Effector-mediated suppression of plant defense against biotrophs through activation of antagonistic defense against necrotrophs

Plant hormones are small molecules involved in the regulation of plant growth, development, reproduction and stress responses. Salicylic acid (SA) and jasmonates (JA) are essential for the activation of defence responses against pathogens. SA signaling is involved in triggering immunity against biotrophic pathogens while JA activates resistance against necrotrophic pathogens. The SA and JA pathways are mostly antagonistic: elevated biotroph resistance correlates with increased necrotroph susceptibility, and vice versa. Using transcriptomics to look for a functional overlap between plant gene silencing and type III-mediated plant responses we found that genes associated to JA signaling were overrepresented in the overlapping set, more so than SA-related genes. We present here the results of the ensuing analysis, showing effector-mediated activation of the JA pathway as a virulence mechanism, and establishing a novel role for gene silencing in the regulation of the JA pathway.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech - See more at: http://riuma.uma.es/xmlui/handle/10630/5393/submit/12176e8e39815b4d6112192a6815221718868c2f.continue#sthash.RYCQTkwy.dpu

PAMP-triggered immunity against Pseudomonas syringae involves microRNA-mediated regulation of several uncharacterized R genes

Two main types of noncoding small RNA molecules have been found in plants: microRNAs (miRNAs) and small interfering RNAs (siRNAs). They differ in their biogenesis and mode of action, but share similar sizes (20-24 nt). Their precursors are processed by Dicer-Like RNase III (dcl) proteins present in Arabidopsis thaliana, and in their mature form can act as negative regulators of gene expression, being involved in a vast array of plant processes, including plant development, genomic integrity or response to stress. Small-RNA mediated regulation can occurs at transcriptional level (TGS) or at post-transcriptional level (PTGS). In recent years, the role of gene silencing in the regulation of expression of genes related to plant defence responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between the expression profiles of different mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes through the generation of siRNAs. We have also found that the corresponding pri-miRNA is down-regulated after PAMP-perception in a SA-dependent manner. We also demonstrate that plants with altered levels of miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated phenotypes, suggesting a role for this miRNA* in this defence response against bacteria. In addition we identify one of the target genes as a negative regulator of defence response against Pseudomonas syringae.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. MINECO, FEDE

Análisis del solapamiento funcional entre la respuesta de la planta frente a efectores secretados por Pseudomonas syringae y el silenciamiento génico

Las plantas están continuamente sometidas a infecciones por diferentes patógenos. Los patógenos bacterianos usan un complejo sistema de secreción para translocar proteínas efectoras al interior del huésped y suprimir así las respuestas de defensas. En los últimos años, se ha demostrado la participación de ciertos microRNAs en respuestas de defensas de la planta frente a patógenos bacterianos, así como la actuación de erectores bacterianos en rutas reguladas por miRNAs. Sin embargo poco se sabe acerca de cómo la regulación por microRNAs afecta a las respuestas de defensas de la planta frente a patógenos bacterianos, y de hecho menos es conocido sobre el impacto de la supresión de defensas mediada por efectores sobre las rutas reguladas por microRNAs. En este estudio, hemos realizado comparaciones entre análisis de microarrays, con el objetivo de diferenciar genes expresados diferencialmente (DEGs) en Arabidopsis, comunes entre la respuesta de defensa frente a efectores de Pseudomonas syringae y mutantes afectados en la biogénesis de miRNAs. El análisis de la anotación funcional de los DEGS comunes identificados, seguidos de un análisis de expresión mediante RT-qPCR nos ha servido para identificar procesos biológicos regulados por rutas de miRNAs en la interacción planta-patógenoUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Junta de Andalucía, Proyectos de Excelencia P06-CVI-02088; MINECO, Plan Nacional BIO2012-3564

