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.

Non-recognition-of-BTH4, an Arabidopsis mediator subunit homolog, is necessary for development and response to salicylic acid.

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.

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.

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 × Laer-0 and Laer-0 × 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.

Structure-function analysis of npr1 alleles in Arabidopsis reveals a role for its paralogs in the perception of salicylic acid.

Salicylic acid (SA) is necessary for plant defence against some pathogens, whereas NPR1 is necessary for SA perception. Plant defence can be induced to an extreme by several applications of benzothiadiazole (BTH), an analogue of SA. Thus, plants that do not perceive BTH grow unaffected, whereas wild-type plants grow stunted. This feature allows us to screen for mutants in Arabidopsis thaliana that show insensitivity to BTH in a high-throughput fashion. Most of the mutants are npr1 alleles, with similar phenotypes in plant weight and pathogen growth. The mutations are clustered in the carboxyl-terminal part of the protein, and no obvious null alleles were recovered. These facts have prompted a search for knockouts in the NPR1 gene. Two of these KO alleles identified are null and have an intermediate phenotype. All the evidence presented lead us to propose a redundancy in SA perception, with the paralogs of NPR1 taking part in this signalling. We show that the mutations recovered in the screening genetically interact with the paralogs preventing their function in SA signalling.

ß-carbonic anhydrases play a role in salicylic acid perception in Arabidopsis.

The plant hormone salicylic acid (SA) is required for defense responses. NON EXPRESSER OF PATHOGENESIS RELATED 1 (NPR1) and NON RECOGNITION OF BTH-4 (NRB4) are required for the response to SA in Arabidopsis (Arabidopsis thaliana). Here, we isolated several interactors of NRB4 using yeast two-hybrid assays. Two of these interactors, ßCA1 and ßCA2, are ß-carbonic anhydrase family proteins. Since double mutant ßca1 ßca2 plants did not show any obvious phenotype, we investigated other ßCAs and found that NRB4 also interacts with ßCA3 and ßCA4. Moreover, several ßCAs interacted with NPR1 in yeast, including one that interacted in a SA-dependent manner. This interaction was abolished in loss-of-function alleles of NPR1. Interactions between ßCAs and both NRB4 and NPR1 were also detected in planta, with evidence for a triple interaction, NRB4-ßCA1-NPR1. The quintuple mutant ßca1 ßca2 ßca3 ßca4 ßca6 showed partial insensitivity to SA. These findings suggest that one of the functions of carbonic anhydrases is to modulate the perception of SA in plants.

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.