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

A bacterial acetyltransferase targets the protein kinase ZIP1, a positive regulator of plant immunity

Pseudomonas syringae is a model bacterial pathogen that penetrates the leaf to reach the plant apoplast, where it replicates causing disease. In order to do that, the pathogen must interfere and suppress a two-tiered plant defense response: PTI (PAMP-Triggered Immunity, or basal resistance) and ETI (Effector-Triggered Immunity). P. syringae uses a type III secretion system to directly deliver effector proteins inside the plant cell cytosol, many of which are known to suppress PTI, some of which are known to trigger ETI, and a handful of which are known to suppress ETI. Bacterial infection can also trigger a systemic plant defense response that protects the plant against additional pathogen attacks known as SAR (Systemic Acquired Resistance). We are particularly interested in the molecular and cellular mechanisms involved in effector-mediated defense evasion by P. syringae, in particular those involved in the suppression of ETI and SAR, and/or mediation of hormone signaling. Here we present data describing effector-mediated interference with plant immunity, by means of acetylation of a key positive regulator of local and systemic responses. Our work identifies a novel plant target for effector function, and characterizes its function. This work illustrates how analyzing the means by which a given effector interferes with its target can provide novel information regarding eukaryotic molecular mechanisms.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. MINECO BIO2015-64391R y FEDE

The bacterial effector HopZ1a acetylates ZIP1 kinase to suppress Arabidopsis defence responses

During the plant-pathogen interaction, disease or resistance are determined in the plant by a series of molecular events. The plant detects Pathogen-Associated Molecular Patterns (PAMPs), such as flagellin, triggering a defence response called PTI (PAMP-Triggered Immunity). Bacterial pathogens can in turn suppress such defence response through the translocation into the plant cell cytosol of virulence proteins, called effectors, via a Type Three Secretion System (T3SS). In resistant plants, intracellular receptors known as R proteins recognize these effectors, triggering a second line of defence, more specific and intense, called ETI (Effector-Triggered Immunity), which usually leads to programmed cell death known as HR (Hypersensitive Response). Pseudomonas syringae is a phytopathogenic bacterium whose virulence depends on a T3SS and its effector repertoire. Some strains include HopZ1a, an unusual effector which is able to suppress in Arabidopsis both local (PTI and ETI), and systemic (SAR, for Systemic Acquired Resistance) defences, by means of its acetyltransferase activity. In resistant Arabidopsis plants, HopZ1a acetylates the ZED1 pseudokinase, which is proposed to function as a decoy mimicking HopZ1a target in the plant: ZED1 modification activates an R-protein (ZAR1) to trigger HopZ1a-dependent ETI. None of the Arabidopsis proteins proposed to date as HopZ1a targets is a kinase, nor fully explains the effector´s defence suppression abilities. In this work we identify an Arabidopsis kinase that functions as a positive regulator of PTI, ETI and SAR, which interacts with HopZ1a and is acetylated by this effector in lysine residues essential for its kinase activity. Further, HopZ1a can specifically suppress the defence phenotypes resulting from ZIP1 expression in Arabidopsis. We propose that ZIP1 acetylation by HopZ1a interferes with its kinase activity, and consequently with positive defence signalling.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

Salmonella typhimurium SifA effector protein requires its membrane-anchoring C-terminal hexapeptide for its biological function

SifA is a Salmonella typhimurium effector protein that is translocated across the membrane of the Salmonella-containing vacuole by the Salmonella pathogenicity island 2-encoded type III secretion system. SifA is necessary for the formation ofSalmonella-induced filaments and for the maintenance of the vacuolar membrane enclosing the pathogen. We have investigated the role of the C-terminal hexapeptide of SifA as a potential site for membrane anchoring. An S. typhimuriumstrain carrying a deletion of the sequence encoding this hexapeptide (sifAΔ6) was found to be attenuated for systemic virulence in mice. In mouse macrophages, sifAΔ6 mutant bacteria displayed a reduced association with vacuolar markers, similar to that of sifA null mutant bacteria, and exhibited a dramatic replication defect. Expression of SifA in epithelial cells results in the mobilization of lysosomal glycoproteins in large vesicular structures and Sif-like tubules. This process requires the presence of the C-terminal hexapeptide domain of SifA. Ectopic expression of truncated or mutated versions of SifA affecting the C-terminal hexapeptide revealed a strong correlation between the membrane binding capability and the biological activity of the protein. Finally, the eleven C-terminal residues of SifA are shown to be sufficient to target the Aequorea green fluorescent protein to membranes. Altogether, our results indicate that membrane anchoring of SifA requires its C-terminal hexapeptide domain, which is important for the biological function of this bacterial effector