Suppression of HopZ Effector-Triggered Plant Immunity in a Natural Pathosystem

Many type III-secreted effectors suppress plant defenses, but can also activate effector-triggered immunity (ETI) in resistant backgrounds. ETI suppression has been shown for a number of type III effectors (T3Es) and ETI-suppressing effectors are considered part of the arms race model for the co-evolution of bacterial virulence and plant defense. However, ETI suppression activities have been shown mostly between effectors not being naturally expressed within the same strain. Furthermore, evolution of effector families is rarely explained taking into account that selective pressure against ETI-triggering effectors may be compensated by ETI-suppressing effector(s) translocated by the same strain. The HopZ effector family is one of the most diverse, displaying a high rate of loss and gain of alleles, which reflects opposing selective pressures. HopZ effectors trigger defense responses in a variety of crops and some have been shown to suppress different plant defenses. Mutational changes in the sequence of ETI-triggering effectors have been proposed to result in the avoidance of detection by their respective hosts, in a process called pathoadaptation. We analyze how deleting or overexpressing HopZ1a and HopZ3 affects virulence of HopZ-encoding and non-encoding strains. We find that both effectors trigger immunity in their plant hosts only when delivered from heterologous strains, while immunity is suppressed when delivered from their native strains. We carried out screens aimed at identifying the determinant(s) suppressing HopZ1a-triggered and HopZ3-triggered immunity within their native strains, and identified several effectors displaying suppression of HopZ3-triggered immunity. We propose effector-mediated cross-suppression of ETI as an additional force driving evolution of the HopZ family

Obtención de plantas compuestas de olivo mediante transformación con Agrobacterium rhizogenes

La transformación con Agrobacterium rhizogenes ha sido utilizada como herramienta para estudios de genómica funcional en raíces (Collier et al. 2005 Plant Journal 43:449-457; Baranski et al. 2006 Plant Cell Reports 25:190-197). La obtención de plantas compuestas de olivo (sistema radicular transgénico y parte aérea no transgénica), mediante esta técnica, sería de gran ayuda para estudiar la interacción de esta especie con los patógenos de suelo Verticillium dahliae y Rosellinia necatrix. En este trabajo, se presentan las primeras aproximaciones para la transformación de brotes micropropagados de olivo mediante A. rhizogenes. Se han utilizado 2 genotipos procedentes de semilla del cv. Picual, uno con baja capacidad de enraizamiento, P1, y otro, con alta capacidad, P138, y dos cepas de A. rhizogenes: A4, que contiene el plásmido silvestre Ri, y K599, con el plásmido binario pKGWFS7.0-35SP, que incluye el gen marcador gfp. En el caso del genotipo P1, en la fase de co-cultivo con la bacteria, se añadieron al medio 3 mg/l AIB, para facilitar la formación de raíces. En el genotipo P138, se obtuvo un 100% de enraizamiento tanto en el tratamiento control como en el de brotes infectados con la cepa A4; sin embargo, aquéllos inoculados con la cepa K599 sólo alcanzaron un 70% de enraizamiento. Asimismo, se observó que el 61% de las raíces obtenidas tras la infección con K599 mostraron fluorescencia verde bajo el microscopio confocal. En el genotipo P1, el 60% de las plantas control formaron raíces, frente al 10% de plantas infectadas con A4 y ninguna con la cepa K599. La naturaleza transgénica de las raíces obtenidas tras la infección con A4, en ambos genotipos, se evaluará mediante amplificación por PCR del gen RolB.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Genetic Analysis of the Individual Contribution to Virulence of the Type III Effector Inventory of Pseudomonas syringae pv. phaseolicola

Several reports have recently contributed to determine the effector inventory of the sequenced strain Pseudomonas syringae pv. phaseolicola (Pph) 1448a. However, the contribution to virulence of most of these effectors remains to be established. Genetic analysis of the contribution to virulence of individual P. syringae effectors has been traditionally hindered by the lack of phenotypes of the corresponding knockout mutants, largely attributed to a high degree of functional redundancy within their effector inventories. In support of this notion, effectors from Pseudomonas syringae pv. tomato (Pto) DC3000 have been classified into redundant effector groups (REGs), analysing virulence of polymutants in the model plant Nicotiana benthamiana. However, using competitive index (CI) as a virulence assay, we were able to establish the individual contribution of AvrPto1PtoDC3000 to Pto DC3000 virulence in tomato, its natural host, even though typically, contribution to virulence of AvrPto1 is only shown in strains also lacking AvrPtoB (also called HopAB2), a member of its REG. This report raised the possibility that even effectors targeting the same defence signalling pathway may have an individual contribution to virulence, and pointed out to CI assays as the means to establish such a contribution for individual effectors. In this work, we have analysed the individual contribution to virulence of the majority of previously uncharacterised Pph 1448a effectors, by monitoring the development of disease symptoms and determining the CI of single knockout mutants at different stages of growth within bean, its natural host. Despite their potential functional redundancy, we have found individual contributions to virulence for six out of the fifteen effectors analysed. In addition, we have analysed the functional relationships between effectors displaying individual contribution to virulence, highlighting the diversity that these relationships may present, and the interest of analysing their functions within the context of the infection

Analysis of the Role of the Type III Effector Inventory of Pseudomonas syringae pv. phaseolicola 1448a in Interaction with the Plant ▿

In Pseudomonas syringae, the type III secretion system (T3SS) is essential for disease in compatible hosts and for eliciting the hypersensitive response in incompatible hosts. P. syringae pathovars secrete a variable number of type III effectors that form their secretomes. The secretome of Pseudomonas syringae pv. phaseolicola 1448a (Pph1448a) currently includes 22 experimentally validated effectors, one HrpL-regulated candidate for which translocation results have been inconsistent, two translocated candidates for which in planta expression has not been established, one bioinformatically identified candidate, and six candidates that have been experimentally discarded. We analyzed the translocation and/or expression of these and other candidates to complete the Pph1448a effector inventory, bringing this inventory to 27 bona fide effectors, including a new one that does not belong to any of the previously described effector families. We developed a simple process for rapidly making single and double knockout mutants and apply it to the generation of an effector mutant collection that includes single knockouts for the majority of the Pph1448a effector inventory. We also generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the single and double mutant strains as well as strains expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to virulence in bean plants, thus validating this approach for dissecting the contribution of the Pph1448a type III effector inventory to virulence. We also analyzed the effect that the expression of these four effectors from Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) has during its interaction with Arabidopsis thaliana, establishing that AvrB4-1, but not the others, determines a restriction of bacterial growth that takes place mostly independently of the salicylic acid (SA)-signaling pathway

Supresión de la respuesta de defensa disparada por efectores de la familia Hopz de Pseudomonas syringae pertenecientes a la familia Hopz

El sistema de secreción tipo III permite a las bacterias fitopatógenas superar los sistemas de defensa de la planta mediante la translocación de efectores tipo III (T3E) al interior de la célula vegetal. Jones y Dangl en 2006 propusieron el modelo de zig-zag, que ha sido aceptado universalmente como patrón de evolución de los efectores bacterianos y la resistencia de las plantas. Sin embargo, la evolución de familias de efectores es raramente explicada teniendo en cuenta que la presión negativa debida a un disparo de defensa ETI (effector-triggered immunity) en un hospedador puede ser compensada por una actividad supresora de ETI de un efector de la misma estirpe. La familia de efectores HopZ de P. syringae es una de las más diversas y mejor caracterizadas desde un punto de vista evolutivo. Esta familia muestra una alta tasa de transferencia horizontal entre patovares, y pequeños cambios en las secuencias de sus miembros permiten evitar el reconocimiento por parte de la planta sin alterar su función, un proceso llamado patoadaptación (Ma et al., 2006). Nuestros resultados muestran que ciertos efectores de la familia HopZ no disparan inmunidad en sus respectivos patosistemas, mientras que su mutación normalmente provoca una pequeña atenuación en el crecimiento bacteriano. Sin embargo, la expresión heteróloga de esos erectores produce una respuesta de defensa en el mismo hospedador, indicando que el hospedador puede detectar su presencia y activar la inmunidad. De esta forma la estirpe que codifica el effector HopZ impide el disparo de inmunidad probablemente mediante la actividad de supresión de otros efectores. Identificamos los erectores que suprimen la resistencia disparada por HopZ3 y proponemos la supresión cruzada de ETI como una fuerza alternativa que dirige la evolución de la familia de efectores HopZ.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. MINECO BIO2012-35641. Plan Propio de Investigación de la UMA. FEDE

