RETINOBLASTOMA RELATED (RBR) interaction with key factors of the RNA-directed DNA methylation (RdDM) pathway

● Transposable elements and other repetitive elements are silenced by the RNA-directed DNA methylation pathway (RdDM). In RdDM, POLIV-derived transcripts are converted into double stranded RNA (dsRNA) by the activity of RDR2 and subsequently processed into 24 nucleotide short interfering RNAs (24-nt siRNAs) by DCL3. 24-nt siRNAs are recruited by AGO4 and serve as guides to direct AGO4-siRNA complexes to chromatin bound POLV-derived transcripts generated from the template/target DNA. The interaction between POLV, AGO4, DMS3, DRD1, RDM1 and DRM2 promotes DRM2-mediated de novo DNA methylation. In silico exploration of Arabidopsis RBR protein partners revealed that several members of the RdDM pathway contain a motif that confers high affinity binding to RBR, including the largest subunits of POLIV and POLV (NRPD1 and NRPE1), the shared second largest subunit of POLIV and POLV (NRPD/E2), RDR1, RDR2, DCL3, DRM2 and SUVR2. We demonstrate that RBR binds to DRM2, DRD1 and SUVR2. We also report that seedlings from loss-of-function mutants in RdDM and in RBR show similar phenotypes in the root apical meristem. Furthermore, we show that RdDM and SUVR2 targets are up-regulated in the 35S::AmiGO-RBR background. Our results suggest a novel mechanism for RBR function in transcriptional gene silencing based on the interaction with key players of the RdDM pathway and opens several new hypotheses, including the convergence of RBR-DRM2 on the transcriptional control of TEs and several cell/tissue and stage-specific target genes.

An atypical class of non-coding small RNAs is produced in rice leaves upon bacterial infection

Non-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant–microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20–22 nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant–microbe interactions.

PTEN differentially regulates endocytosis, migration, and proliferation in the enteric protozoan parasite Entamoeba histolytica

PTEN is a lipid phosphatase that is highly conserved and involved in a broad range of biological processes includingcytoskeletal reorganization, endocytosis, signal transduction, and cell migration in all eukaryotes. Although regulation of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] signaling via PTEN has been well established in model organisms and mammals, it remains elusive in the parasitic protist E. histolytica, which heavily relies on PtdIns phosphate(s)-dependent membrane traffic, migration, and phago- and trogocytosis for its pathogenesis. In this study, we characterized the major PTEN from E. histolytica, EhPTEN1, which shows the highest expression at the transcript level in the trophozoite stage among 6 possible PTENs, to understand the significance of PtdIns(3,4,5)P3 signaling in this parasite. Live imaging of GFP-EhPTEN1 expressing amebic trophozoites showed localization mainly in the cytosol with a higher concentration at pseudopods and the extending edge of the phago- and trogocytic cups. Furthermore, quantitative analysis of phago- and trogocytosis using a confocal image cytometer showed that overexpression of EhPTEN1 caused reduction in trogo- and phagocytosis while transcriptional gene silencing of EhPTEN1 gene caused opposite phenotypes. These data suggest that EhPTEN1 has an inhibitory role in these biological processes. Conversely, EhPTEN1 acts as a positive regulator for fluid-phase and receptor-mediated endocytosis in E. histolytica trophozoites. Moreover, we showed that EhPTEN1 was required for optimal growth and migration of this parasite. Finally, the phosphatase activity of EhPTEN1 towards PtdIns(3,4,5)P3 was demonstrated, suggesting that the biological roles of EhPTEN1 are likely linked to its catalytic function. Taken together, these results indicate that EhPTEN1 differentially regulates multiple cellular activities essential for proliferation and pathogenesis of the organism, via PtdIns(3,4,5)P3 signaling. Elucidation of biological roles of PTEN and PtdIns(3,4,5)P3 signaling at the molecular levels promotes our understanding of the pathogenesis of this parasite and potentially leads to the design of novel therapeutics against amebiasis.

