Silenced Suppressor Genes Research Articles

Production of active human iduronate-2-sulfatase (IDS) enzyme in Nicotiana benthamiana

Many strategies have been developed to produce high levels of biologically active recombinant proteins in plants for biopharmaceutical purposes. However, the production of an active form of human iduronate-2-sulfatase (hIDS) for the treatment of Hunter syndrome by enzyme replacement therapy (ERT) is challenging due to the requirement for cotranslational modification by a formylglycine-producing enzyme encoded by sulfatase modifying factor 1 (hSUMF1) at the Cys84 residue, which converts it to C(alpha)-formylglycine. In this study, we have shown that hIDS can be highly expressed in N. benthamiana by using different constructs. Among them, BiP-GB1-L-dCBD1-2L-8xHis-L-6xHis-3L-EK-hIDS-HDEL (GB1-CBD1-hIDS) showed a high expression level when transiently co-expressed with the turnip crinkle virus gene silencing suppressor P38 and GB1-fused human calreticulin (GB1-CRT1) as a folding enhancer. The hSUMF1 was co-expressed with hIDS for cotranslational modification. The full-length recombinant proteins were purified using Ni2+-NTA affinity resin followed by enterokinase treatment to obtain tag-free hIDS. The N-terminal fragment was removed using microcrystalline cellulose (MCC) beads. The purified active form of hIDS can successfully cleave the sulfate group from an artificial substrate, 4-nitrocatechol sulfate, at a similar level to commercial hIDS expressed in animal cells. These results suggest that plants could be a promising platform for the production of recombinant hIDS.

The C5 protein of euphorbia leaf curl virus is a virulence factor and gene silencing suppressor

The genome of a monopartite begomovirus, or the DNA-A component of a bipartite begomovirus, typically encodes six proteins: two on the viral strand (AV1/V1 and AV2/V2) and four on the complementary strand (AC1/C1, AC2/C2, AC3/C3, AC4/C4). Recent studies, however, have identified additional begomoviral proteins with various functions. This paper reports that euphorbia leaf curl virus (EuLCV), a monopartite begomovirus, encodes a seventh protein, C5. Promoter activity of the upstream fragment of the EuLCV C5 gene was shown using a GUS expression vector. EuLCV C5 also enhanced the pathogenicity and accumulation of potato virus X (PVX) in Nicotiana benthamiana. Localization studies revealed that EuLCV C5 localizes to the cytoplasm and nucleus, forming granular structures on the cell membrane. Additionally, C5 acts as a post-transcriptional gene silencing (PTGS) suppressor. A C5 deletion mutant of EuLCV (EuLCV-ΔC5) exhibited reduced pathogenicity and viral accumulation compared to wild-type EuLCV in N. benthamiana.

High production of recombinant protein using geminivirus-based deconstructed vectors in Nicotiana benthamiana.

We focused on the geminiviral vector systems to develop an efficient vector system for plant biotechnology. Begomoviruses and curtoviruses, which belong to the Geminiviridae family, contain an intergenic region (IR) and four genes involved in replication, including replication-associated protein (Rep, C1), transcriptional activator (TrAP, C2), and replication enhancer (REn, C3). Geminiviruses can amplify thousands of copies of viral DNA using plant DNA polymerase and viral replication-related enzymes and accumulate viral proteins at high concentrations. In this study, we optimized geminiviral DNA replicon vectors based on tomato yellow leaf curl virus (TYLCV), honeysuckle yellow vein virus (HYVV), and mild curly top virus (BMCTV) for the rapid, high-yield plant-based production of recombinant proteins. Confirmation of the optimal combination by co-delivery of each replication-related gene and each IR harboring the Pontellina plumata-derived turbo green fluorescence protein (tGFP) gene via agroinfiltration in Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 3 days. Co-expression with the p19 protein of the tomato bush stunt virus, a gene-silencing suppressor, further enhanced tGFP accumulation by stabilizing mRNA. With this system, tGFP protein was produced at 0.7-1.2 mg/g leaf fresh weight, corresponding to 6.9-12.1% in total soluble protein. These results demonstrate the advantages of rapid and high-level production of recombinant proteins using the geminiviral DNA replicon system for transient expression in plants.

