DCL1 Research Articles

Resistance gene Ty-1 restricts TYLCV infection in tomato by increasing RNA silencing

A major antiviral mechanism in plants is mediated by RNA silencing through the action of DICER-like (DCL) proteins, which cleave dsRNA into discrete small RNA fragments, and ARGONAUTE (AGO) proteins, which use the small RNAs to target single-stranded RNA. RNA silencing can also be amplified through the action of RNA-dependent RNA polymerases (RDRs), which use single stranded RNA to generate dsRNA that in turn is targeted by DCL proteins. As a counter-defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different components in the RNA silencing pathway. The tomato Ty-1 gene confers resistance to the DNA virus tomato yellow leaf curl virus (TYLCV) and has been reported to encode an RDRγ protein. However, the molecular mechanisms by which Ty-1 controls TYLCV infection, including whether Ty-1 is involved in RNA silencing, are unknown. Here, by using a transient expression assay, we have confirmed that Ty-1 shows antiviral activity against TYLCV in Nicotiana benthamiana. Also, in transient expression-based silencing assays, Ty-1 augmented systemic transgene silencing in GFP transgenic N. benthamiana plants. Furthermore, co-expression of Ty-1 or other RDRγ proteins from N. benthamiana or Arabidopsis with various proteins resulted in lower protein expression. These results are consistent with a model wherein Ty-1-mediated resistance to TYLCV is due, at least in part, to an increase in RNA silencing activity.

Structural determinants in the miRNA/miRNA* duplex and the DCL1 PAZ domain for precise and efficient plant miRNA processing.

The accessory proteins Hyponastic-like 1 (HYL1) and Serrated (SE) enhance the precise and efficient processing of miRNAs by Dicer-like 1 (DCL1), which is important for proper miRNA function. However, other factors determining the precision and efficiency of miRNA biogenesis are not well-known. Here, we found that an asymmetric bulge (AB) at the 3' end of miR-5p (produced from the 5' arm of the pre-miRNA) reduced the precision of the second cleavage, whereas an AB at other sites of miR-5p mainly affected the accumulation level of miR-5p in transient expression in Nicotiana benthamiana. In contrast, many ABs in miR-3p (produced from the 3' arm of the pre-miRNA) impose strong negative impact on the processing precision and the accumulation level of miR-5p in N. benthamiana. Arabidopsis DCL1/SE/HYL1 complex-mediated miRNA processing was reconstituted in Saccharomyces cerevisiae to further investigate AB-mediated interference with DCL1 processing. With this system, the positional effect of AB on miRNA processing was tested. The results showed that ABs on the middle of miR-5p have less of an impact on DCL1 cleavage efficiency and precision, whereas those on miR-3p or near the ends of miR-5p strongly reduce DCL1 cleavage activity, precision or both. Studies using the yeast miRNA processing system and transgenic Arabidopsis also revealed the importance of the interaction between the 2-nt 3' overhang of pre-miRNA and the 3' overhang binding pocket (3'BP) on the precision of the second cleavage reaction for many endogenous miRNAs. These findings provide new insights into the mechanism of miRNA biogenesis.

Genome-wide identification of gene families related to miRNA biogenesis in Mangifera indica L. and their possible role during heat stress.

Mango is a popular tropical fruit that requires quarantine hot water treatment (QHWT) for postharvest sanitation, which can cause abiotic stress. Plants have various defense mechanisms to cope with stress; miRNAs mainly regulate the expression of these defense responses. Proteins involved in the biogenesis of miRNAs include DICER-like (DCL), ARGONAUTE (AGO), HYPONASTIC LEAVES 1 (HYL1), SERRATE (SE), HUA ENHANCER1 (HEN1), HASTY (HST), and HEAT-SHOCK PROTEIN 90 (HSP90), among others. According to our analysis, the mango genome contains five DCL, thirteen AGO, six HYL, two SE, one HEN1, one HST, and five putative HSP90 genes. Gene structure prediction and domain identification indicate that sequences contain key domains for their respective gene families, including the RNase III domain in DCL and PAZ and PIWI domains for AGOs. In addition, phylogenetic analysis indicates the formation of clades that include the mango sequences and their respective orthologs in other flowering plant species, supporting the idea these are functional orthologs. The analysis of cis-regulatory elements of these genes allowed the identification of MYB, ABRE, GARE, MYC, and MeJA-responsive elements involved in stress responses. Gene expression analysis showed that most genes are induced between 3 to 6 h after QHWT, supporting the early role of miRNAs in stress response. Interestingly, our results suggest that mango rapidly induces the production of miRNAs after heat stress. This research will enable us to investigate further the regulation of gene expression and its effects on commercially cultivated fruits, such as mango, while maintaining sanitary standards.

Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence.

Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs effectively silenced a β-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes showed increased amounts of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs.

Widespread changes to the translational landscape in a maize microRNA biogenesis mutant.

MicroRNAs are short, non-coding RNAs that repress gene expression in both plants and animals and have diverse functions related to growth, development, and stress responses. The ribonuclease, DICER-LIKE1 (DCL1) is required for two steps in plant miRNA biogenesis: cleavage of the primary miRNAs (pri-miRNAs) to release a hairpin structure, called the precursor miRNA (pre-miRNA) and cleavage of the pre-miRNA to generate the miRNA/miRNA* duplex. The mature miRNA guides the RNA-induced silencing complex to target RNAs with complementary sequences, resulting in translational repression and/or RNA cleavage of target mRNAs. However, the relative contribution of translational repression versus mRNA degradation by miRNAs remains unknown at the genome-level in crops, especially in maize. The maize fuzzy tassel (fzt) mutant contains a hypomorphic mutation in DCL1 resulting in broad developmental defects. While most miRNAs are reduced in fzt, the levels of miRNA-targeted mRNAs are not dramatically increased, suggesting that translational regulation by miRNAs may be common. To gain insight into the repression mechanism of plant miRNAs, we combined ribosome profiling and RNA-sequencing to globally survey miRNA activities in maize. Our data indicate that translational repression contributes significantly to regulation of most miRNA targets and that approximately one-third of miRNA targets are regulated primarily at the translational level. Surprisingly, ribosomes appear altered in fzt mutants suggesting that DCL1 may also have a role in ribosome biogenesis. Thus, DICER-LIKE1 shapes the translational landscape in plants through both miRNA-dependent and miRNA-independent mechanisms.

РОЛЬ МИКРОРНК В РАЗВИТИИ УСТОЙЧИВОСТИ РАСТЕНИЙ К ХОЛОДУ

Plants, as sessile organisms, are forced to adapt to changes in the environment, including temperature changes that limit growth, development and productivity). Abiotic stresses have become a serious problem due to their widespread nature and destructive effects on plants. However, plants have developed complex ways to perceive and respond to environmental changes. The mechanisms of plant resistance to hypothermia have been studied for a long time and transcriptional control of expression of genes sensitive to cold has been studied in some detail. To activate protective mechanisms, plants trigger a network of genetic regulators, including changing the expression of a significant part of genes using transcriptional and/or translational regulators. microRNAs can be considered important participants in the response of plants to cold stress. The key to understanding the involvement of microRNAs in plant stress reactions was studies that showed the regulation of their expression under stress. microRNAs are small non-coding RNAs that manifest themselves as important regulatory components in plants. microRNA expression includes transcription of microRNA genes by RNA polymerase II, multistage processing of primary transcripts using the enzyme DICER-LIKE1 (DCL1) and the formation of an effector complex consisting of miRNAs and proteins of the ARGONAUTE (AGO) family. Such complexes interact with complementary RNA targets, suppressing their expression at the transcriptional and post-transcriptional levels. Thus, microRNAs regulate a variety of biological processes, including responses to changes in the environment. Currently, microRNAs are considered as an important tool for regulating the work of genes. Such significant processes in plants as maintaining homeostasis, growth and development, transition from the vegetative to the reproductive phase, signaling and response to various stresses are regulated by microRNAs. The article describes the role of microRNAs in the response of plants to cold stress, analyzes microRNA targets, and discusses the prospects for using microRNAs in the practice of improving plant resistance to external influences.

Principles of miRNA/miRNA* function in plant MIRNA processing.

MicroRNAs (miRNAs) are essential regulators of gene expression, defined by their unique biogenesis, which requires the precise excision of the small RNA from an imperfect fold-back precursor. Unlike their animal counterparts, plant miRNA precursors exhibit variations in sizes and shapes. Plant MIRNAs can undergo processing in a base-to-loop or loop-to-base direction, with DICER-LIKE1 (DCL1) releasing the miRNA after two cuts (two-step MIRNAs) or more (sequential MIRNAs). In this study, we demonstrate the critical role of the miRNA/miRNA* duplex region in the processing of miRNA precursors. We observed that endogenous MIRNAs frequently experience suboptimal processing in vivo due to mismatches in the miRNA/miRNA* duplex, a key region that fine-tunes miRNA levels. Enhancing the interaction energy of the miRNA/miRNA* duplex in two-step MIRNAs results in a substantial increase in miRNA levels. Conversely, sequential MIRNAs display distinct and specific requirements for the miRNA/miRNA* duplexes along their foldback structure. Our work establishes a connection between the miRNA/miRNA* structure and precursor processing mechanisms. Furthermore, we reveal a link between the biological function of miRNAs and the processing mechanism of their precursors with the evolution of plant miRNA/miRNA* duplex structures.

Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery.

Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors in vivo. Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.

Evidence for an RNAi-independent role of Arabidopsis DICER-LIKE2 in growth inhibition and basal antiviral resistance.

Flowering plant genomes encode four or five DICER-LIKE (DCL) enzymes that produce small interfering RNAs (siRNAs) and microRNAs, which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense, and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense. However, DCL2 may also counteract DCL4 since knockout of DCL4 causes growth defects that are suppressed by DCL2 inactivation. Current models maintain that RNAi via DCL2-dependent siRNAs is the biochemical basis of both effects. Here, we report that DCL2-mediated antiviral resistance and growth defects cannot be explained by the silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either with specific point mutations in DCL2 or with reduced DCL2 dosage because of heterozygosity for dcl2 knockout alleles. Intriguingly, all DCL2 functions require its catalytic activity, and the penetrance of DCL2-dependent growth phenotypes in dcl4 mutants correlates with DCL2 protein levels but not with levels of major DCL2-dependent siRNAs. We discuss this requirement and correlation with catalytic activity but not with resulting siRNAs, in light of other findings that reveal a DCL2 function in innate immunity activation triggered by cytoplasmic double-stranded RNA.

Knockout of SlDCL2b attenuates the resistance of tomato to potato spindle tuber viroid infection.

RNA interference, or RNA silencing, is an important defence mechanism against viroid infection in plants. Plants encode multiple DICER-LIKE (DCL) proteins that are key components of the RNA silencing pathway. However, the roles of different DCLs in defence responses against viroid infection remain unclear. Here, we determined the function of tomato DCL2b (SlDCL2b) in defence responses against potato spindle tuber viroid (PSTVd) infection using SlDCL2b loss-of-function tomato mutant plants. Compared with wild-type plants, mutant plants were more susceptible to PSTVd infection, developing more severe symptoms earlier and accumulating higher levels of PSTVd RNAs. Moreover, we verified the feedback mechanism for the regulation of SlDCL2b expression by miR6026. Functional blocking of tomato miR6026, by expressing its target mimics, can enhance resistance to PSTVd infection in tomato plants. These findings deepen the current understanding of RNAi-based resistance against viroid infection and provide a potentially new strategy for viroid control.

Transcriptome and small RNAome profiling uncovers how a recombinant begomovirus evades RDRγ-mediated silencing of viral genes and outcompetes its parental virus in mixed infection.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) causes severe disease of cultivated tomatoes. Geminiviruses replicate circular single-stranded genomic DNA via rolling-circle and recombination-dependent mechanisms, frequently generating recombinants in mixed infections. Circular double-stranded intermediates of replication also serve as templates for Pol II bidirectional transcription. IS76, a recombinant derivative of TYLCV with a short sequence in the bidirectional promoter/origin-of-replication region acquired from a related begomovirus, outcompetes TYLCV in mixed infection and breaks disease resistance in tomato Ty-1 cultivars. Ty-1 encodes a γ-clade RNA-dependent RNA polymerase (RDRγ) implicated in Dicer-like (DCL)-mediated biogenesis of small interfering (si)RNAs directing gene silencing. Here, we profiled transcriptome and small RNAome of Ty-1 resistant and control susceptible plants infected with TYLCV, IS76 or their combination at early and late infection stages. We found that RDRγ boosts production rates of 21, 22 and 24 nt siRNAs from entire genomes of both viruses and modulates DCL activities in favour of 22 and 24 nt siRNAs. Compared to parental TYLCV, IS76 undergoes faster transition to the infection stage favouring rightward transcription of silencing suppressor and coat protein genes, thereby evading RDRγ activity and facilitating its DNA accumulation in both single and mixed infections. In coinfected Ty-1 plants, IS76 efficiently competes for host replication and transcription machineries, thereby impairing TYLCV replication and transcription and forcing its elimination associated with further increased siRNA production. RDRγ is constitutively overexpressed in Ty-1 plants, which correlates with begomovirus resistance, while siRNA-generating DCLs (DCL2b/d, DCL3, DCL4) and genes implicated in siRNA amplification (α-clade RDR1) and function (Argonaute2) are upregulated to similar levels in TYLCV- and IS76-infected susceptible plants. Collectively, IS76 recombination facilitates replication and promotes expression of silencing suppressor and coat proteins, which allows the recombinant virus to evade the negative impact of RDRγ-boosted production of viral siRNAs directing transcriptional and posttranscriptional silencing.

Dicer-Like Protein 4 and RNA-Dependent RNA Polymerase 6 Are Involved in Tomato Torrado Virus Pathogenesis in Nicotiana benthamiana.

Tomato torrado virus (ToTV) is a type member of the Torradovirus genus in the Secoviridae family known to cause severe necrosis in susceptible tomato varieties. ToTV also infects other Solanaceae plants, including Nicotiana benthamiana, where it induces distinctive disease symptoms: plant growth drop with the emergence of spoon-like malformed systemic leaves. Virus-induced post-transcriptional gene silencing (PTGS) is significant among plant defense mechanisms activated upon virus invasion. The PTGS, however, can be counteracted by suppressors of RNA silencing commonly found in viruses, which efficiently disrupt the antiviral defense of their host. Here, we addressed the question of PTGS antiviral activity and its suppression in N. benthamiana during ToTV infection-a phenomenon not described for any representative from the Torradovirus genus so far. First, we showed that neither the Vp26-a necrosis-inducing pathogenicity determinant of ToTV-nor other structural viral proteins limited the locally induced PTGS similar to p19, a well-characterized potent suppressor of RNA silencing of tombusviruses. Moreover, by employing wild-type and transgenic lines of N. benthamiana with suppressed Dicer-like 2 (DCL2), Dicer-like 4 (DCL4), Argonaute 2 and RNA-dependent RNA polymerase 6 (RDR6) proteins, we proved their involvement in anti-ToTV defense. Additionally, we identified DCL4 as the major processor of ToTV-derived siRNA. More importantly, our results indicate the essential role of the Suppressor of Gene Silencing 3 (SGS3)/RDR6 pathway in anti-ToTV defense. Finally, we conclude that ToTV might not require a potent RNA silencing suppressor during infection of the model plant N. benthamiana.

