Javier García-Andrade

The RNA silencing enzyme RNA polymerase v is required for plant immunity.

RNA–directed DNA methylation (RdDM) is an epigenetic control mechanism driven by small interfering RNAs (siRNAs) that influence gene function. In plants, little is known of the involvement of the RdDM pathway in regulating traits related to immune responses. In a genetic screen designed to reveal factors regulating immunity in Arabidopsis thaliana, we identified NRPD2 as the OVEREXPRESSOR OF CATIONIC PEROXIDASE 1 (OCP1). NRPD2 encodes the second largest subunit of the plant-specific RNA Polymerases IV and V (Pol IV and Pol V), which are crucial for the RdDM pathway. The ocp1 and nrpd2 mutants showed increases in disease susceptibility when confronted with the necrotrophic fungal pathogens Botrytis cinerea and Plectosphaerella cucumerina. Studies were extended to other mutants affected in different steps of the RdDM pathway, such as nrpd1, nrpe1, ago4, drd1, rdr2, and drm1drm2 mutants. Our results indicate that all the mutants studied, with the exception of nrpd1, phenocopy the nrpd2 mutants; and they suggest that, while Pol V complex is required for plant immunity, Pol IV appears dispensable. Moreover, Pol V defective mutants, but not Pol IV mutants, show enhanced disease resistance towards the bacterial pathogen Pseudomonas syringae DC3000. Interestingly, salicylic acid (SA)–mediated defenses effective against PsDC3000 are enhanced in Pol V defective mutants, whereas jasmonic acid (JA)–mediated defenses that protect against fungi are reduced. Chromatin immunoprecipitation analysis revealed that, through differential histone modifications, SA–related defense genes are poised for enhanced activation in Pol V defective mutants and provide clues for understanding the regulation of gene priming during defense. Our results highlight the importance of epigenetic control as an additional layer of complexity in the regulation of plant immunity and point towards multiple components of the RdDM pathway being involved in plant immunity based on genetic evidence, but whether this is a direct or indirect effect on disease-related genes is unclear.

Arabidopsis ocp3 mutant reveals a mechanism linking ABA and JA to pathogen‐induced callose deposition

In the present study, we evaluated the role of the defense-related gene OCP3 in callose deposition as a response to two necrotrophic fungal pathogens, Botrytis cinerea and Plectosphaerella cucumerina. ocp3 plants exhibited accelerated and intensified callose deposition in response to fungal infection associated with enhanced disease resistance to the two pathogens. A series of double mutant analyses showed potentiation of callose deposition and the heightened disease resistance phenotype in ocp3 plants required the plant hormone abscisic acid (ABA) and the PMR4 gene encoding a callose synthase. This finding was congruent with an observation that ocp3 plants exhibited increased ABA accumulation, and ABA was rapidly synthesized following fungal infection in wild-type plants. Furthermore, we determined that potentiation of callose deposition in ocp3 plants, including enhanced disease resistance, also required jasmonic acid (JA) recognition though a COI1 receptor, however JA was not required for basal callose deposition following fungal infection. In addition, potentiation of callose deposition in ocp3 plants appeared to follow a different mechanism than that proposed for callose β-amino-butyric acid (BABA)-induced resistance and priming, because ocp3 plants responded to BABA-induced priming for callose deposition and induced resistance of a magnitude similar to that observed in wild-type plants. Our results point to a model in which OCP3 represents a specific control point for callose deposition regulated by JA yet ultimately requiring ABA. These results provide new insights into the mechanism of callose deposition regulation in response to pathogen attack; however the complexities of the processes remain poorly understood.

Priming Plant Defence Against Pathogens by Arbuscular Mycorrhizal Fungi

Root colonisation by arbuscular mycorrhizal fungi (AMF) can improve plant resistance/tolerance to biotic stresses. Although this bioprotection has been amply described in different plant systems, the underlying mechanisms remain largely unknown. Besides mechanisms such as improved plant nutrition and competition, experimental evidence supports the involvement of plant defence mechanisms in the observed protection. During mycorrhiza establishment, modulation of plant defence responses occurs upon recognition of the AMF in order to achieve a functional symbiosis. As a consequence of this modulation, a mild, but effective activation of the plant immune responses may occur, not only locally but also systemically. This activation leads to a primed state of the plant that allows a more efficient activation of defence mechanisms in response to attack by potential enemies.

MYB46 Modulates Disease Susceptibility to Botrytis cinerea in Arabidopsis

In this study, we show that the Arabidopsis (Arabidopsis thaliana) transcription factor MYB46, previously described to regulate secondary cell wall biosynthesis in the vascular tissue of the stem, is pivotal for mediating disease susceptibility to the fungal pathogen Botrytis cinerea. We identified MYB46 by its ability to bind to a new cis-element located in the 5′ promoter region of the pathogen-induced Ep5C gene, which encodes a type III cell wall-bound peroxidase. We present genetic and molecular evidence indicating that MYB46 modulates the magnitude of Ep5C gene induction following pathogenic insults. Moreover, we demonstrate that different myb46 knockdown mutant plants exhibit increased disease resistance to B. cinerea, a phenotype that is accompanied by selective transcriptional reprogramming of a set of genes encoding cell wall proteins and enzymes, of which extracellular type III peroxidases are conspicuous. In essence, our results substantiate that defense-related signaling pathways and cell wall integrity are interconnected and that MYB46 likely functions as a disease susceptibility modulator to B. cinerea through the integration of cell wall remodeling and downstream activation of secondary lines of defense.

A deletion in NRT2.1 attenuates Pseudomonas syringae-induced hormonal perturbation, resulting in primed plant defenses

For an efficient defense response against pathogens, plants must coordinate rapid genetic reprogramming to produce an incompatible interaction. Nitrate Trasnporter2 (NRT2) gene family members are sentinels of nitrate availability. In this study, we present an additional role for NRT2.1 linked to plant resistance against pathogens. This gene antagonizes the priming of plant defenses against the bacterial pathogen Pseudomonas syringae pv tomato DC3000 (Pst). The nrt2 mutant (which is deficient in two genes, NRT2.1 and NRT2.2) displays reduced susceptibility to this bacterium. We demonstrate that modifying environmental conditions that stimulate the derepression of the NRT2.1 gene influences resistance to Pst independently of the total level of endogenous nitrogen. Additionally, hormonal homeostasis seemed to be affected in nrt2, which displays priming of salicylic acid signaling and concomitant irregular functioning of the jasmonic acid and abscisic acid pathways upon infection. Effector-triggered susceptibility and hormonal perturbation by the bacterium seem to be altered in nrt2, probably due to reduced sensitivity to the bacterial phytotoxin coronatine. The main genetic and metabolic targets of coronatine in Arabidopsis (Arabidopsis thaliana) remain largely unstimulated in nrt2 mutants. In addition, a P. syringae strain defective in coronatine synthesis showed the same virulence toward nrt2 as the coronatine-producing strain. Taken together, the reduced susceptibility of nrt2 mutants seems to be a combination of priming of salicylic acid-dependent defenses and reduced sensitivity to the bacterial effector coronatine. These results suggest additional functions for NRT2.1 that may influence plant disease resistance by down-regulating biotic stress defense mechanisms and favoring abiotic stress responses.

OCP3 is an important modulator of NPR1-mediated jasmonic acid-dependent induced defenses in Arabidopsis

BackgroundUpon appropriate stimulation, plants increase their level of resistance against future pathogen attack. This phenomenon, known as induced resistance, presents an adaptive advantage due to its reduced fitness costs and its systemic and broad-spectrum nature. In Arabidopsis, different types of induced resistance have been defined based on the signaling pathways involved, particularly those dependent on salicylic acid (SA) and/or jasmonic acid (JA).ResultsHere, we have assessed the implication of the transcriptional regulator OCP3 in SA- and JA-dependent induced defenses. Through a series of double mutant analyses, we conclude that SA-dependent defense signaling does not require OCP3. However, we found that ocp3 plants are impaired in a Pseudomonas fluorescens WCS417r-triggered induced systemic resistance (ISR) against both Pseudomonas syrinagae DC3000 and Hyaloperonospora arabidopsidis, and we show that this impairment is not due to a defect in JA-perception. Likewise, exogenous application of JA failed to induce defenses in ocp3 plants. In addition, we provide evidence showing that the over-expression of an engineered cytosolic isoform of the disease resistance regulator NPR1 restores the impaired JA-induced disease resistance in ocp3 plants.ConclusionsOur findings point to a model in which OCP3 may modulate the nucleocytosolic function of NPR1 in the regulation of JA-dependent induced defense responses.

