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In eukaryotic genomes, gene expression and DNA recombination are affected by structural chromatin traits. Chromatin structure is shaped by the activity of enzymes that either introduce covalent modifications in DNA and histone proteins or use energy from ATP to disrupt histone-DNA interactions. The genomic 'marks' that are generated by covalent modifications of histones and DNA, or by the deposition of histone variants, are susceptible to being altered in response to stress. Recent evidence has suggested that proteins generating these epigenetic marks play crucial roles in the defence against pathogens. Histone deacetylases are involved in the activation of jasmonic acid- and ethylene-sensitive defence mechanisms. ATP-dependent chromatin remodellers mediate the constitutive repression of the salicylic acid-dependent pathway, whereas histone methylation at the WRKY70 gene promoter affects the activation of this pathway. Interestingly, bacterial-infected tissues show a net reduction in DNA methylation, which may affect the disease resistance genes responsible for the surveillance against pathogens. As some epigenetic marks can be erased or maintained and transmitted to offspring, epigenetic mechanisms may provide plasticity for the dynamic control of emerging pathogens without the generation of genomic lesions.

Post-transcriptional gene silencing mediated by microRNAs (miRNAs) modulates numerous developmental and stress response pathways. For the last two decades, HASTY (HST), the ortholog of human EX-PORTIN 5, was considered to be a candidate protein that exports plant miRNAs from the nucleus to the cytoplasm. Here, we report that HST functions in the miRNA pathway independent of its cargo-exporting activity in Arabidopsis. We found that Arabidopsis mutants with impaired HST shuttling exhibit normal subcellular distribution of miRNAs. Interestingly, protein-protein interaction and microscopy assays showed that HST directly interacts with the microprocessor core component DCL1 through its N-terminal domain. Moreover, mass spectrometry analysis revealed that HST also interacts independently of its N-terminal domain with the mediator complex subunit MED37. Further experiments revealed that HST could act as a scaffold to facilitate the recruitment of DCL1 to genomic MIRNA loci by stabilizing the DCL1-MED37 complex, which in turn promotes the transcription and proper processing of primary miRNA transcripts (pri-miRNAs). Taken together, these results suggest that HST is likely associated with the formation of the miRNA biogenesis complex at MIRNA genes, promoting the transcription and processing of pri-miRNAs rather than the direct export of processed miRNAs from the nucleus.

Pattern recognition receptors (PRR) and nucleotide-binding leucine-rich repeat proteins (NLR) are major components of the plant immune system responsible for pathogen detection. To date, the transcriptional regulation of PRR/NLR genes is poorly understood. Some PRR/NLR genes are affected by epigenetic changes of neighboring transposable elements (TEs) (cis regulation). We analyzed whether these genes can also respond to changes in the epigenetic marks of distal pericentromeric TEs (trans regulation). We found that Arabidopsis tissues infected with Pseudomonas syringae pv. tomato (Pst) initially induced the expression of pericentromeric TEs, and then repressed it by RNA-directed DNA methylation (RdDM). The latter response was accompanied by the accumulation of small RNAs (sRNAs) mapping to the TEs. Curiously these sRNAs also mapped to distal PRR/NLR genes, which were controlled by RdDM but remained induced in the infected tissues. Then, we used non-infected mom1 (Morpheus' molecule 1) mutants that expressed pericentromeric TEs to test if they lose repression of PRR/NLR genes. mom1 plants activated several PRR/ NLR genes that were unlinked to MOM1-targeted TEs, and showed enhanced resistance to Pst. Remarkably, the increased defenses of mom1 were abolished when MOM1/RdDM-mediated pericentromeric TEs silencing was re-established. Therefore, common sRNAs could control PRR/NLR genes and distal pericentromeric TEs and preferentially silence TEs when they are activated.

In most organisms, the maturation of nascent RNAs is coupled to transcription. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNA genes (MIRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs coupled to transcription. We found that the entire biogenesis occurs co-transcriptionally for pri-miRNAs processed from the loop of the hairpin but requires a second nucleoplasmic step for those processed from the base. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops, formed near the transcription start site region of MIRNAs, promote co-transcriptional pri-miRNA processing. Furthermore, our results suggest the neofunctionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

Transposable elements (TEs) are extremely abundant in complex plant genomes. siRNAs of 24 nucleotides in length control transposon activity in a process that involves de novo methylation of targeted loci. Usually, these epigenetic modifications trigger nucleosome condensation and a permanent silencing of the affected loci. Here, we show that a TE-derived inverted repeat (IR) element, inserted near the sunflower HaWRKY6 locus, dynamically regulates the expression of the gene by altering chromatin topology. The transcripts of this IR element are processed into 24-nt siRNAs, triggering DNA methylation on its locus. These epigenetic marks stabilize the formation of tissue-specific loops in the chromatin. In leaves, an intragenic loop is formed, blocking HaWRKY6 transcription. While in cotyledons (Cots), formation of an alternative loop, encompassing the whole HaWRKY6 gene, enhances transcription of the gene. The formation of this loop changes the promoter directionality, reducing IR transcription, and ultimately releasing the loop. Our results provide evidence that TEs can act as active and dynamic regulatory elements within coding loci in a mechanism that combines RNA silencing, epigenetic modification, and chromatin remodeling machineries.

An intrinsically disordered protein interacts with HYPONASTIC LEAVES1 and ARGONAUTE1 in a post-miRNA processing complex to promote ARGONAUTE1 stability and miRNA activity.

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