Accumulation dynamics of ARGONAUTE proteins during meiosis in Arabidopsis

Meiosis is a specialized cell division that is key for reproduction and genetic diversity in sexually reproducing plants. Recently, different RNA silencing pathways have been proposed to carry a specific activity during meiosis, but the pathways involved during this process remain unclear. Here, we explored the subcellular localization of different ARGONAUTE (AGO) proteins, the main effectors of RNA silencing, during male meiosis in Arabidopsis thaliana using immunolocalizations with commercially available antibodies. We detected the presence of AGO proteins associated with posttranscriptional gene silencing (AGO1, 2, and 5) in the cytoplasm and the nucleus, while AGOs associated with transcriptional gene silencing (AGO4 and 9) localized exclusively in the nucleus. These results indicate that the localization of different AGOs correlates with their predicted roles at the transcriptional and posttranscriptional levels and provide an overview of their timing and potential role during meiosis.

Small RNA Profiling of Susceptible and Resistant Ty-1 Encoding Tomato Plants Upon Tomato Yellow Leaf Curl Virus Infection

Ty-1 presents an atypical dominant resistance gene that codes for an RNA-dependent RNA polymerase (RDR) of the gamma class and confers resistance to tomato yellow leaf curl virus (TYLCV) and other geminiviruses. Tomato lines bearing Ty-1 not only produce relatively higher amounts of viral small interfering (vsi)RNAs, but viral DNA also exhibits a higher amount of cytosine methylation. Whether Ty-1 specifically enhances posttranscriptional gene silencing (PTGS), leading to a degradation of RNA target molecules and primarily relying on 21–22 nucleotides (nts) siRNAs, and/or transcriptional gene silencing (TGS), leading to the methylation of cytosines within DNA target sequences and relying on 24-nts siRNAs, was unknown. In this study, small RNAs were isolated from systemically TYLCV-infected leaves of Ty-1 encoding tomato plants and susceptible tomato Moneymaker (MM) and sequence analyzed. While in susceptible tomato plants vsiRNAs of the 21-nt size class were predominant, their amount was drastically reduced in tomato containing Ty-1. The latter, instead, revealed elevated levels of vsiRNAs of the 22- and 24-nt size classes. In addition, the genomic distribution profiles of the vsiRNAs were changed in Ty-1 plants compared with those from susceptible MM. In MM three clear hotspots were seen, but these were less pronounced in Ty-1 plants, likely due to enhanced transitive silencing to neighboring viral genomic sequences. The largest increase in the amount of vsiRNAs was observed in the intergenic region and the V1 viral gene. The results suggest that Ty-1 enhances an antiviral TGS response. Whether the elevated levels of 22 nts vsiRNAs contribute to an enhanced PTGS response or an additional TGS response involving a noncanonical pathway of RNA dependent DNA methylation remains to be investigated.

Evolutionary history of the vertebrate Piwi gene family

PIWIs are regulatory proteins that belong to the Argonaute family. Piwis are primarily expressed in gonads and protect the germline against the mobilization and propagation of transposable elements (TEs) through transcriptional gene silencing. Vertebrate genomes encode up to four Piwi genes: Piwil1, Piwil2, Piwil3 and Piwil4, but their duplication history is unresolved. We leveraged phylogenetics, synteny and expression analyses to address this void. Our phylogenetic analysis suggests Piwil1 and Piwil2 were retained in all vertebrate members. Piwil4 was the result of Piwil1 duplication in the ancestor of gnathostomes, but was independently lost in ray-finned fishes and birds. Further, Piwil3 was derived from a tandem Piwil1 duplication in the common ancestor of marsupial and placental mammals, but was secondarily lost in Atlantogenata (Xenarthra and Afrotheria) and some rodents. The evolutionary rate of Piwil3 is considerably faster than any Piwi among all lineages, but an explanation is lacking. Our expression analyses suggest Piwi expression has mostly been constrained to gonads throughout vertebrate evolution. Vertebrate evolution is marked by two early rounds of whole genome duplication and many multigene families are linked to these events. However, our analyses suggest Piwi expansion was independent of whole genome duplications.

