Cucumber mosaic virus suppressor 2b binds to AGO4-related small RNAs and impairs AGO4 activities

Cucumber mosaic virus suppressor 2b (CMV2b) is a nuclear viral suppressor that interferes with local and systemic silencing and inhibits AGO1 slicer activity. CMV2b-mediated transgene hypomethylation and its localization in Cajal bodies suggests a role of CMV2b in RNA-directed DNA methylation (RdDM). However, its direct involvement in RdDM, or its binding with small RNAs (sRNAs) in vivo is not yet established. Here, we show that CMV2b binds both microRNAs (miRNAs) and small interfering RNAs (siRNAs) in vivo. sRNA sequencing data from the CMV2b immunocomplex revealed its preferential binding with 24-nt repeat-associated siRNAs. We provide evidence that CMV2b also has direct interaction with the AGO4 protein by recognizing its PAZ and PIWI domains. Subsequent analysis of AGO4 functions revealed that CMV2b reduced AGO4 slicer activity and the methylation of several loci, accompanied by the augmented accumulation of 24-nt siRNAs in Arabidopsis inflorescences. Intriguingly, CMV2b also regulated an AGO4-related epiallele independently of its catalytic potential, which further reinforces the repressive effects of CMV2b on AGO4 activity. Collectively, our results demonstrate that CMV2b can counteract AGO4-related functions. We propose that by adopting novel counter-host defense strategies against AGO1 and AGO4 proteins, CMV creates a favorable cellular niche for its proliferation.     

High-throughput sequence analysis of small RNAs in skotomorphogenic seedlings of Brassica rapa ssp. rapa

Skotomorphogenic development is the process by which seedlings adapt to a stressful dark environment. Such metabolic responses to abiotic stresses in plants are known to be regulated in part by microRNAs (miRNAs); however, little is known about the involvement of miRNAs in the regulation of skotomorphogenesis. To identify miRNAs at the genome-wide level in skotomorphogenic seedlings of turnip (Brassica rapa subsp. rapa), an important worldwide root vegetable, we used Solexa sequencing to sequence a small RNA library from seedlings grown in the dark for 4 days. Deep sequencing showed that the small RNAs (sRNAs) were predominantly 21 to 24 nucleotides long. Specifically, 13,319,035 reads produced 359,531 unique sRNAs including rRNA, tRNA, miRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and unannotated sRNAs. Sequence analysis identified 96 conserved miRNAs belonging to 36 miRNA families and 576 novel miRNAs. qRT-PCR confirmed that the miRNAs were expressed during skotomorphogenesis similar to the trends shown by the Solexa sequencing results. A total of 2013 potential targets were predicted, and the targets of BrmiR157, BrmiR159 and BrmiR160 were proved to be regulated by miRNA-guided cleavage. These results show that specific regulatory miRNAs are present in skotomorphogenic seedlings of turnip and may play important roles in growth, development, and response to dark environment.     

Exploring the interaction between small RNAs and R genes during Brachypodium response to Fusarium culmorum infection

The present study aims to investigate small RNA interactions with putative disease response genes in the model grass species Brachypodium distachyon. The fungal pathogen Fusarium culmorum (Fusarium herein) and phytohormone salicylic acid treatment were used to induce the disease response in Brachypodium. Initially, 121 different putative disease response genes were identified using bioinformatic and homology based approaches. Computational prediction was used to identify 33 candidate new miRNA coding sequences, of which 9 were verified by analysis of small RNA sequence libraries. Putative Brachypodium miRNA target sites were identified in the disease response genes, and a subset of which were screened for expression and possible miRNA interactions in 5 different Brachypodium lines infected with Fusarium. An NBS-LRR family gene, 1g34430, was polymorphic among the lines, forming two major genotypes, one of which has its miRNA target sites deleted, resulting in altered gene expression during infection. There were siRNAs putatively involved in regulation of this gene, indicating a role of small RNAs in the B. distachyon disease response.     

Genome-wide identification of cytosine-5 DNA methyltransferases and demethylases in Solanum lycopersicum

Recent studies have reported that decreased level of DNA cytosine methylation in the global genome was closely related to the initiation of tomato (Solanum lycopersicum) fruit ripening. However, genome-scale analysis of cytosine-5 DNA methyltransferases (C5-MTases) and demethylases in tomato has not been engaged. In this study, 7 C5-MTases and 3 demethylases were identified in tomato genome, which probably contributed to DNA cytosine methylation level in tomato. The 7 C5-MTases were categorized into 4 subgroups, and the 3 demethylases were classified into 2 subgroups based on phylogenetic analyses. Comprehensive analysis of their structure and genomic localization was also performed in this paper. According to online RNA-seq data, 4 S. lycopersicum C5-MTase (SlC5-MTase) genes (SlMET, SlDRM1L1, SlDRM5, SlMET3L) were expressed higher than others, and one DNA demethylase gene (SlDML) was significantly changed during tomato fruit development and ripening. Furthermore, all these five gene expressions at breaker (BK) stage changed with 1-methylcyclopropene (1-MCP) treatment, indicating that they were regulated by ethylene directly or indirectly in tomato fruit. In addition, subcellular localization analysis indicated that SlDRM1L1 and SlDRM5 located in the nucleus might have responsibility for RNA-directed DNA methylation (RdDM). Collectively, this paper provided a framework for gene discovery and functional characterization of C5-MTases and DNA demethylases in other Solanaceae species.     

Surviving an identity crisis: A revised view of chromatin insulators in the genomics era

The control of complex, developmentally regulated loci and partitioning of the genome into active and silent domains is in part accomplished through the activity of DNA-protein complexes termed chromatin insulators. Together, the multiple, well-studied classes of insulators in Drosophila melanogaster appear to be generally functionally conserved. In this review, we discuss recent genomic-scale experiments and attempt to reconcile these newer findings in the context of previously defined insulator characteristics based on classical genetic analyses and transgenic approaches. Finally, we discuss the emerging understanding of mechanisms of chromatin insulator regulation. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.