The bacterial effector HopAF1 interacts with Arabidopsis MKK4/5

Pseudomonas syringae is a phytopathogenic bacterium whose virulence depends on a Type III Secretion System and its effector (T3E) repertoire, which is translocated into the host cell cytosol suppressing both basal and effector-triggered Immunity (ETI). HopAF1 is a T3E that suppresses several plant immunity phenotypes, including flg22-induced ethylene (ET) production. HopAF1 is mainly located in the plasma membrane (PM), where it interacts with plant proteins MTN1/2 that participate in ET biosynthesis. However, PM localization is not essential for HopAF1 ability to repress ET accumulation. While seeking for additional HopAF1 interactors in the plant, we have identified MAP kinase kinases MKK4/5. The MAP kinase module including MKK4/5 and MPK3/6 positively regulates 1-aminocyclopropane-1-carboxylic acid synthase (ACS) activity, a rate-limiting and major regulatory step in stress-induced ET production. MKK4/5-dependent phosphorylation directly stabilizes ACS2/6 and indirectly induces ACS transcription. We originally identified HopAF1-MKK4 interaction through an MS-based screening, and have now confirmed it by pull-down and BIFC, while obtaining variable results using FRET-FLIM. Moreover, we are monitoring ACS2/6 expression levels by qRT-PCR, in the presence and absence of HopAF1. We propose that HopAF1 interference with MKK4/5 is likely to contribute to the suppression of ET production, by altering MKK4/5-dependent regulation of ACS activity.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

El efector bacteriano HopZ1a acetila ZIP1 para suprimir las respuestas de defensa en Arabidopsis

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

El regulador positivo de defensas ZIP1 es acetilado por el efector bacteriano HopZ1a para suprimir las respuestas de defensa en Arabidopsis

Durante la interacción planta-patógeno, el éxito en la colonización y desarrollo de la enfermedad del patógeno viene determinado por su capacidad de evadir y/o suprimir las respuestas de defensas disparadas por la planta. La planta detecta patrones moleculares, como por ejemplo flagelina o EF-Tu, desencadenando una defensa llamada PTI , que algunas bacteriaspatógenas pueden suprimir mediante la proteínas de virulencia (efectores) . En plantas resistentes, la célula detecta dichos efectores mediante receptores intracelulares, proteínas R, desencadenando una segunda línea de defensa, llamada ETI , que conduce a una muerte celular programada conocida como HR. La activación de estas respuestas de defensa a nivel local inducen la Respuesta Sistémica Adquirida (SAR) en hojas distales. Pseudomonas syringae es una bacteria fitopatógena, capaz de infectar una gran variedad de plantas, incluyendo Arabidopsis. Su virulencia depende de su capacidad de translocar efectores Tipo III (T3Es), directamente al citosol de la célula hospedadora mediante un Sistema de Secreción Tipo III (T3SS). HopZ1a es un efector capaz de suprimir en Arabidopsis defensas locales (PTI y ETI) y sistémicas (SAR) por medio de su actividad acetiltransferasa. En bacterias patógenas de animales, los homólogos de HopZ1a inactivan por acetilación a quinasas del hospedador implicadas en señalización positiva de defensa. En plantas resistentes, HopZ1a acetila la pseudoquinasa ZED1, propuesta como señuelo que imita al objetivo HopZ1a en la planta, para desencadenar la ETI dependiente de HopZ1a. Estos datos sugieren una quinasa reguladora positiva de defensa, tanto a nivel local como sistémico, como probable diana de HopZ1a. En este trabajo identificamos a ZIP1, una que funciona como un regulador positivo de PTI, ETI y SAR, que interactúa con HopZ1a y es acetilada por este efector en una lisina esencial para su actividad quinasa.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Bistable expression of relevant genes for Pseudomonas syringae virulence

Backgrounds Heterogeneity or phenotypic variation has been known to take place in microbial clonal populations for decades. Under certain regulatory circuits, heterogeneity in gene expression can be enhanced leading to a bimodal expression profile in homogeneous environments. This process is known as bistability. The relevance of these processes has been demonstrated in Salmonella enterica and other human pathogen in the establishment of antibiotic persistence, and it has been shown to affect virulence genes, and to be linked to the establishment of chronic persistence. Nevertheless, little is known about the occurrence or impact of these processes in the adaptation of bacteria to non-animal host. Objectives To address the question of whether there is phenotypic heterogeneity in the expression of genes relevant for adaptation to a non-animal host of the model plant pathogen Pseudomonas syringae. Methods Transcriptional chromosome-located fusion to reporter genes encoding fluorescent protein were constructed in P. syringae pv. phaseolicola. Genes selected were several encoding different elements of a type III secretion system (T3SS) and flagellin, since motility has been reported as counter-regulated with the T3SS, Expression from these genes was analyzed using single-cell analysis methods, such as flow cytometry and fluorescent microscopy. Conclusions We recently showed expression of T3SS genes is phenotypically heterogeneous in planta and becomes bistable under certain laboratory conditions through the action of a double regulatory loop on the transcriptional activator HrpL. We present here single cell analyses of the gene encoding flagellin, as well as its responses to different regulatory proteins.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec