Homology-dependent Gene Silencing Research Articles

Current scenario of RNAi-based hemipteran control.

RNA interference (RNAi) is an homology-dependent gene silencing mechanism that is a feasible and sustainable avenue for the management of hemipteran pests. Commercial implementation of RNAi-based control strategies is impeded by limited knowledge about the mechanism of double-stranded RNA (dsRNA) uptake, the function of core RNAi genes and systemic RNAi mechanisms in hemipteran insects. This review briefly summarizes recent progress in RNAi-based studies aimed to reduce insect populations, viral transmission and insecticide resistance focusing on hemipteran pests. This review explores RNAi-mediated management of hemipteran insects and offers potential solutions, including in silico approaches coupled with laboratory-based toxicity assays to circumvent potential off-target effects against beneficial organisms. We further explore ways to mitigate degradation of dsRNA in the environment and the insect such as stacking and formulation of dsRNA effectors. Finally, we conclude by considering nontransformative RNAi approaches, concatomerization of RNAi sequences and pyramiding RNAi with active constituents to reduce dsRNA production and application cost, and to improve broad-spectrum hemipteran pest control. © 2020 Society of Chemical Industry.

Targeted de novo promoter methylation for gene silencing is maintained in the next generation of a transgenic tobacco plant

Homology-dependent gene silencing (HDGS) is a resistance mechanism against homologous transgene introduction or virus infection in various organisms. In plants in particular, HDGS phenomena in homologous DNA or RNA sequences can cause gene silencing. In this study, GUS-silenced transgenic lines were developed by agroinfiltration with the i35S binary vector for inducing targeted de novo CaMV 35S promoter methylation. Agroinfiltration with i35S triggered inheritable de novo methylation of the CaMV 35S promoter and CAAT box region by HDGS. Moreover, targeted de novo CaMV 35S promoter methylation appeared to be controlled by the expression of SUVH9, which is involved in endogenous DNA methylation and hyper-methylation of the introduced promoter. In summary, this study demonstrated that targeted de novo CaMV 35S promoter methylation may be subjected to an HDGS mechanism that can downregulate gene expression and maintain the heritable stability of the methylated promoter.

RNAi (RNA Interference) Vectors for Functional Genomics Study in Plants

The discovery of RNA interference (RNAi) has revolutionized gene silencing for basic as well as applied studies. RNAi is a homology-dependent gene silencing mechanism, triggered by the introduction of double-stranded RNA (dsRNA) molecule. Since it can specifically suppress function of the targeted gene, the technique has been very useful in functional genomic studies. For efficient knock-down of the targeted gene, Gateway cloning based RNAi Silencing (pRISI) vector was developed. When a target gene-specific trigger fragment is cloned as inverted repeats in the pRISI vector, it produces a hairpin containing RNA transcribed from a CaMV35S (in case of pRISI-Ca) or from an ubiquitin (in case of pRISI-Ub) promoter. Function of the pRISI-Ca vector was confirmed by RNAi-mediated gene silencing experiment in Arabidopsis thaliana for At2g30350 gene which was found to be involved in repair of methylated DNA. A similar vector, pRISI-Ub was also developed for gene silencing studies in monocotyledonous plants. Its function was successfully demonstrated by transient suppression of GUS (UidA) gene co-bombarded with GUS-RNAi construct in wheat leaf. The vectors would be useful in rapid and easy preparation of RNAi constructs for reverse genetic studies for functional genomics in plants.

A family of small tyrosine rich proteins is essential for oogonial and oospore cell wall development of the mycoparasitic oomycete Pythium oligandrum

The mycoparasitic oomycete Pythium oligandrum is homothallic, producing an abundance of thick-walled spiny oospores in culture. After mining a cDNA sequence dataset, we identified a family of genes that code for small tyrosine rich (Pythium oligandrumsmall tyrosine rich (PoStr)) proteins. Sequence analysis identified similarity between the PoStr proteins and putative glycine-rich cell wall proteins from the related plant pathogenic oomycete Pythium ultimum, and mating-induced genes from the oomycete Phytophthora infestans. Expression analysis showed that PoStr transcripts accumulate during oospore production in culture and immunolocalisation indicates the presence of these proteins in oogonial and oospore cell walls. PoStr protein abundance correlated positively with production of oogonia as determined by antibiotic-mediated oogonia suppression. To further characterise the role of PoStr proteins in P. oligandrum oospore production, we silenced this gene family using homology-dependent gene silencing. This represents the first characterisation of genes using gene silencing in a Pythium species. Oospores from silenced strains displayed major ultrastructural changes and were sensitive to degradative enzyme treatment. Oogonia of silenced strains either appeared to be arrested at the mature oosphere stage of development or in around 40 % of the structures, showed a complete suppression of oospore formation. Suppressed oogonia were highly vacuolated and the oogonium wall was thickened by a new inner wall layer. Our data suggest PoStr proteins are probably integral structural components of the normal oospore cell wall and play a key role in oospore formation.

