Significance: We described the high-throughput identification of DNA-binding specificities of 63 plant transcription factors (TFs) and their relevance as cis- regulatory elements in vivo. Almost half of the TFs recognized secondary motifs partially or completely differing from their corresponding primary ones. Analysis of coregulated genes, transcriptomic data, and chromatin hypersensitive regions revealed the biological relevance of more than 80% of the binding sites identified. Our combined analysis allows the prediction of the function of a particular TF as activator or repressor through a particular DNA sequence. The data support the correlation between cis- regulatory elements in vivo and the sequence determined in vitro. Moreover, it provides a framework to explore regulatory networks in plants and contributes to decipher the transcriptional regulatory code.

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.

Here, we report the characterization of the Arabidopsis thaliana ocp11 (for overexpressor of cationic peroxidase11) mutant, in which a β-glucuronidase reporter gene under the control of the H2O2-responsive Ep5C promoter is constitutively expressed. ocp11 plants show enhanced disease susceptibility to the virulent bacterium Pseudomonas syringae pv tomato DC3000 (P.s.t. DC3000) and also to the avirulent P.s.t. DC3000 carrying the effector avrRpm1 gene. In addition, ocp11 plants are also compromised in resistance to the nonhost pathogen P. syringae pv tabaci. Genetic and molecular analyses reveal that ocp11 plants are not affected in salicylic acid perception. We cloned OCP11 and show that it encodes ARGONAUTE4 (AGO4), a component of the pathway that mediates the transcriptional gene silencing associated with small interfering RNAs that direct DNA methylation at specific loci, a phenomenon known as RNA-directed DNA methylation (RdDM). Thus, we renamed our ocp11 mutant ago4-2, as it represents a different allele to the previously characterized recessive ago4-1. Both mutants decrease the extent of DNA cytosine methylation at CpNpG and CpHpH (asymmetric) positions present at different DNA loci and show commonalities in all of the molecular and phenotypic aspects that we have considered. Interestingly, we show that AGO4 works independently of other components of the RdDM pathway in mediating resistance to P.s.t. DC3000, and loss of function in other components of the pathway operating upstream of AGO4, such as RDR2 and DCL3, or operating downstream, such as DRD1, CMT3, DRM1, and DRM2, does not compromise resistance to this pathogen.

A 69-kDa proteinase (P69), a member of the pathogenesis-related proteins, is induced and accumulates in tomato (Lycopersicon esculentum) plants as a consequence of pathogen attack. We have used the polymerase chain reaction to identify and clone a cDNA from tomato plants that represent the pathogenesis-related P69 proteinase. The nucleotide sequence analysis revealed that P69 is synthesized in a preproenzyme form, a 745-amino acid polypeptide with a 22-amino acid signal peptide, a 92-amino acid propolypeptide, and a 631-amino acid mature polypeptide. Within the mature region the most salient feature was the presence of domains homologous to the subtilisin serine protease family. The amino acid sequences surrounding Asp-146, His-203, and Ser-532 of P69 are closely related to the catalytic sites (catalytic triad) of the subtilisin-like proteases. Northern blot analysis revealed that the 2.4-kb P69 mRNA accumulates abundantly in leaves and stem tissues from viroid-infected plants, whereas the mRNA levels in tissues from healthy plants were undetectable. Our results indicate that P69, a secreted calcium-activated endopeptidase, is a plant pathogenesis-related subtilisin-like proteinase that may collaborate with other defensive proteins in a general mechanism of active defense against attacking pathogens.

Pathogenesis-related (PR) proteins form a heterogeneous family of plant proteins that are likely to be involved in defense and are inducible by pathogen attacks. One group of PRs, represented by the subfamily PR-1, are low-molecular-weight proteins of unknown biochemical function. Here we describe the cloning and characterization of two closely related genes encoding a basic and an acidic PR-1 protein (PR1b1 and PR1a2) from tomato (Lycopersicon esculentum). We present a comparative study of the mode of transcriptional regulation of these two genes in transgenic tobacco plants using a series of promoter-GUS fusions. Unexpectedly, the chimeric PR1a2/GUS gene is not induced by pathogenic signals but instead shows constitutive expression with a reproducible developmental expression pattern. It is expressed in shoot meristems, trichomes, and cortical cells as well as in vascular and nearby tissues of the mature stem. This constitutive expression pattern may represent preemption of plant defenses against potential pathogens. Conversely, the chimeric PR1b1/GUS gene does not show any constitutive expression in the plant, but it is transcriptionally activated following pathogen attack. Upon infection by tobacco mosaic virus, the PR1b1 gene is strongly activated locally in tissues undergoing the hypersensitive response but not systemically in uninoculated tissues. Furthermore, its expression is induced by both salicylic acid and ethylene precursors, two signals that coexist and apparently mediate the activation of local defenses during the hypersensitive response. We speculate that the different mode of expression of the two genes presented here, together with that reported previously for the induction of other PR-1 genes in systemic, uninoculated tissues, may all be complementary and necessary for the plant to acquire an efficient refractory state to resist pathogen attacks.

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.