomic profile of olive cultivars in response to Vd infection and the three climate SRES scenarios was assessed using a 12-plex microarray of 37,449 olive unigenes (OLEAGEN project). Main differences in the expression profiles were due to olive genotype, followed by the climatic conditions at the three SRES scenarios and in a lesser extent by Vd infection. Thus, plants growing at current climate showed higher number of differentially expressed genes compared to plants growing at SRES-IPCC A2 and B2 scenarios with a general trend to decrease the number of significantly transcribed genes as temperature and CO2 increased. Furthermore, a higher number of genes (up to 10x) were expressed differentially in 'Arbequina' than in 'Picual' in response to environmental changes as well as to infection by the D pathotype with a low number of genes being common for both cultivars.