Genome-wide association studies meta-analysis uncovers NOJO and SGS3 novel genes involved in Arabidopsis thaliana primary root development and plasticity

BackgroundArabidopsis thaliana primary root growth has become a model for evo-devo studies due to its simplicity and facility to record cell proliferation and differentiation. To identify new genetic components relevant to primary root growth, we used a Genome-Wide Association Studies (GWAS) meta-analysis approach using data published in the last decade. In this work, we performed intra and inter-studies analyses to discover new genetic components that could participate in primary root growth.Methods and resultsWe used 639 accessions from nine different studies under control conditions and performed different GWAS tests. We found that primary root growth changes were associated with 41 genes, of which six (14.6%) have been previously described as inhibitors or promoters of primary root growth. The knockdown lines of two genes, Suppressor of Gene Silencing (SGS3), involved in tasiRNA processing, and a gene with a Sterile Alpha Motif (SAM) motif named NOJOCH MOOTS (NOJO), confirmed their role as repressors of primary root growth, none has been shown to participate in this developmental process before.ConclusionsIn summary, our GWAS analysis of different available studies identified new genes that participate in primary root growth; two of them were identified as repressors of primary root growth.

Pepper vein yellow virus P0 protein triggers NbHERC3, NbBax, and NbCRR mediated hypersensitive response.

P0 proteins encoded by the pepper vein yellow virus (PeVYV) are pathogenic factors that cause hypersensitive response (HR). However, the host gene expression related to PeVYV P0-induced HR has not been thoroughly studied. Transcriptomic technology was used to investigate the host pathways mediated by the PeVYV P0 protein to explore the molecular mechanisms underlying its function. We found 12,638 differentially expressed genes (DEGs); 6784 and 5854 genes were significantly upregulated and downregulated, respectively. Transcriptomic and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analyses revealed that salicylic acid (SA) and jasmonic acid (JA) synthesis-related gene expression was upregulated, and ethylene synthesis-related gene expression was downregulated. Ultrahigh performance liquid chromatography-tandem mass spectrometry was used to quantify SA and JA concentrations in Nicotiana benthamiana, and the P0 protein induced SA and JA biosynthesis. We then hypothesized that the pathogenic activity of the P0 protein might be owing to proteins related to host hormones in the SA and JA pathways, modulating host resistance at different times. Viral gene silencing suppression technology was used in N. benthamiana to characterize candidate proteins, and downregulating NbHERC3 (Homologous to E6-AP carboxy-terminus domain and regulator of choromosome condensation-1 dmain protein 3) accelerated cell necrosis in the host. The downregulation of NbCRR reduced cell death, while that of NbBax induced necrosis and curled heart leaves. Our findings indicate that NbHERC3, NbBax, and NbCRR are involved in P0 protein-driven cell necrosis.

Knockdown of orthotospovirus-derived silencing suppressor gene by plant-mediated RNAi approach induces viral resistance in tomato

RNA interference (RNAi) shows significant effectiveness in conferring resistance to viral infections in various plants. The current study was conducted to develop transgenic tomato (Solanum lycopersicum cv. Rio Grande) lines expressing a 320 bp conserved inverted region of the integral 35S promoter region. The molecular cloning was done for the construction of expression vectors in a hairpin structure by the Tomato spotted wilt orthotospovirus (TSWV) species Orthotospovirus tomatomaculae, silencing suppressor (NSs) gene. It contains both sense and antisense orientation in a binary vector pFGC5941. Short hairpin RNA (shRNA) structures were employed to reduce the possibility of inducing endogenous non-specific antiviral responses, which usually target longer double-stranded RNAs (dsRNAs). To confirm the transgenic plants, molecular analysis was performed using TSWV gene-specific primers (TSWV-NSs). Based on the EHA105 Agrobacterium strain and the pFGC5941 vector carrying NSs and nptII genes in the T-DNA region, the results validate the insertion of kanamycin resistance into the regenerated transgenic plants. The virus source harboring TSWV was used to inoculate putative transgenic plants for evaluating the confirmation of successful transformation strategy for inducing virus resistance. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed by using qRT-NSs primer pairs to determine the relative gene expression of all constructs (TSWV-NSs both sense and antisense orientation) after inoculations. In comparison to the control groups, which contained wild type control (non-transgenic tomato plant), empty pFGC5941 vector and the pFGC5941+sense construct, the expression of the NSs gene was noticeably reduced in the full clone (pFGC5941+sense + antisense) construct. For the validation of RNAi effectiveness, Nicotiana tabacum plants were also inoculated with viral constructs. Specifically, they displayed severe symptoms of TSWV in plants containing the pFGC5941+sense, pFGC5941 empty vector, and in wild-type tomato plants that are correlated with bioassay results. The lack of TSWV symptoms validated the hypothesis after careful observation of the expression of NSs genes in terms of pathogenicity. This research demonstrates the RNA interference effectiveness by targeting NSs gene as a reliable technique for achieving long lasting antiviral protection in tomatoes. The findings have practical relevance and the potential to be a tool for limiting tomato crop losses caused by TSWV worldwide.

Distinct tomato yellow leaf curl Chuxiong virus isolated from whiteflies and plants in China and its symptom determinant and suppressor of post-transcriptional gene silencing

A begomovirus isolated from whiteflies (Bemisia tabaci) and tomato, sweet potato in China was found to be representative of a distinct begomovirus species, for which the name tomato yellow leaf curl Chuxiong virus (TYLCCxV) is proposed. The results of genomic identification and sequence comparison showed that TYLCCxV shares the highest complete nucleotide sequence identity (88.3%) with croton yellow vein mosaic virus (CroYVMV), and may have originated from the recombination between synedrella leaf curl virus (SyLCV) and squash leaf curl Yunnan virus (SLCuYV). Agrobacterium-mediated inoculation showed that TYLCCxV is highly infectious for a range of plant species, producing upward leaf curling, leaf crumpling, chlorosis, distortion, and stunt symptoms in Solanum lycopersicum plants. The results of Southern blot indicated that TYLCCxV is capable of efficiently replicating two heterologous betasatellites. The inoculation of PVX::C4 on Nicotiana benthamiana induced upward leaf curling and stem elongation symptoms, suggesting that TYLCCxV C4 functions as a symptom determinant. TYLCCxV V2 is an important virulence factor that induces downward leaf curling symptoms, elicits systemic necrosis, and suppresses local and systemic GFP silencing in co-agroinfiltrated N. benthamiana and transgenic 16c plants. Considering the multifunctional virulence proteins V2 and C4, the possibility of TYLCCxV causing devastating epidemics on tomato in China is discussed.

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.

Exploring Potential Epigenetic Biomarkers for Colorectal Cancer Metastasis.

Metastatic progression is a complex, multistep process and the leading cause of cancer mortality. There is growing evidence that emphasises the significance of epigenetic modification, specifically DNA methylation and histone modifications, in influencing colorectal (CRC) metastasis. Epigenetic modifications influence the expression of genes involved in various cellular processes, including the pathways associated with metastasis. These modifications could contribute to metastatic progression by enhancing oncogenes and silencing tumour suppressor genes. Moreover, specific epigenetic alterations enable cancer cells to acquire invasive and metastatic characteristics by altering cell adhesion, migration, and invasion-related pathways. Exploring the involvement of DNA methylation and histone modification is crucial for identifying biomarkers that impact cancer prediction for metastasis in CRC. This review provides a summary of the potential epigenetic biomarkers associated with metastasis in CRC, particularly DNA methylation and histone modifications, and examines the pathways associated with these biomarkers.

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.

PDL1-dependent trans-acting siRNAs regulate lateral organ polarity development in rice

Leaves and glumes act as lateral organs and have essential effects on photosynthesis and seed morphology, thus affecting yield. However, the molecular mechanisms controlling their polarity development in rice is still worth further analysis. Here, we isolated a polarity defect of lateral organs 1 (pdl1) mutant in rice, which exhibits twisted/filamentous-shaped leaves and cracked/filamentous-shaped lemmas caused by defects in polarity development. PDL1 encodes a SUPPRESSOR OF GENE SILENCING 3 protein localized in the cytoplasm granules. PDL1 is expressed in the shoot apical meristem, inflorescence meristem, floral meristem, and lateral organs including leaf and floral organs. PDL1 is involved in the synthesis of tasiR-ARF, which may subsequently modulate the expression of OsARFs. Meanwhile, the expression of abaxial miR165/166 and the adaxial identity genes OSHBs was increased and decreased significantly, respectively. The results of this study clarified the molecular mechanism that the PDL1-mediated tasiR-ARF synthesis regulates the lateral organ polarity development in rice.

Self-interaction of Tomato spotted wilt virus NSs protein enhances gene silencing suppressor activity, but is dispensable as avirulence determinant on pepper

Self-interaction of Tomato spotted wilt virus NSs protein enhances gene silencing suppressor activity, but is dispensable as avirulence determinant on pepper

The interaction between the lemon ribosomal protein ClRPS9-2 and citrus yellow vein clearing virus coat protein affects viral infection and gene silencing suppressor activity.

Citrus yellow vein clearing virus (CYVCV) is an emerging virus that causes serious economic damage to the lemon industry worldwide. The coat protein (CP) of CYVCV is a strong RNA silencing suppressor and is associated with the severity of symptoms in citrus, yet the interaction between CP and host factors remains unknown. In this study, the 40S ribosomal subunit protein S9-2 (ClRPS9-2) was identified as a CP-binding partner using the yeast two-hybrid system from a lemon (cv. Eureka) cDNA library, and the interaction between CP and ClRPS9-2 was demonstrated by in vivo methods. The results suggest that the N-terminal 8-108 amino acid sequence of ClRPS9-2 is crucial for its interaction with CP and may be associated with the nuclear localization of ClRPS9-2. The accumulation and silencing suppressor activity of CP were reduced by transient expression of ClRPS9-2 in Nicotiana benthamiana. Reverse transcription-quantitative PCR analysis showed that the content of CYVCV in ClRPS9-2 transgenic Eureka lemon plants was approximately 50% of that in CYVCV-infected wild-type plants 1 month after inoculation, and mild yellowing and vein clearing symptoms were observed in the transgenic plants. These findings demonstrate that ClRPS9-2 plays a role in host defensive reactions, and the enhanced resistance of transgenic plants to CYVCV may be associated with the up-regulation of salicylic acid-related and R genes.

ADAR1 Biology Can Hinder Effective Antiviral RNA Interference.

Viruses constantly evolve and adapt to the antiviral defenses of their hosts. The biology of viral circumvention of these selective pressures can often be attributed to the acquisition of novel antagonistic gene products or by rapid genome change that prevents host recognition. To study viral evasion of RNA interference (RNAi)-based defenses, we established a robust antiviral system in mammalian cells using recombinant Sendai virus designed to be targeted by endogenous host microRNAs (miRNAs) with perfect complementarity. Using this system, we previously demonstrated the intrinsic ability of positive-strand RNA viruses to escape this selective pressure via homologous recombination, which was not observed in negative-strand RNA viruses. Here, we show that given extensive time, escape of miRNA-targeted Sendai virus was enabled by host adenosine deaminase acting on RNA 1 (ADAR1). Independent of the viral transcript targeted, ADAR1 editing resulted in disruption of the miRNA-silencing motif, suggesting an intolerance for extensive RNA-RNA interactions necessary for antiviral RNAi. This was further supported in Nicotiana benthamiana, where exogenous expression of ADAR1 interfered with endogenous RNAi. Together, these results suggest that ADAR1 diminishes the effectiveness of RNAi and may explain why it is absent in species that utilize this antiviral defense system. IMPORTANCE All life at the cellular level has the capacity to induce an antiviral response. Here, we examine the result of imposing the antiviral response of one branch of life onto another and find evidence for conflict. To determine the consequences of eliciting an RNAi-like defense in mammals, we applied this pressure to a recombinant Sendai virus in cell culture. We find that ADAR1, a host gene involved in regulation of the mammalian response to virus, prevented RNAi-mediated silencing and subsequently allowed for viral replication. In addition, the expression of ADAR1 in Nicotiana benthamiana, which lacks ADARs and has an endogenous RNAi system, suppresses gene silencing. These data indicate that ADAR1 is disruptive to RNAi biology and provide insight into the evolutionary relationship between ADARs and antiviral defenses in eukaryotic life.

Naturally occurring substitution of an amino acid in a plant virus gene-silencing suppressor enhances viral adaptation to increasing thermal stress.

Cereal yellow dwarf virus (CYDV-RPV) encodes a P0 protein that functions as a viral suppressor of RNA silencing (VSR). The strength of silencing suppression is highly variable among CYDV-RPV isolates. In this study, comparison of the P0 sequences of CYDV-RPV isolates and mutational analysis identified a single C-terminal amino acid that influenced P0 RNA-silencing suppressor activity. A serine at position 247 was associated with strong suppressor activity, whereas a proline at position 247 was associated with weak suppressor activity. Amino acid changes at position 247 did not affect the interaction of P0 with SKP1 proteins from Hordeum vulgare (barley) or Nicotiana benthamiana. Subsequent studies found P0 proteins containing a P247 residue were less stable than the P0 proteins containing an S247 residue. Higher temperatures contributed to the lower stability and in planta and the P247 P0 proteins were subject to degradation via the autophagy-mediated pathway. A P247S amino acid residue substitution in P0 increased CYDV-RPV replication after expression in agroinfiltrated plant leaves and increased viral pathogenicity of P0 generated from the heterologous Potato virus X expression vector system. Moreover, an S247 CYDV-RPV could outcompete the P247 CYDV-RPV in a mixed infection in natural host at higher temperature. These traits contributed to increased transmission by aphid vectors and could play a significant role in virus competition in warming climates. Our findings underscore the capacity of a plant RNA virus to adapt to climate warming through minor genetic changes in gene-silencing suppressor, resulting in the potential for disease persistence and prevalence.

First report of Cotton leafroll dwarf virus infecting cacao (Theobroma cacao L.) trees in Brazil.

Cotton leafroll dwarf virus (genus Polerovirus, family Solemoviridae) has been commonly reported affecting cotton plants (Gossypium spp., family Malvaceae) and several weed species (Ramos-Sobrinho et al., 2021; Sedhain et al., 2021). During a recent survey, cacao (Theobroma cacao L.) trees exhibiting virus-like symptoms such as leaf mosaic, vein clearing, and yellow spot were observed in the south part of the state of Bahia, northeastern Brazil, in 2022. Leaf samples were randomly collected from symptomatic cacao plants (n=30) growing in an affected area of approximately 30 ha. Total RNA obtained from pooled cacao samples were subjected to Illumina HiSeq 2500 sequencing as previously described (Keith et al., 2021), and partial sequences of cotton leafroll dwarf virus (CLRDV), and other virus-specific sequence contigs, were de novo assembled according to Ramos-Sobrinho et al. (2021). To further investigate the presence of CLRDV in cacao leaves, total RNA was individually extracted using a modified silica protocol (Rott and Jelkmann, 2001) and used as template for cDNA synthesis with random hexamers using the SuperScript™ IV First-Strand Synthesis System (Invitrogen, CA, USA) following the manufacturer´s protocol. Detection of CLRDV was carried out by reverse transcription-polymerase chain reaction (RT-PCR) with the primers PL4F and o3-R, which amplify the open reading frame 3 (ORF3) encoding the capsid protein (Corrêa et al., 2005). Expected size amplicons (~0.6 kb) were observed from 16 out of 30 symptomatic plants, indicating ~53% of the cacao trees were infected by CLRDV. Considering 14 symptomatic plants tested negative for CLRDV, the symptoms observed here could also be caused by other viral groups or abiotic stress. To confirm the detection of CLRDV, the first half (~3.5kb) of the viral genome was amplified from two representative cacao samples using the primers P20F and P22R (Avelar et al., 2020). The RT-PCR products were gel-purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, WI, USA) and Sanger sequenced. The RNA Illumina sequencing from pooled cacao samples (n=30) yielded 34,610,572 million trimmed reads. Two contigs of 868 and 839 nucleotides (nt) in length, and sharing high nt identity with CLRDV isolates, were assembled from 6,903 and 10,271 reads, at a coverage depth of 795 and 1,224x, respectively. Together, these contigs represent ~29% of the complete viral genome and included part of the 5´-untraslated region, ORF0 and the second half of ORF1-ORF2. Additional CLRDV-like contigs were observed across the viral genome, but they were not considered for further analyses due to the poor sequence quality. The Illumina- and Sanger-derived ORF0 and partial ORF1-ORF2 sequences shared >97% nt identity, suggesting they were congruent. Pairwise sequence comparisons for ORF0, encoding the gene silencing suppressor P0, indicated the cacao-associated isolates shared 99.7 and 99.2% nt and amino acid (aa) identity one with another, respectively. The ORF0 nt sequences showed 91.9-93.8 and 90.7-93.6% identity, while the aa sequences shared 85.8-88.5 and 86.2-90.0% similarity, with CLRDV isolates previously reported in South America and the USA, respectively. Finally, the ~3.5kb nt sequences of cacao-infecting CLRDV isolates shared 92.9-95.8% identity with CLRDV genomes deposited in NCBI-GenBank. The Bayesian phylogenetic tree reconstructed based on ORF0 nt sequences showed the new sequences were more closely related to CLRDV-atypical isolates (GenBank accession nos. KF359946, KF359947, KF906260, and KF906261). Together, these results suggest the new ORF0 sequences belong to CLRDV and were deposited in GenBank under accession nos. ON954058-ON954059. To our knowledge, this is the first report of CLRDV infecting cacao plants, expanding the range of malvaceous hosts of this polerovirus. CLRDV is largely known for causing yield losses in cotton crops, but additional studies are needed to determine if CLRDV infection is detrimental to cacao production.

Grapevine red blotch virus C2 and V2 are suppressors of post-transcriptional gene silencing

Grapevine red blotch virus (GRBV) is the causative agent of grapevine red blotch disease (GRBD) which is one of the major threats faced by grapevine industry in the United States. Since its initial identification in 2011, the disease has rapidly spread in the major US grape-growing regions of the Pacific Northwest, causing major economic impacts. Geminiviruses, the largest family of plant viruses, can induce and be targeted by host post-transcriptional gene-silencing (PTGS) anti-viral mechanisms. As a counter-defense mechanism, viruses have evolved viral silencing suppressor proteins to combat PTGS mechanisms and establish a successful infection in host plants. Here we provide characterization of two ORFs of GRBV, C2 and V2 as viral silencing suppressors. In Nicotiana benthamiana line 16c GFP marker plants, synergism or additive effects of C2 and V2 suppressors was observed at the mRNA level when they are expressed together transiently. Additionally, we showed there is no evidence by yeast two-hybrid of self-interaction (dimerization) of C2 or V2 proteins, and no evidence of physical interaction between these two suppressors.