A Comparative Transcriptome Analysis of Avocado Embryogenic Lines Susceptible or Resistant to Rosellinia necatrix Exudate

Avocado embryogenic cultures were selected for resistance to the culture filtrate (CF) of Rosellinia necatrix, the causal agent of White Root Rot disease. A resistant callus line was obtained through recurrent selections in progressively increasing concentrations of fungal CF (from 60% to 80%). RNA sequencing (RNA-Seq) technology was used to compare the transcriptomic profiles of the avocado embryogenic-callus-resistant line L3 (capable to survive in the presence of 80% CF) and control line AN-9 (not exposed to CF), after 24 h of growth in a medium containing 40% CF. A total of 25,211 transcripts were obtained, of which 4,918 and 5,716 were differentially expressed in the resistant and control line, respectively. Interestingly, exposure of embryogenic callus lines to 40% of R. necatrix exudates induced genes previously reported to be related to avocado defense against fungal diseases (lignin biosynthesis, Pathogenesis Related (PR) proteins, WRKY (WRKYGQK) Transcription Factor (TF), NAC (NAM, ATAF1/2, and CUC2) TF, proteinase inhibitors and Ethylene Response Transcription Factor (ERF), among others), which were accumulated in greater amounts in the resistant line in comparison to the susceptible one. This research will contribute to the understanding of avocado defense against this pathogen, thereby aiding in the selection of resistant avocado rootstocks

Papel del silenciamiento genético en la regulación de genes R durante la interacción con Pseudomonas syringae

El silenciamiento génico es un mecanismo de regulación de la expresión génica mediado por pequeños RNAs. En plantas, los precursores de estos pequeños RNAs, son procesados por proteinas Dicer-Like (RNAse III). Existen dos tipos principales de pequeños RNAs involucrados en silenciamiento génico: Los pequeños RNAs interferentes (siRNAs) y los microRNAs (miRNAs), difiriendo estos en su biogénesis y modo de acción pero compartiendo tamaños similares (20-24 nt). Esta regulación mediada por pequeños RNAs puede ocurrir tanto a nivel transcripcional (TGS) como post-transcripcional(PTGS). Durante el proceso de infección, las plantas modulan la expresión de una variedad de genes implicados en la respuesta de defensa, donde recientemente se ha demostrado que los pequeños RNAs desempeñan un papel fundamental. El análisis de los perfiles de expresión de diferentes mutantes afectados en la biogénesis de pequeños RNAs y plantas infectadas con Pseudomonas syringae pathovar tomato DC3000, nos ha permitido identificar una serie de genes “R” no caracterizados (TIR-NBS-LRR) expresados diferencialmente en ambas condiciones. Con el uso de diferentes herramientas bioinformáticas, identificamos un miRNA* de 22 nt como potencial regulador de la expresión de dichos genes “R” a través de la generación de siRNAs. Hemos demostrado que la expresión del precursor de dicho miRNA* (pri-miRNA) es reprimida durante la infección tras la detección de Patrones Moleculares Asociados a Patogenos (PAMPs) y de una forma dependiente del Ácido Salicílico (SA). Además hemos observado que plantas con niveles alterados de dicho miRNA* muestran fenotipos alterados de PTI, lo cual sugiere un papel del miRNA* en este mecanismo de defensa frente a la bacteria. Finalmente hemos identificado uno de los genes diana de este miRNA como un regulador negativo de a respuesta de defensa frente a Pseudomonas syringae.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech