Non-Recognition-of-BTH4, an Arabidopsis Mediator Subunit Homolog, Is Necessary for Development and Response to Salicylic Acid

[EN] Salicylic acid (SA) signaling acts in defense and plant development. The only gene demonstrated to be required for the response to SA is Arabidopsis thaliana NON-EXPRESSER OF PATHOGENESIS-RELATED GENE 1 (NPR1), and npr1 mutants are insensitive to SA. By focusing on the effect of analogs of SA on plant development, we identified mutants in additional genes acting in the SA response. In this work, we describe a gene necessary for the SA Non-Recognition-of-BTH4 (NRB4). Three nrb4 alleles recovered from the screen cause phenotypes similar to the wild type in the tested conditions, except for SA-related phenotypes. Plants with NRB4 null alleles express profound insensitivity to SA, even more than npr1. NRB4 null mutants are also sterile and their growth is compromised. Plants carrying weaker nrb4 alleles are also insensitive to SA, with some quantitative differences in some phenotypes, like systemic acquired resistance or pathogen growth restriction. When weak alleles are used, NPR1 and NRB4 mutations produce an additive phenotype, but we did not find evidence of a genetic interaction in F1 nor biochemical interaction in yeast or in planta. NRB4 is predicted to be a subunit of Mediator, the ortholog of MED15 in Arabidopsis. Mechanistically, NRB4 functions downstream of NPR1 to regulate the SA response.This work was supported by the "Ministerio de Economia y Competitividad" (MINECO) of Spain (Grant BIO201018896 to P.T., a Junta de Ampliacion de Estudios-Consejo Superior de Investigaciones Cientificas Fellowship to J.V.C., and a Formacion del Personal Investigador-MINECO to A.D.) and "Generalitat Valenciana" of Spain (Grant ACOMP/2012/105 to P.T.). We appreciate the opinions and generous help of Jeff Dangl and Pablo Vera with the article as well as the revision of Philippa Borrill.Canet Perez, JV.; Dobón Alonso, A.; Tornero Feliciano, P. (2012). Non-Recognition-of-BTH4, an Arabidopsis Mediator Subunit Homolog, Is Necessary for Development and Response to Salicylic Acid. Plant Cell. 24(10):4220-4235. https://doi.org/10.1105/tpc.112.103028S42204235241

The Blade-On-Petiole genes of Arabidopsis are essential for resistance induced by methyl jasmonate

Background: NPR1 is a gene of Arabidopsis thaliana required for the perception of salicylic acid. This perception triggers a defense response and negatively regulates the perception of jasmonates. Surprisingly, the application of methyl jasmonate also induces resistance, and NPR1 is also suspected to be relevant. Since an allelic series of npr1 was recently described, the behavior of these alleles was tested in response to methyl jasmonate. Results: The response to methyl jasmonate of different npr1s alleles and NPR1 paralogs null mutants was measured by the growth of a pathogen. We have also tested the subcellular localization of some npr1s, along with the protein-protein interactions that can be measured in yeast. The localization of the protein in npr1 alleles does not affect the response to methyl jasmonate. In fact, NPR1 is not required. The genes that are required in a redundant fashion are the BOPs. The BOPs are paralogs of NPR1, and they physically interact with the TGA family of transcription factors. Conclusions: Some npr1 alleles have a phenotype in this response likely because they are affecting the interaction between BOPs and TGAs, and these two families of proteins are responsible for the resistance induced by methyl jasmonate in wild type plants.This work was supported by the "Ministerio de Economia y Competitividad" (MINECO) of Spain (grant BIO201018896 to PT, a JAE-CSIC Fellowship to JVC and a FPI-MINECO to AD) and "Generalitat Valenciana" of Spain (grant ACOMP/2012/105 to PT). Thanks to Dr. Xinnian Dong for NPR1 overexpression lines and to Dr. Ove Nilsson for BOPs overexpression lines. We appreciate the opinions and generous help of Drs. Vicente Ramirez, Pablo Vera, and Shelley Hepworth about the manuscript.Dobón Alonso, A.; Fajmonova, J.; Tornero Feliciano, P.; Canet, JV. (2012). 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Molecular analysis of menadione-induced resistance against biotic stress in Arabidopsis

19 páginas, 6 figuras, 2 tablas.Menadione sodium bisulphite (MSB) is a water-soluble derivative of vitamin K3, or menadione, and has been previously demonstrated to function as a plant defence activator against several pathogens in several plant species. However, there are no reports of the role of this vitamin in the induction of resistance in the plant model Arabidopsis thaliana. In the current study, we demonstrate that MSB induces resistance by priming in Arabidopsis against the virulent strain Pseudomonas syringae pv. tomato DC3000 (Pto) without inducing necrosis or visible damage. Changes in gene expression in response to 0.2 mm MSB were analysed in Arabidopsis at 3, 6 and 24 h post-treatment using microarray technology. In general, the treatment with MSB does not correlate with other publicly available data, thus MSB produces a unique molecular footprint. We observed 158 differentially regulated genes among all the possible trends. More up-regulated genes are included in categories such as 'response to stress' than the background, and the behaviour of these genes in different treatments confirms their role in response to biotic and abiotic stress. In addition, there is an over-representation of the G-box in their promoters. Some interesting functions are represented among the individual up-regulated genes, such as glutathione S-transferases, transcription factors (including putative regulators of the G-box) and cytochrome P450s. This work provides a wide insight into the molecular cues underlying the effect of MSB as a plant resistance inducer.This work was partially funded by an INVESCAN, S.L. grant (No.OTT2001438) to the CSIC and by a BIO2006-02168 grant of MICINN to PT. The microarrays were funded in part by the “Genome España” Foundation. MER was supported by a research contract (ID-TF-06/002) from the Consejería de Industria, Comercio y Nuevas Tecnologías (Gobierno de Canarias). The authors thank CajaCanarias for their research support. We also thank Lorena Perales for her help in performing the bacterial growth curves, Dr. Héctor Cabrera for his useful advice on writing the manuscript, the English translation service of the Universidad Politécnica de Valencia and Mrs. Pauline Agnew whose endeavoured to edit the English translation of this paper.Peer reviewe

An allele of Arabidopsis COI1 with hypo- and hypermorphic phenotypes in plant growth, defence and fertility

Resistance to biotrophic pathogens is largely dependent on the hormone salicylic acid (SA) while jasmonic acid (JA) regulates resistance against necrotrophs. JA negatively regulates SA and is, in itself, negatively regulated by SA. A key component of the JA signal transduction pathway is its receptor, the COI1 gene. Mutations in this gene can affect all the JA phenotypes, whereas mutations in other genes, either in JA signal transduction or in JA biosynthesis, lack this general effect. To identify components of the part of the resistance against biotrophs independent of SA, a mutagenised population of NahG plants (severely depleted of SA) was screened for suppression of susceptibility. The screen resulted in the identification of intragenic and extragenic suppressors, and the results presented here correspond to the characterization of one extragenic suppressor, coi1-40. coi1-40 is quite different from previously described coi1 alleles, and it represents a strategy for enhancing resistance to biotrophs with low levels of SA, likely suppressing NahG by increasing the perception to the remaining SA. The phenotypes of coi1-40 lead us to speculate about a modular function for COI1, since we have recovered a mutation in COI1 which has a number of JA-related phenotypes reduced while others are equal to or above wild type levels.This work was supported by grant BIO201018896 from "Ministerio de Economia y Competitividad" (MINECO) of Spain and by grant ACOMP/2012/105 from "Generalitat Valenciana" to PT, a JAE-CSIC Fellowship to JVC, a FPI-MINECO to AD, and Fellowships from the European Molecular Biology Organization and the Human Frontier Science Program to BBHW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Dobón Alonso, A.; Wulff, BBH.; Canet Perez, JV.; Fort Rausell, P.; Tornero Feliciano, P. (2013). An allele of Arabidopsis COI1 with hypo- and hypermorphic phenotypes in plant growth, defence and fertility. PLoS ONE. 1(8):55115-55115. https://doi.org/10.1371/journal.pone.0055115S551155511518Vlot, A. C., Dempsey, D. A., & Klessig, D. F. (2009). Salicylic Acid, a Multifaceted Hormone to Combat Disease. Annual Review of Phytopathology, 47(1), 177-206. doi:10.1146/annurev.phyto.050908.135202Mauch, F., Mauch-Mani, B., Gaille, C., Kull, B., Haas, D., & Reimmann, C. (2001). Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase. The Plant Journal, 25(1), 67-77. doi:10.1046/j.1365-313x.2001.00940.xGaffney, T., Friedrich, L., Vernooij, B., Negrotto, D., Nye, G., Uknes, S., … Ryals, J. (1993). 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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.Funding: This work was supported by the Spanish MINECO (BFU2012 to PV), and Generalitat Valenciana (Prometeo2014/020 to PV). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Dobón Alonso, A.; Canet Perez, JV.; García-Andrade Serrano, J.; Angulo, C.; Neumetzler, L.; Persson, S.; Vera Vera, P. (2015). Novel Disease Susceptibility Factors for Fungal Necrotrophic Pathogens in Arabidopsis. PLoS Pathogens. 11(4):1-30. https://doi.org/10.1371/journal.ppat.1004800S13011

Interacción de LRA5 con genes asociados a defensa en Arabidopsis

Tesis doctoral del Departamento de Biotecnología de la Universidad Politécnica de Valencia, realizada en el Instituto de Biología Molecular y Celular de PlantasLa interacción planta‐patógeno es una de las relaciones más interesantes que se pueden establecer entre las plantas y los organismos que las rodean. El estudio de mutantes afectados en respuestas de defensa constituye una importante fuente de información acerca de los mecanimos que la componen. LRA5 fue descrito previamente como un mutante de Arabidopsis thaliana (Arabidopsis) afectado en varios tipos de resistencia y que presenta niveles anormalmente bajos de ácido salicílico (SA), hormona relacionada con la resistencia a patógenos biotrofos. Se ha intentado cartografiar su posición en el genoma de Arabidopsis, reduciendo el intervalo de mapeo a tres únicos genes en los que no se ha encontrado alteración en la secuencia ni en la expresón que pudiera explicar el fenotipo de LRA5, descubriendo finalmente que se trata de un transgén NahG (que codifica para una enzima salicilato hidroxilasa) de origen desconocidoPara poder caracterizar componentes de la resistencia a biotrofos independientes de la hormona SA, se ha realizado un rastreo de supresores de LRA5 que restauraran, al menos parcialmente, la resistencia frente a Pseudomonas syringae pv tomato (Pto). La mayor parte del trabajo se ha centrado en la caracterización de dos mutantes extragénicos, escogidos por su carácter opuesto. esh1 (de EDWARD SCISSOR HANDS 1) posee un peculiar fenotipo de hojas abigarradas y de silicuas agrupadas en los extremos de las inflorescencias, dependiente del número de días pasados en día corto. Es además capaz de suprimir la susceptibilidad a Pto y a otros patógenos, y expresa de manera constitutiva PR1. Por otro lado, jrc1 (de JAROCKS 1) solo restaura la resistencia a Pto y expresa PR1 de manera exacerbada tras la infección con patógeno. Además presenta fenotipos de insensibilidad al ácido jasmónico (JA).Es especialmente llamativo el fenotipo de raíces laterales, cuyo número excede con mucho los niveles de coi1‐1 y jin1, otros mutantes en la señalización por JA. Finalmente, el cartografiado ha permitido descubrir que jrc1 es un alelo de COI1 no nulo. COI1 es un gen crucial en las repuestas basadas en la hormona JA, explicando la supresión de NahG por la regulación negativa existente entre SA y JA.Peer reviewe

Quantitative genetic analysis of salicylic acid perception in Arabidopsis

Salicylic acid (SA) is a phytohormone required for a full resistance against some pathogens in Arabidopsis, and NPR1 (Non-Expressor of Pathogenesis Related Genes 1) is the only gene with a strong effect on resistance induced by SA which has been described. There can be additional components of SA perception that escape the traditional approach of mutagenesis. An alternative to that approach is searching in the natural variation of Arabidopsis. Different methods of analyzing the variation between ecotypes have been tried and it has been found that measuring the growth of a virulent isolate of Pseudomonas syringae after the exogenous application of SA is the most effective one. Two ecotypes, Edi-0 and Stw-0, have been crossed, and their F2 has been studied. There are two significant Quantitative Trait Loci (QTLs) in this population, and there is one QTL in each one of the existing mapping populations Col-4 x Laer-0 and Laer-0 x No-0. They have different characteristics: while one QTL is only detectable at low concentrations of SA, the other acts after the point of crosstalk with methyl jasmonate signalling. Three of the QTLs have candidates described in SA perception as NPR1, its interactors, and a calmodulin binding protein.This work was supported by the Ministerio de Ciencia e Innovación (MICINN) of Spain (grant BIO201018896 to PT, a JAE-CSIC Fellowship to JVC and a FPI-MICINN to AD). We appreciate the BTH provided by Syngenta and the genotyping by CEGEN (Fundación Genoma España). The authors declare that they have no conflict of interest.Peer reviewe

Resistance and biomass in Arabidopsis: a new model for Salicylic acid perception.

Salicylic acid (SA) is an essential hormone for plant defence and development SA perception is usually measured by counting the number of pathogens that grow in planta upon an exogenous application of the hormone. A biological SA perception model based on plant fresh weight reduction caused by disease resistance in Arabidopsis thaliana is proposed This effect is more noticeable when a chemical analogue of SA is used, like Benzothiadiazole (BTH) By spraying BTH several times, a Substantial difference in plant biomass is observed when compared with the mock treatment Such difference is dose-dependent and does not require pathogen inoculation The model is robust and allows for the comparison of different Arabidopsis ecotypes, recombinant inbreed lines, and mutants Our results show that two mutants, non-expresser of pathogenesis-related genes 1 (npr1) and auxin resistant 3 (axr3), fail to lose biomass when BTH is applied to them Further experiments show that axr3 responds to SA and BTH in terms of defence induction. NPR1-related genotypes also confirm the pivotal role of NPR1 in SA perception, and suggest an active program of depletion of resources in the infected tissuesMinisteno de Ciencia e Innovacion (MICINN) BIO2006-02168 JAE-CSIC Fellowship FPI-MICINNPeer reviewe

Molecular analysis of menadione-induced resistance against biotic stress in Arabidopsis

19 páginas, 6 figuras, 2 tablas.Menadione sodium bisulphite (MSB) is a water-soluble derivative of vitamin K3, or menadione, and has been previously demonstrated to function as a plant defence activator against several pathogens in several plant species. However, there are no reports of the role of this vitamin in the induction of resistance in the plant model Arabidopsis thaliana. In the current study, we demonstrate that MSB induces resistance by priming in Arabidopsis against the virulent strain Pseudomonas syringae pv. tomato DC3000 (Pto) without inducing necrosis or visible damage. Changes in gene expression in response to 0.2 mm MSB were analysed in Arabidopsis at 3, 6 and 24 h post-treatment using microarray technology. In general, the treatment with MSB does not correlate with other publicly available data, thus MSB produces a unique molecular footprint. We observed 158 differentially regulated genes among all the possible trends. More up-regulated genes are included in categories such as 'response to stress' than the background, and the behaviour of these genes in different treatments confirms their role in response to biotic and abiotic stress. In addition, there is an over-representation of the G-box in their promoters. Some interesting functions are represented among the individual up-regulated genes, such as glutathione S-transferases, transcription factors (including putative regulators of the G-box) and cytochrome P450s. This work provides a wide insight into the molecular cues underlying the effect of MSB as a plant resistance inducer.This work was partially funded by an INVESCAN, S.L. grant (No.OTT2001438) to the CSIC and by a BIO2006-02168 grant of MICINN to PT. The microarrays were funded in part by the “Genome España” Foundation. MER was supported by a research contract (ID-TF-06/002) from the Consejería de Industria, Comercio y Nuevas Tecnologías (Gobierno de Canarias). The authors thank CajaCanarias for their research support. We also thank Lorena Perales for her help in performing the bacterial growth curves, Dr. Héctor Cabrera for his useful advice on writing the manuscript, the English translation service of the Universidad Politécnica de Valencia and Mrs. Pauline Agnew whose endeavoured to edit the English translation of this paper.Peer reviewe