Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes and our knowledge of the evolution of chromatin related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from 3 vascular plants 400 Mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional organization of the genome of early land plants.Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and repeats in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres, and a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that chromatin regions belonging to repressed heterochromatin were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing. C C A A CG A G Centromeric repeat Centromeric repeat Centromeric repeat CENP-B box (RC) CENP-B box (RC) CENP-B box (RC) 54 0 17 17 109 162 T

Background Posttranscriptional gene silencing (PTGS) is one of the most important mechanisms for plants during viral infection. However, viruses have also developed viral suppressors to negatively control PTGS by inhibiting microRNA (miRNA) and short-interfering RNA (siRNA) regulation in plants. The first identified viral suppressor, P1/HC-Pro, is a fusion protein that was translated from potyviral RNA. Upon infecting plants, the P1 protein itself is released from HC-Pro by the self-cleaving activity of P1. P1 has an unknown function in enhancing HC-Pro-mediated PTGS suppression. We performed proteomics to identify P1-interacting proteins. We also performed transcriptomics that were generated from Col-0 and various P1/HC-Pro-related transgenic plants to identify novel genes. The results showed several novel genes were identified through the comparative network analysis that might be involved in P1/HC-Pro-mediated PTGS suppression. Results First, we demonstrated that P1 enhances HC-Pro function and that the mechanism might work through P1 binding to VERNALIZATION INDEPENDENCE 3/SUPERKILLER 8 (VIP3/SKI8), a subunit of the exosome, to interfere with the 5 ′ -fragment of the PTGS-cleaved RNA degradation product. Second, the AGO1 was specifically posttranslationally degraded in transgenic Arabidopsis expressing P1/HC - Pro of turnip mosaic virus (TuMV) ( P1/HC Tu plant). Third, the comparative network highlighted potentially critical genes in PTGS, including miRNA targets, calcium signaling, hormone (JA, ET, and ABA) signaling, and defense response. Conclusion Through these genetic and omics approaches, we revealed an overall perspective to identify many critical genes involved in PTGS. These new findings significantly impact in our understanding of P1/HC-Pro-mediated PTGS suppression.

Genomes of potyviruses, the largest group of plant viruses, encode HC-Pro proteins that mediate RNA silencing suppression. HC-Pros may exhibit only 40% similarity between species, and induce different levels in autophagic ARGONAUTE1 (AGO1) degradation. Our data indicated that HC-Pro of turnip mosaic virus (HC-ProTu) could efficiently trigger AGO1 degradation through autophagy compared with HC-Pros of zucchini yellow mosaic virus (HC-ProZy) and tobacco etch virus (HC-ProTe). Furthermore, HC-ProTu, but not in HC-ProZy, forms a suppression body (S-body) to recruit AGO1 and HEN1, preventing those components from translocating into the nucleus. HC-ProTu, but not HC-ProZy and HC-ProTe, specifically inhibits HEN1 activity, resulting in unmethylated microRNAs (miRNAs) accumulating in the cytoplasm without loading into AGO1. Therefore, we hypothesize that HC-ProTu could enhance the autophagic AGO1 degradation due to the unique HEN1 inhibition interfering with RNA-inducing silencing complex (RISC) assembly.

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Background To investigate the mechanism of RNA silencing suppression, the genetic transformation of viral suppressors of RNA silencing (VSRs) in Arabidopsis integrates ectopic VSR expression at steady state, which overcomes the VSR variations caused by different virus infections or limitations of host range. Moreover, identifying the insertion of the transgenic VSR gene is necessary to establish a model transgenic plant for the functional study of VSR. Methods Developing an endogenous AGO1-based in vitro RNA-inducing silencing complex (RISC) assay prompts further investigation into VSR-mediated suppression. Three P1/HC-Pro plants from turnip mosaic virus (TuMV) (P1/HC-ProTu), zucchini yellow mosaic virus (ZYMV) (P1/HC-ProZy), and tobacco etch virus (TEV) (P1/HC-ProTe) were identified by T-DNA Finder and used as materials for investigations of the RISC cleavage efficiency. Results Our results indicated that the P1/HC-ProTu plant has slightly lower RISC activity than P1/HC-ProZy plants. In addition, the phenomena are consistent with those observed in TuMV-infected Arabidopsis plants, which implies that HC-ProTu could directly interfere with RISC activity. Conclusions In this study, we demonstrated the application of various plant materials in an in vitro RISC assay of VSR-mediated RNA silencing suppression.