Dysregulation of RNA interference components in COVID-19 patients

Objective Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus causing severe respiratory illness (COVID-19). This virus was initially identified in Wuhan city, a populated area of the Hubei province in China, and still remains one of the major global health challenges. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing that plays a crucial role in innate viral defense mechanisms by inhibiting the virus replication as well as expression of various viral proteins. Dicer, Drosha, Ago2, and DGCR8 are essential components of the RNAi system, which is supposed to be dysregulated in COVID-19 patients. This study aimed to assess the expression level of the mentioned mRNAs in COVID-19patients compared to healthy individuals. Results Our findings demonstrated that the expression of Dicer, Drosha, and Ago2 was statistically altered in COVID-19 patients compared to healthy subjects. Ultimately, the RNA interference mechanism as a crucial antiviral defense system was suggested to be dysregulated in COVID-19 patients.

Mastrevirus Rep and RepA Proteins Suppress de novo Transcriptional Gene Silencing

Transcriptional gene silencing (TGS) in plants is a defense mechanism against DNA virus infection. The genomes of viruses in the Geminiviridae family encode several TGS suppressors. In this study, we induced de novo TGS against the transgenic GFP gene encoding green fluorescent protein by expressing a hairpin-shaped self-complementary RNA corresponding to the enhancer region of the 35S promoter (hpE35S). In addition, we examined the TGS suppression activity of proteins encoded in the genome of Tobacco yellow dwarf virus (TYDV, genus Mastrevirus). The results show that the replication-associated protein (Rep) and RepA encoded by TYDV have TGS suppressor activity and lead to decreased accumulation of 24-nt siRNAs. These results suggest that Rep and RepA can block the steps before the loading of siRNAs into Argonaute (AGO) proteins. This is the first report of TGS suppressors in the genus Mastrevirus.

Arabidopsis ASYMMETRIC LEAVES2 (AS2): roles in plant morphogenesis, cell division, and pathogenesis

The ASYMMETRIC LEAVES2 (AS2) gene in Arabidopsis thaliana is responsible for the development of flat, symmetric, and extended leaf laminae and their vein systems. AS2 protein is a member of the plant-specific AS2/LOB protein family, which includes 42 members comprising the conserved amino-terminal domain referred to as the AS2/LOB domain, and the variable carboxyl-terminal region. Among the members, AS2 has been most intensively investigated on both genetic and molecular levels. AS2 forms a complex with the myb protein AS1, and is involved in epigenetic repression of the abaxial genes ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3), ARF4, and class 1 KNOX homeobox genes. The repressed expression of these genes by AS2 is markedly enhanced by the cooperative action of various modifier genes, some of which encode nucleolar proteins. Further downstream, progression of the cell division cycle in the developing organs is stimulated; meristematic states are suppressed in determinate leaf primordia; and the extension of leaf primordia is induced. AS2 binds the specific sequence in exon 1 of ETT/ARF3 and maintains methylated CpGs in several exons of ETT/ARF3. AS2 forms bodies (designated as AS2 bodies) at nucleolar peripheries. AS2 bodies partially overlap chromocenters, including inactive 45S ribosomal DNA repeats, suggesting the presence of molecular and functional links among AS2, the 45S rDNAs, and the nucleolus to exert the repressive regulation of ETT/ARF3. The AS2/LOB domain is characterized by three subdomains, the zinc finger (ZF) motif, the internally conserved-glycine containing (ICG) region, and the leucine-zipper-like (LZL) region. Each of these subdomains is essential for the formation of AS2 bodies. ICG to LZL are required for nuclear localization, but ZF is not. LZL intrinsically has the potential to be exported to the cytoplasm. In addition to its nuclear function, it has been reported that AS2 plays a positive role in geminivirus infection: its protein BV1 stimulates the expression of AS2 and recruits AS2 to the cytoplasm, which enhances virus infectivity by suppression of cytoplasmic post transcriptional gene silencing.