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. The influence of epigenetic regulation in controlling the adaptive responses of living organisms to changes in the environment is becoming a common theme in biology. RNA–directed DNA methylation (RdDM) is an epigenetic control mechanism driven by a subset of noncoding small interfering RNAs (siRNAs) that influence gene function without changing DNA sequence by inducing de novo methylation of cytosines, or by modification of histones, at their target genomic regions. The implication and roles of the RdDM mechanism in the orchestration of plant immune responses still remains to be characterized. A recent study in the model plant Arabidopsis showed that ARGONAUTE4, one of the characteristic components of the RdDM pathway, was required for plant immunity against bacterial pathogens. Here, in a genetic screen aiming to identify cellular factors integral in regulating immunity in Arabidopsis, we further identified that the RNA polymerases V, another crucial component of the RdDM pathway, is pivotal for plant immunity against fungal pathogens. Similarly, we identified that additional components of the RdDM pathway, but surprisingly not RNA polymerase IV, are similarly required for plant immunity. Based on genetic evidence, our results highlight the importance of RdDM as an additional layer of complexity in the regulation of plant immune responses.

Plant β-glucanases are enzymes involved in the synthesis, remodelling and turnover of cell wall components during multiple physiological processes. Based on the type of the glycoside bond they cleave, plant β-glucanases have been grouped into three categories: (i) β-1,4-glucanases degrade cellulose and other polysaccharides containing 1,4-glycosidic bonds to remodel and disassemble the wall during cell growth. (ii) β-1,3-glucanases are responsible for the mobilization of callose, governing the symplastic trafficking through plasmodesmata. (iii) β-1,3-1,4-glucanases degrade mixed linkage glucan, a transient wall polysaccharide found in cereals, which is broken down to obtain energy during rapid seedling growth. In addition to their roles in the turnover of self-glucan structures, plant β-glucanases are crucial in regulating the outcome in symbiotic and hostile plant–microbe interactions by degrading non-self glucan structures. Plants use these enzymes to hydrolyse β-glucans found in the walls of microbes, not only by contributing to a local antimicrobial defence barrier, but also by generating signalling glucans triggering the activation of global responses. As a counterpart, microbes developed strategies to hijack plant β-glucanases to their advantage to successfully colonize plant tissues. This review outlines our current understanding on plant β-glucanases, with a particular focus on the latest advances on their roles in adaptative responses.

Contents Summary 901 I. Introduction 901 II. Biochemistry and structure of plant SBTs 902 III. Phylogeny of plant SBTs and family organization 903 IV. Physiological roles of plant SBTs 905 V. Conclusions and outlook 911 Acknowledgements 912 References 912 Summary: Subtilases (SBTs) are serine peptidases that are found in all three domains of life. As compared with homologs in other Eucarya, plant SBTs are more closely related to archaeal and bacterial SBTs, with which they share many biochemical and structural features. However, in the course of evolution, functional diversification led to the acquisition of novel, plant‐specific functions, resulting in the present‐day complexity of the plant SBT family. SBTs are much more numerous in plants than in any other organism, and include enzymes involved in general proteolysis as well as highly specific processing proteases. Most SBTs are targeted to the cell wall, where they contribute to the control of growth and development by regulating the properties of the cell wall and the activity of extracellular signaling molecules. Plant SBTs affect all stages of the life cycle as they contribute to embryogenesis, seed development and germination, cuticle formation and epidermal patterning, vascular development, programmed cell death, organ abscission, senescence, and plant responses to their biotic and abiotic environments. In this article we provide a comprehensive picture of SBT structure and function in plants.

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.

In higher eukaryotes, induced resistance associates with acquisition of a priming state of the cells for a more effective activation of innate immunity; however, the nature of the components for mounting this type of immunological memory is not well known. We identified an extracellular subtilase from Arabidopsis, SBT3.3, the overexpression of which enhances innate immune responses while the loss of function compromises them. SBT3.3 expression initiates a durable autoinduction mechanism that promotes chromatin remodeling and activates a salicylic acid(SA)-dependent mechanism of priming of defense genes for amplified response. Moreover, SBT3.3 expression-sensitized plants for enhanced expression of the OXI1 kinase gene and activation of MAP kinases following pathogen attack, providing additional clues for the regulation of immune priming by SBT3.3. Conversely, in sbt3.3 mutant plants pathogen-mediated induction of SA-related defense gene expression is drastically reduced and activation of MAP kinases inhibited. Moreover, chromatin remodeling of defense-related genes normally associated with activation of an immune priming response appear inhibited in sbt3.3 plants, further indicating the importance of the extracellular SBT3.3 subtilase in the establishment of immune priming. Our results also point to an epigenetic control in the regulation of plant immunity, since SBT3.3 is up-regulated and priming activated when epigenetic control is impeded. SBT3.3 represents a new regulator of primed immunity. Following a first encounter with a pathogen, higher eukaryotes develop enhanced resistance to subsequent infections by a broad spectrum of pathogens. This type of induced resistance (IR) exhibits memory characteristics after the first encounter with a pathogen —training effect— and appears evolutionarily conserved. IR response components must reside in a plant's capacity to reprogram gene expression. Among the mechanisms involved in immune-related transcriptional reprogramming, the importance of chromatin remodelling is emerging. Recent studies indicated a causal link between priming and chromatin remodelling, pointing to a histone memory for information storage in the plant immune response. These results emphasized the importance of epigenetic control as an additional layer of complexity in plant immunity. However, the nature of the components for activating this type of immunological memory remains elusive. Here, in a search aiming to identify cellular factors integral in regulating immunity in Arabidopsis, we found that the SBT3.3 gene, encoding an extracellular subtilase enzyme, is pivotal for establishing plant immune priming. Moreover, based on molecular and genetic evidences, our results indicate that SBT3.3 expression is under epigenetic control thus highlighting the importance of this mechanism of gene regulation in the control of plant immunity and IR.