Mediated Plastid RNA Editing in Plant Immunity

Plant regulatory circuits coordinating nuclear and plastid gene expression have evolved in response to external stimuli. RNA editing is one of such control mechanisms. We determined the Arabidopsis nuclear-encoded homeodomain-containing protein OCP3 is incorporated into the chloroplast, and contributes to control over the extent of ndhB transcript editing. ndhB encodes the B subunit of the chloroplast NADH dehydrogenase-like complex (NDH) involved in cyclic electron flow (CEF) around photosystem I. In ocp3 mutant strains, ndhB editing efficiency decays, CEF is impaired and disease resistance to fungal pathogens substantially enhanced, a process recapitulated in plants defective in editing plastid RNAs encoding NDH complex subunits due to mutations in previously described nuclear-encoded pentatricopeptide-related proteins (i.e. CRR21, CRR2). Furthermore, we observed that following a pathogenic challenge, wild type plants respond with editing inhibition of ndhB transcript. In parallel, rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue. Therefore, NDH complex activity and plant immunity appear as interlinked processes.

Enhanced disease resistance to Botrytis cinerea in myb46 Arabidopsis plants is associated to an early down-regulation of CesA genes

The cell wall is a protective barrier of paramount importance for the survival of plant cells. Monitoring the integrity of cell wall allows plants to quickly activate defence pathways to minimize pathogen entry and reduce the spread of disease. Counterintuitively, however, pharmacological effects as well as genetic lesions that affect cellulose biosynthesis and content confer plants with enhanced resistance against necrotrophic fungi. This kind of pathogens target cellulose for degradation to facilitate penetration and to generate glucose units as a food source. Our results points towards the existence of a transcriptional reprogramming mechanism in genes encoding cellulose synthases (CesAs) that occurs very soon after Botrytis cinerea attack and that results in a temporarily shut down of some CesA genes. Interestingly, the observed coordinated down-regulation of CesA genes is more pronounced, and occurs earlier, in myb46 mutant plants. In the resistant myb46 plants, pathogen infection induces transient down-regulation of CesA genes that concurs with a selective transcriptional reprogramming in a set of genes encoding structural cell wall proteins and extracellular remodelling enzymes. Together with previous indications, our results favour the hypothesis that CesAs are part of a surveillance system of the cell wall integrity that senses the presence of a pathogen and transduces that signal into a rapid transcriptional reprogramming of the affected cell.

Novel disease susceptibility factors for fungal necrotrophic pathogens in Arabidopsis.

Host cells use an intricate signaling system to respond to invasions by pathogenic microorganisms. Although several signaling components of disease resistance against necrotrophic fungal pathogens have been identified, our understanding for how molecular components and host processes contribute to plant disease susceptibility is rather sparse. Here, we identified four transcription factors (TFs) from Arabidopsis that limit pathogen spread. Arabidopsis mutants defective in any of these TFs displayed increased disease susceptibility to Botrytis cinerea and Plectosphaerella cucumerina, and a general activation of non-immune host processes that contribute to plant disease susceptibility. Transcriptome analyses revealed that the mutants share a common transcriptional signature of 77 up-regulated genes. We characterized several of the up-regulated genes that encode peptides with a secretion signal, which we named PROVIR (for provirulence) factors. Forward and reverse genetic analyses revealed that many of the PROVIRs are important for disease susceptibility of the host to fungal necrotrophs. The TFs and PROVIRs identified in our work thus represent novel genetic determinants for plant disease susceptibility to necrotrophic fungal pathogens.

Reciprocal regulation between AtNRT2.1 and AtAMT1.1 expression and the kinetics of NH4+ and NO3− influxes

Our results show that AtNRT2.1 expression has a positive effect on the NH(4)(+) ion influx, mediated by the HATS, as also occurs with AtAMT1.1 expression on the NO(3)(-) ion influx. AtNRT2.1 expression plays a key role in the regulation of AtAMT1.1 expression and in the NH(4)(+) ion influx, differentiating the nitrogen source, and particularly, the lack of it. Nitrogen starvation produces a compensatory effect by AtAMT1.1 when there is an absence of the AtNRT2.1 gene. Our results also show that, in the atnrt2 mutant lacking both AtNRT2.1 and AtNRT2.2, gene functions present different kinetic parameters on the NH(4)(+) ion influx mediated by the HATS, according to the source and availability of nitrogen. Finally, the absence of AMT1.1 also produces changes in the kinetic parameters of the NO(3)(-) influx, showing different V(max) values depending on the source of nitrogen available.