Transgene Induced Co-Suppression during Vegetative Growth in Cryptococcus neoformans

Introduction of DNA sequences into the genome often results in homology-dependent gene silencing in organisms as diverse as plants, fungi, flies, nematodes, and mammals. We previously showed in Cryptococcus neoformans that a repeat transgene array can induce gene silencing at a high frequency during mating (∼50%), but at a much lower frequency during vegetative growth (∼0.2%). Here we report a robust asexual co-suppression phenomenon triggered by the introduction of a cpa1::ADE2 transgene. Multiple copies of the cpa1::ADE2 transgene were ectopically integrated into the genome, leading to silencing of the endogenous CPA1 and CPA2 genes encoding the cyclosporine A target protein cyclophilin A. Given that CPA1-derived antisense siRNAs were detected in the silenced isolates, and that RNAi components (Rdp1, Ago1, and Dcr2) are required for silencing, we hypothesize that an RNAi pathway is involved, in which siRNAs function as trans factors to silence both the CPA1 and the CPA2 genes. The silencing efficiency of the CPA1 and CPA2 genes is correlated with the transgene copy number and reached ∼90% in the presence of >25 copies of the transgene. We term this transgene silencing phenomenon asexual co-suppression to distinguish it from the related sex-induced silencing (SIS) process. We further show that replication protein A (RPA), a single-stranded DNA binding complex, is required for transgene silencing, suggesting that RPA might play a similar role in aberrant RNA production as observed for quelling in Neurospora crassa. Interestingly, we also observed that silencing of the ADE2 gene occurred at a much lower frequency than the CPA1/2 genes even though it is present in the same transgene array, suggesting that factors in addition to copy number influence silencing. Taken together, our results illustrate that a transgene induced co-suppression process operates during C. neoformans vegetative growth that shares mechanistic features with quelling.

PnPMA1, an atypical plasma membrane H+-ATPase, is required for zoospore development in Phytophthora parasitica

Biflagellate zoospores are the major infective agents that initiate plant infection for most Phytophthora species. Once released from sporangia, zoospores swim and use a number of tactic responses to actively target host tissues. However, the molecular mechanisms controlling zoospore development and behaviour are largely unknown. Previous studies have shown that the PnPMA1 gene is highly expressed in zoospores and germinated cysts of Phytophthora parasitica and encodes an atypical plasma membrane H+-ATPase containing an insertion of ∼155 amino acid residues at the C terminus. Using topology determination we now show that the C-terminal insertion loop in the PnPMA1 protein is located in the extracellular space. To elucidate the biological function of PnPMA1, PnPMA1-deficient transformants were generated by homology-dependent gene silencing and were confirmed by quantitative PCR of PnPMA1 transcripts and detection of associated small interfering RNAs (siRNAs). High levels of PnPMA1 silencing in P. parasitica resulted in production of nonflagellate and large aberrant zoospores, rapid transition from zoospores to cysts, and a decreased germination rate of cysts. These results indicate that PnPMA1 plays important roles in zoospore development.

Recent advances in development of marker-free transgenic plants: Regulation and biosafety concern

During the efficient genetic transformation of plants with the gene of interest, some selectable marker genes are also used in order to identify the transgenic plant cells or tissues. Usually, antibiotic- or herbicide-selective agents and their corresponding resistance genes are used to introduce economically valuable genes into crop plants. From the biosafety authority and consumer viewpoints, the presence of selectable marker genes in released transgenic crops may be transferred to weeds or pathogenic microorganisms in the gastrointestinal tract or soil, making them resistant to treatment with herbicides or antibiotics, respectively. Sexual crossing also raises the problem of transgene expression because redundancy of transgenes in the genome may trigger homology-dependent gene silencing. The future potential of transgenic technologies for crop improvement depends greatly on our abilities to engineer stable expression of multiple transgenic traits in a predictable fashion and to prevent the transfer of undesirable transgenic material to non-transgenic crops and related species. Therefore, it is now essential to develop an efficient marker-free transgenic system. These considerations underline the development of various approaches designed to facilitate timely elimination of transgenes when their function is no longer needed. Due to the limiting number of available selectable marker genes, in future the stacking of transgenes will be increasingly desirable. The production of marker-free transgenic plants is now a critical requisite for their commercial deployment and also for engineering multiple and complex trait. Here we describe the current technologies to eliminate the selectable marker genes (SMG) in order to develop marker-free transgenic plants and also discuss the regulation and biosafety concern of genetically modified (GM) crops.

Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3-9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana.

The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host's defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3-9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.

Small RNA-Mediated Epigenetic Myostatin Silencing

Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

RNAi induced gene silencing in crop improvement.

The RNA silencing is one of the innovative and efficient molecular biology tools to harness the down-regulation of expression of gene(s) specifically. To accomplish such selective modification of gene expression of a particular trait, homology dependent gene silencing uses a stunning variety of gene silencing viz. co-suppression, post-transcriptional gene silencing, virus-induced gene silencing etc. This family of diverse molecular phenomena has a common exciting feature of gene silencing which is collectively called RNA interference abbreviated to as RNAi. This molecular phenomenon has become a focal point of plant biology and medical research throughout the world. As a result, this technology has turned out to be a powerful tool in understanding the function of individual gene and has ultimately led to the tremendous use in crop improvement. This review article illustrates the application of RNAi in a broad area of crop improvement where this technology has been successfully used. It also provides historical perspective of RNAi discovery and its contemporary phenomena, mechanism of RNAi pathway.

Genome-wide identification of Phytophthora sojae SNARE genes and functional characterization of the conserved SNARE PsYKT6

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating membrane fusion and key factors for vesicular trafficking in all eukaryotic cells. Taking advantage of the available whole genome sequence of the oomycete plant pathogen Phytophthora sojae, 35 genes encoding putative SNARE proteins were identified in the genome of this organism. PsYKT6, one of the most conserved SNARE proteins, was functionally characterized by homology-dependent gene silencing. The phenotype analysis showed that PsYKT6 is important for proper asexual development, sexual reproduction, and pathogenesis on host soybean cultivars.

Trans-acting antisense RNAs mediate transcriptional gene cosuppression in S. cerevisiae

Homology-dependent gene silencing, a phenomenon described as cosuppression in plants, depends on siRNAs. We provide evidence that in Saccharomyces cerevisiae, which is missing the RNAi machinery, protein coding gene cosuppression exists. Indeed, introduction of an additional copy of PHO84 on a plasmid or within the genome results in the cosilencing of both the transgene and the endogenous gene. This repression is transcriptional and position-independent and requires trans-acting antisense RNAs. Antisense RNAs induce transcriptional gene silencing both in cis and in trans, and the two pathways differ by the implication of the Hda1/2/3 complex. We also show that trans-silencing is influenced by the Set1 histone methyltransferase, which promotes antisense RNA production. Finally we show that although antisense-mediated cis-silencing occurs in other genes, trans-silencing so far depends on features specific to PHO84. All together our data highlight the importance of noncoding RNAs in mediating RNAi-independent transcriptional gene silencing.

RNA interference: Emerging diagnostics and therapeutics tool

RNA interference has been recognized to be an efficient mechanism for the sequence specific inhibition of gene-expression caused by 21–23 bp long dsRNA. This is a potent tool to study gene function and is ready to lend a hand in the treatment of disease. Two main classes of dsRNA involved are short-interfering RNA and micro RNA which differ in biogenesis and modes of target regulation. The discovery of RNAi has led to the development in the field of biomedical research. The evolution of RNAi has led to diverse applications such as identification of gene function, identification of disease causing genes and therapeutics. This homology dependent gene silencing, hence has a great latent in transcriptional regulation and medicine. This review summarizes the role of RNAi in human health and diseases.