Phase separation of SGS3 drives siRNA body formation and promotes endogenous gene silencing

The generation of small interfering RNA (siRNA) involves many RNA processing components, including SUPPRESSOR OF GENE SILENCING 3 (SGS3), RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), and DICER-LIKE proteins (DCLs). Nonetheless, how these components are coordinated to produce siRNAs is unclear. Here, we show that SGS3 forms condensates via phase separation invivo and invitro. SGS3 interacts with RDR6 and drives it to form siRNA bodies in cytoplasm, which is promoted by SGS3-targeted RNAs. Disrupting SGS3 phase separation abrogates siRNA body assembly and siRNA biogenesis, whereas coexpression of SGS3 and RDR6 induces siRNA body formation in tobacco and yeast cells. Dysfunction in translation and mRNA decay increases the number of siRNA bodies, whereas DCL2/4 mutations enhance their size. Purification of SGS3 condensates identifies numerous RNA-binding proteins and siRNA processing components. Together, our findings reveal that SGS3 phase separation-mediated formation of siRNA bodies is essential for siRNA production and gene silencing.

Investigation of the effects of the royal jelly on genomic demethylation and tumor suppressor genes in human cancer cells.

Royal jelly is a gelatinous nutrient secretion produced by the mandibular glands of young worker honey bees and has a critical role in honey bee life. In the honey bee colonies, queen and worker honey bees have very different morphologies and behaviors due to their diet in the larval period, despite having the same genome. In comparison, queen bees formed from larvae that feed royal jelly exclusively, and worker bees formed from larvae that feed on much less royal jelly. DNA methylation has been shown to play a critical role in the development of queen and worker honeybees. Alterations in DNA methylation, one of the epigenetic mechanisms defined as hereditable nucleotide modifications that occur in gene expression without changes in the DNA sequence, are closely related to many diseases, especially cancer. Hypermethylation of CpG islands located in the promoter regions of genes causes gene silencing and tumor suppressor genes epigenetically have silenced in cancer. The inactivation of tumor suppressor genes disrupts nearly all cellular pathways in cancer. In contrast to genetic alterations, gene silencing by epigenetic modifications may potentially be reversed and used in cancer treatment. Royal jelly, which causes epigenetic changes in bee colonies, has the potential to cause a change in cancer cells. In our study, royal jelly's effects on DNA methyltransferase enzyme and gene methylation of RASSF1A tumor suppressor were investigated in human cancer cell lines (HeLa, HT29, and A549), and modifications in the gene expression profile of royal jelly were determined by next generation sequencing.

Turnip crinkle virus-encoded suppressor of RNA silencing interacts with Arabidopsis SGS3 to enhance virus infection.

Most plant viruses encode suppressors of RNA silencing (VSRs) to protect themselves from antiviral RNA silencing in host plants. The capsid protein (CP) of Turnip crinkle virus (TCV) is a well-characterized VSR, whereas SUPPRESSOR OF GENE SILENCING 3 (SGS3) is an important plant-encoded component of the RNA silencing pathways. Whether the VSR activity of TCV CP requires it to engage SGS3 in plant cells has yet to be investigated. Here, we report that TCV CP interacts with SGS3 of Arabidopsis in both yeast and plant cells. The interaction was identified with the yeast two-hybrid system, and corroborated with bimolecular fluorescence complementation and intracellular co-localization assays in Nicotiana benthamiana cells. While multiple partial TCV CP fragments could independently interact with SGS3, its hinge domain connecting the surface and protruding domains appears to be essential for this interaction. Conversely, SGS3 enlists its N-terminal domain and the XS rice gene X and SGS3 (XS) domain as the primary CP-interacting sites. Interestingly, SGS3 appears to stimulate TCV accumulation because viral RNA levels of a TCV mutant with low VSR activities decreased in the sgs3 knockout mutants, but increased in the SGS3-overexpressing transgenic plants. Transgenic Arabidopsis plants overexpressing TCV CP exhibited developmental abnormalities that resembled sgs3 knockout mutants and caused similar defects in the biogenesis of trans-acting small interfering RNAs. Our data suggest that TCV CP interacts with multiple RNA silencing pathway components that include SGS3, as well as previously reported DRB4 (dsRNA-binding protein 4) and AGO2 (ARGONAUTE protein2), to achieve efficient suppression of RNA silencing-mediated antiviral defence.