Comprehensive analysis of Dicer-like, Argonaute, and RNA-dependent RNA polymerase gene families and their expression analysis in response to abiotic stresses in Jatropha curcas

ABSTRACT Eukaryotic gene expression is regulated by RNA interference (RNAi). Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) are essential elements involved in the process of RNAi. However, Jatropha (J.) curcas, an oil-producing plant, has not undergone any previous systematic analysis or expression profiling of these genes. In this study, a total of four JcDCL, nine JcAGO, and seven JcRDR genes were identified in the J. curcas genome. Based on phylogenetic analysis, each gene family was classified into four clades and randomly distributed on nine J. curcas chromosomes. Protein sequence alignment revealed that the classical PAZ, Piwi, and RdRP domains were present in all JcDCLs, JcAGOs, and JcRDRs, respectively. Comprehensive expression pattern analysis using RNA-seq data showed various expression patterns in different organs and under different abiotic stresses. In addition, special attention should be paid to JcDCL1, JcAGO1, JcAGO4, and JcRDR5, which play important roles in response to various stresses.

Cannabis Virome Reconstruction and Antiviral RNAi Characterization through Small RNA Sequencing

Viral infections pose an emerging threat to hemp (Cannabis sativa) cultivation. We used Illumina small (s)RNA sequencing for virome reconstruction and characterization of antiviral RNA interference (RNAi) in monoecious and dioecious hemp varieties, which exhibited different virus-like symptoms. Through de novo and reference-based sRNA assembly, we identified and reconstructed Cannabis cryptic virus (family Partitiviridae), Cannabis sativa mitovirus 1 (Mitoviridae) and Grapevine line pattern virus (Bromoviridae) as well as a novel virus tentatively classified into Partitiviridae. Members of both Partitiviridae and Bromoviridae were targeted by antiviral RNAi, generating 21 nt and, less abundant, 22 nt sRNAs from both strands of the entire virus genome, suggesting the involvement of Dicer-like (DCL) 4 and DCL2 in viral sRNA biogenesis, respectively. Mitovirus sRNAs represented predominantly the positive-sense strand and had a wider size range, with the 21 nt class being most abundant on both strands. For all viruses, 21 and 22 nt sRNAs had predominantly 5′-terminal uridine or cytosine, suggesting their binding to antiviral Argonaute (AGO) 1 and AGO5, respectively. As no clear association of any virus with symptoms was observed, further studies should clarify if these viruses individually or in combination can cause hemp diseases.

Identification and Analysis of Protein Family Associated with RNA Interference Pathway in Juglandaceae.

One of the crucial processes for small RNA synthesis and plant disease resistance is RNA interference (RNAi). Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), double-stranded RNA binding (DRB), and Argonaute are important proteins implicated in RNAi (AGO). Numerous significant woody plants belong to the Juglandaceae; walnut is one of the four groups of woody plants on earth and one of the four groups of dried fruits. In order to correlate walnuts and their homologues, this work integrated numerous web resources from structural analysis and transcriptome data collected from gene families in order to elucidate the evolution and functional differentiation of RNA-related proteins in the walnut (Juglans rega) genome. 5 DCL genes, 13 RDR genes, 15 DRB genes, and 15 AGO genes are found in the walnut genome and encode conserved protein domains and motifs with similar subcellular distribution.There are three classes and seven subclasses of walnut AGO proteins. RDRS are primarily split into four categories, whereas DRBs can be divided into six. DCLs are separated into four groups. The walnut RDR1 copy number of 9 is the exception, with 7 of those copies being dispersed in clusters on chromosome 16. Proteins are susceptible to various levels of purification selection, but in walnut, purification selection drives gene creation. These findings also indicated some resemblance in other plants belonging to the walnut family. Under various tissues and stresses, many RNA-related genes in walnut produced abundant, selective expression. In this study, the genome of the Juglandaceae's DCL, RDR, DRB, and AGO gene families were discovered and analysed for the first time. The evolution, structure, and expression characteristics of these families were also preliminary studied, offering a foundation for the development and breeding of the walnut RNAi pathway.

Some Type III effectors of Ralstonia solanacearum may suppress host RNA silencing for function

Ralstonia solanacearum is a devastating bacterial pathogen responsible for bacterial wilt in various crops. It employs the Type III Secretion System (T3SS) to deliver effectors known as R. solanacearum injected proteins (Rips) into host plant cells. These Rips play a critical role in the pathogenicity of the bacterium, yet their mechanisms of action remain incompletely understood. In this study, we screened 13 Rips from R. solanacearum strain FJ1003 and discovered that RipAW and RipAU possess the ability to suppress RNA silencing, an essential gene regulatory mechanism in plants. Moreover, we demonstrated heightened susceptibility to R. solanacearum and potato virus X in transgenic Nicotiana benthamiana overexpressing RipAW. Silencing of Dicer-like 1 (DCL1) or RNA-dependent RNA polymerase 6 (RDR6) promoted the infection of R. solanacearum in N. benthamiana. Arabidopsis with defects in Argonaute 1 (AGO1), DCL1 or RDR6 also showed increased susceptibility to R. solanacearum compared to wildtype Arabidopsis. These findings collectively suggest a scenario in which certain Rips interfere with host plant RNA silencing, and RNA silencing suppression may be one of the mechanisms employed by Rips to promote virulence.

Dicer-like2b suppresses the wiry leaf phenotype in tomato induced by tobacco mosaic virus.

Dicer-like (DCL) proteins are principal components of RNA silencing, a major defense mechanism against plant virus infections. However, their functions in suppressing virus-induced disease phenotypes remain largely unknown. Here, we identified a role for tomato (Solanum lycopersicum) DCL2b in regulating the wiry leaf phenotype during defense against tobacco mosaic virus (TMV). Knocking out SlyDCL2b promoted TMV accumulation in the leaf primordium, resulting in a wiry phenotype in distal leaves. Biochemical and bioinformatics analyses showed that 22-nt virus-derived small interfering RNAs (vsiRNAs) accumulated less abundantly in slydcl2b mutants than in wild-type plants, suggesting that SlyDCL2b-dependent 22-nt vsiRNAs are required to exclude virus from leaf primordia. Moreover, the wiry leaf phenotype was accompanied by upregulation of Auxin Response Factors (ARFs), resulting from a reduction in trans-acting siRNAs targeting ARFs (tasiARFs) in TMV-infected slydcl2b mutants. Loss of tasiARF production in the slydcl2b mutant was in turn caused by inhibition of miRNA390b function. Importantly, silencing SlyARF3 and SlyARF4 largely restored the wiry phenotype in TMV-infected slydcl2b mutants. Our work exemplifies the complex relationship between RNA viruses and the endogenous RNA silencing machinery, whereby SlyDCL2b protects the normal development of newly emerging organs by excluding virus from these regions and thus maintaining developmental silencing.

Peptidyl-prolyl isomerase Cyclophilin71 promotes SERRATE phase separation and miRNA processing in Arabidopsis

MicroRNAs (miRNAs) play an important role in gene regulation. In Arabidopsis, mature miRNAs are processed from primary miRNA transcripts by the Dicing complex that contains Dicer-like 1 (DCL1), SERRATE (SE), and Hyponastic Leaves 1 (HYL1). The Dicing complex can form nuclear dicing bodies (D-bodies) through SE phase separation. Here, we report that Cyclophilin71 (CYP71), a peptidyl-prolyl isomerase (PPIase), positively regulates miRNA processing. We show that CYP71 directly interacts with SE and enhances its phase separation, thereby promoting the formation of D-body and increasing the activity of the Dicing complex. We further show that the PPIase activity is important for the function of CYP71 in miRNA production. Our findings reveal orchestration of miRNA processing by a cyclophilin protein and suggest the involvement of peptidyl-prolyl cis-trans isomerization, a structural mechanism, in SE phase separation and miRNA processing.

Epigenetic regulation of female germline development through ERECTA signaling pathway.

Germline development is a key step in sexual reproduction. Sexual plant reproduction begins with the formation of haploid spores by meiosis of megaspore mother cells (MMCs). Although many evidences, directly or indirectly, show that epigenetics plays an important role in MMC specification, how it controls the commitment of the MMC to downstream stages of germline development is still unclear. Electrophoretic mobility shift assay (EMSA), western blot, immunofluorescence, and chromatin immunoprecipitation coupled with quantitative PCR analyses were performed. Genetic interactions between BZR1 transcription factor family and the SWR1-SDG2-ER pathway in the control of female germline development were further studied. The present findings showed in Arabidopsis that two epigenetic factors, the chromatin remodeling complex SWI2/SNF2-RELATED 1 (SWR1) and a writer for H3K4me3 histone modification SET DOMAIN GROUP 2 (SDG2), genetically interact with the ERECTA (ER) receptor kinase signaling pathway and regulate female germline development by restricting the MMC cell fate to a single cell in the ovule primordium and ensure that only that single cell undergoes meiosis and subsequent megaspore degeneration. We also showed that SWR1-SDG2-ER signaling module regulates female germline development by promoting the protein accumulation of BZR1 transcription factor family on the promoters of primary miRNA processing factors, HYPONASTIC LEAVES 1 (HYL1), DICER-LIKE 1 (DCL1), and SERRATE (SE) to activate their expression. Our study elucidated a Gene Regulation Network that provides new insights for understanding how epigenetic factors and receptor kinase signaling pathways function in concert to control female germline development in Arabidopsis.

Genome-wide identification of DCL, AGO and RDR gene families and their associated functional regulatory element analyses in sunflower (Helianthus annuus).

RNA interference (RNAi) regulates a variety of eukaryotic gene expressions that are engaged in response to stress, growth, and the conservation of genomic stability during developmental phases. It is also intimately connected to the post-transcriptional gene silencing (PTGS) process and chromatin modification levels. The entire process of RNA interference (RNAi) pathway gene families mediates RNA silencing. The main factors of RNA silencing are the Dicer-Like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) gene families. To the best of our knowledge, genome-wide identification of RNAi gene families like DCL, AGO, and RDR in sunflower (Helianthus annuus) has not yet been studied despite being discovered in some species. So, the goal of this study is to find the RNAi gene families like DCL, AGO, and RDR in sunflower based on bioinformatics approaches. Therefore, we accomplished an inclusive in silico investigation for genome-wide identification of RNAi pathway gene families DCL, AGO, and RDR through bioinformatics approaches such as (sequence homogeneity, phylogenetic relationship, gene structure, chromosomal localization, PPIs, GO, sub-cellular localization). In this study, we have identified five DCL (HaDCLs), fifteen AGO (HaAGOs), and ten RDR (HaRDRs) in the sunflower genome database corresponding to the RNAi genes of model plant Arabidopsis thaliana based on genome-wide analysis and a phylogenetic method. The analysis of the gene structure that contains exon-intron numbers, conserved domain, and motif composition analyses for all HaDCL, HaAGO, and HaRDR gene families indicated almost homogeneity among the same gene family. The protein-protein interaction (PPI) network analysis illustrated that there exists interconnection among identified three gene families. The analysis of the Gene Ontology (GO) enrichment showed that the detected genes directly contribute to the RNA gene-silencing and were involved in crucial pathways. It was observed that the cis-acting regulatory components connected to the identified genes were shown to be responsive to hormone, light, stress, and other functions. That was found in HaDCL, HaAGO, and HaRDR genes associated with the development and growth of plants. Finally, we are able to provide some essential information about the components of sunflower RNA silencing through our genome-wide comparison and integrated bioinformatics analysis, which open the door for further research into the functional mechanisms of the identified genes and their regulatory elements.