Investigation of P1/HC-Pro-Mediated ABA/Calcium Signaling Responses via Gene Silencing through High- and Low-Throughput RNA-seq Approaches

The P1/HC-Pro viral suppressor of potyvirus suppresses posttranscriptional gene silencing (PTGS). The fusion protein of P1/HC-Pro can be cleaved into P1 and HC-Pro through the P1 self-cleavage activity, and P1 is necessary and sufficient to enhance PTGS suppression of HC-Pro. To address the modulation of gene regulatory relationships induced by turnip mosaic virus (TuMV) P1/HC-Pro (P1/HC-ProTu), a comparative transcriptome analysis of three types of transgenic plants (P1Tu, HC-ProTu, and P1/HC-ProTu) were conducted using both high-throughput (HTP) and low-throughput (LTP) RNA-Seq strategies. The results showed that P1/HC-ProTu disturbed the endogenous abscisic acid (ABA) accumulation and genes in the signaling pathway. Additionally, the integrated responses of stress-related genes, in particular to drought stress, cold stress, senescence, and stomatal dynamics, altered the expressions by the ABA/calcium signaling. Crosstalk among the ABA, jasmonic acid, and salicylic acid pathways might simultaneously modulate the stress responses triggered by P1/HC-ProTu. Furthermore, the LTP network analysis revealed crucial genes in common with those identified by the HTP network in this study, demonstrating the effectiveness of the miniaturization of the HTP profile. Overall, our findings indicate that P1/HC-ProTu-mediated suppression in RNA silencing altered the ABA/calcium signaling and a wide range of stress responses.

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.

Expression 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 or 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.

The epigenetic factor FVE orchestrates cytoplasmic SGS3-DRB4-DCL4 activities to promote transgene silencing in Arabidopsis

Posttranscriptional gene silencing (PTGS) is a regulatory mechanism to suppress undesired transcripts. Here, we identified Flowering locus VE (FVE), a well-known epigenetic component, as a new player in cytoplasmic PTGS. Loss-of-function fve mutations substantially reduced the accumulation of transgene-derived small interfering RNAs (siRNAs). FVE interacts with suppressor of gene silencing 3 (SGS3), a master component in PTGS. FVE promotes SGS3 homodimerization that is essential for its function. FVE can bind to single-stranded RNA and double-stranded RNA (dsRNA) with moderate affinities, while its truncated form FVE-8 has a significantly increased binding affinity to dsRNA. These affinities affect the association and channeling of SGS3-RNA to downstream dsRNA binding protein 4 (DRB4)/Dicer-like protein 2/4 (DCL2/4) complexes. Hence, FVE, but not FVE-8, biochemically enhances the DRB4/DCL2/4 activity in vitro. We surmise that FVE promotes production of transgene-derived siRNAs through concertedly tuning SGS3-DRB4/DCL2/4 functions. Thus, this study revealed a noncanonical role of FVE in PTGS.

Investigation of the effects of P1 on HC-Pro-mediated gene silencing suppression through genetics and omics approaches

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. 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, specifically the AGO1 was specifically posttranslationally degraded in transgenic Arabidopsis expressing P1/HC-Pro of turnip mosaic virus (TuMV) (P1/HCTu 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.

Investigation of the Effects of P1 on Hc-Pro-Mediated Gene Silencing Suppression Through Genetics and Omics Approaches

Background: Viral suppressor negatively controls posttranscriptional gene silencing (PTGS) byinhibiting microRNA (miRNA) and short-interfering RNA (siRNA)regulation in plants. The first identified viral suppressor-P1/HC-Pro is afusion protein. Upon infecting plants, theP1 protein itself gets released from HC-Pro bythe self-cleaving activity of P1. P1 has an unknown function in enhancing HC-Pro-mediated PTGS suppression.We performed proteomics to identifyP1-interacting proteins and observedwhole gene correlationsin P1/HC-Pro-mediated PTGS suppression through transcriptomic studied comparative networks.Results: First, we demonstrated that P1 enhances HC-Pro function and that the mechanism might workthrough the P1 binding to VERNALIZATION INDEPENDENCE 3/ SUPERKILLER8 (VIP3/SKI8),a subunit of the exosome, to interferewith the 5'-fragment of the PTGS-cleaved RNA degradation product.Second,specifically the AGO1 wasposttranslationaldegraded in transgenic Arabidopsis expressing P1/HC-Pro of turnip mosaic virus (TuMV) (P1/HCTu plant).Third, transcriptomic comparative networkshighlighted critical genes inPTGS, including miRNA targets,calcium signaling, hormone (JA, ET, and ABA) signaling, and defense response.Conclusion: Through thesetransgenic and omics approaches, we revealed an overall perspective and new findings in our understandingof the mechanism of P1/HC-Pro-mediated PTGS suppression. Many of the criticalgenes that weresignificantlyimpacted in the omics profiles will be further investigated by CRISPR-knockoutor gain-of-function to understand PTGS in plant better.

An efficient virus-induced gene silencing (VIGS) system for functional genomics in Brassicas using a cabbage leaf curl virus (CaLCuV)-based vector

Background: Virus-induced gene silencing (VIGS), a posttranscriptional gene silencing method, represents an effective technology for the analysis gene functions in plants. However, no VIGS vectors are available for Brassica oleracea till now. The gene silencing vectors TRV, pTYs and CaLCuV were chose to improve the VIGS system in cabbage, using phytoene desaturase (PDS) gene as an efficient visual indicator for VIGS.Results: We successfully silenced the expression of PDS and observed the photobleaching phenomenon in cabbage by pTYs and CaLCuV, with the latter displaying higher efficiency and lower cost. Then, the factors potentially affecting the silencing efficiency of VIGS by CaLCuV in cabbage, including targeting fragment strategy, inoculation method and incubation temperature, were compared. The optimized CaLCuV-based VIGS system is as follows: approximately 500 bp insert sequence, Agrobacterium OD600=1.0, vacuum osmosis method at the bud stage, and the incubation temperature of 22 °C, through which we achieved a stable silencing efficiency of 65%. To further test the effectiveness of the system, we selected two other genes in cabbage including Mg-chelataseH subunit (ChlH) and lobed-leaf 1 (LL1) to knock down their expression, and we observed the expected yellow or lobed leaves. In addition, we successfully applied the CaLCuV-based VIGS system in two other representative Brassica crops including B. rapa and B. nigra and thus expanded its application scope.Conclusion: These results demonstrate that CaLCuV-based VIGS effectively work in cabbage and can be used for analyzing gene function. Our VIGS system described here will contribute to efficient functional genomics research in Brassica crops.

Small RNA In Situ Hybridizations on Sections of Arabidopsis Embryos

Small RNAs mediate posttranscriptional gene silencing in plants and animals. This often occurs in specific cell or tissue types and can be necessary for their differentiation. Determining small RNA (sRNA) localization patterns at cellular resolution can therefore provide information on the corresponding gene regulatory processes they are involved in. Recent improvements with in situ hybridization methods have allowed them to be applied to sRNAs. Here we describe an in situ hybridization protocol to detect sRNAs from sections of early staged Arabidopsis thaliana (Arabidopsis) embryos.

The viral F-box protein P0 induces an ER-derived autophagy degradation pathway for the clearance of membrane-bound AGO1

RNA silencing is a major antiviral defense mechanism in plants and invertebrates. Plant ARGONAUTE1 (AGO1) is pivotal in RNA silencing, and hence is a major target for counteracting viral suppressors of RNA-silencing proteins (VSRs). P0 from Turnip yellows virus (TuYV) is a VSR that was previously shown to trigger AGO1 degradation via an autophagy-like process. However, the identity of host proteins involved and the cellular site at which AGO1 and P0 interact were unknown. Here we report that P0 and AGO1 associate on the endoplasmic reticulum (ER), resulting in their loading into ER-associated vesicles that are mobilized to the vacuole in an ATG5- and ATG7-dependent manner. We further identified ATG8-Interacting proteins 1 and 2 (ATI1 and ATI2) as proteins that associate with P0 and interact with AGO1 on the ER up to the vacuole. Notably, ATI1 and ATI2 belong to an endogenous degradation pathway of ER-associated AGO1 that is significantly induced following P0 expression. Accordingly, ATI1 and ATI2 deficiency causes a significant increase in posttranscriptional gene silencing (PTGS) activity. Collectively, we identify ATI1 and ATI2 as components of an ER-associated AGO1 turnover and proper PTGS maintenance and further show how the VSR P0 manipulates this pathway.