RNAi Vectors for Manipulation of Gene Expression in Higher Plants

RNA interference (RNAi) is a homology-dependent gene silencing technology in which small interfering RNAs (siRNAs) direct RNA cleavage or DNA methylation. After transcription of an RNAi cassette including inverted re- peat sequences against the target gene and a spacer fragment, the resultant transcript forms a hairpin-like structure. The stem region of hairpin RNAs is processed into siRNAs. Here we focus on the structural properties of RNAi vectors that affect the silencing efficiency, and caveats in the evaluation of RNAi phenotype are discussed. Subsequently, several RNAi applications including simultaneous silencing of multiple gene sequences and specific silencing of a member in the gene family were discussed. In addition a newly developed RNAi technology, artificial microRNA, is also introduced.

Arabidopsis HOG1 gene and its petunia homolog PETCBP act as key regulators of yield parameters

Plant hormones influence the key parameters that contribute to crop yield, including biomass, branching and seed number. We tested manipulation of cytokinin signaling as an avenue for influencing these growth parameters. Here we report a full-length cDNA coding for a cytokinin binding protein, Petunia cytokinin binding protein (PETCBP) from Petunia hybrida cv. Mitchell. PETCBP encodes for a protein that exhibits high sequence similarity to S-adenosyl-L-homocysteine hydrolase (SAHH). Transgenic petunia plants expressing this gene in antisense orientation displayed profuse branching, delayed flowering and delayed shoot bud induction from leaf explants in vitro. Homologs were also isolated from Arabidopsis thaliana homology-dependent gene silencing 1 (HOG1) and Orzya sativa (OsCBP). Arabidopsis HOG1 showed high affinity cytokinin binding activity and modified plant architecture similar to PETCBP. Transgenic Arabidopsis plants overexpressing HOG1 showed early flowering with a significantly reduced plant biomass and number of leaves. In contrast, profuse branching, delayed flowering, increased leaf size and higher seed yield were the major phenotypes observed in the antisense suppression lines. These results suggest that genetic manipulation of this cytokinin binding protein or its orthologs could be used for improving crop biomass and seed yield.

Overproduction of non‐translatable mRNA silences. The transcription of Ty1 retrotransposons in S. cerevisiae via functional inactivation of the nuclear cap‐binding complex and subsequent hyperstimulation of the TORC1 pathway

Co-suppression is high gene copy number-triggered homology-dependent gene silencing, and co-suppression may have evolved in eukaryotes to counter invasive molecular parasites, such as viruses and transposons. We previously reported 'Ty1 transcriptional co-suppression'-high Ty1 copy number-triggered transient transcriptional silencing of Ty1 retrotransposons in S. cerevisiae. We report here that this phenomenon is unlikely to be homology-dependent, despite the copy number dependence. The Ty1 mRNA is an extremely poor template for translation, and overproduction of non-translatable mRNA without Ty1 homology is sufficient to initiate the transient Ty1 transcriptional silencing. We present genetic evidence that overproduction of non-translatable mRNA may functionally inactivate the nuclear cap-binding complex (CBC), and inactivation of CBC may then hyperstimulate the TORC1 pathway to mediate Ty1 transcriptional silencing. Our results point to a potent regulatory function of non-translatable mRNA in vivo (via CBC and TORC1) to potentially modulate a variety of intracellular activities, such as Ty1 transcription. Although overproduction of non-translatable mRNA causes transient Ty1 transcriptional silencing, it does not play a detectable role in controlling Ty1 retrotransposition.

A Single Chimeric Transgene Derived from Two Distinct Viruses for Multiple Virus Reslstance

A chimeric transgene composed of two different viral gene segments, was employed to confer multiple virus resistance in transformed tomato through homology-dependent gene silencing. Two important viruses of tomato, Cucumber mosaic virus (CMV) and Tomato leaf curl virus (ToLCV) were targeted. Comparison of 657 nucleotide coat protein (CP) region of CMV isolate (CMV -B) with those of five other CMV-Indian isolates showed more than 95% identity both at the nucleotide as well as amino acid level. Our previous studies showed the presence of conserved domain in the replicase gene of ToLCV comprising of 319 nts at 3’ of the gene. With this in view, gene encoding full-length transcript of CP of CMV-B was fused with the conserved domain of replicase gene of ToLCV. This is a first attempt of the transcriptional fusion of viral gene segments derived from a DNA virus (ToLCV) and a RNA virus (CMV). The chimeric gene construct was transformed in L. esculentum for multiple virus resistance, which is expected to work through homology - dependent gene silencing.

Organization, not duplication, triggers silencing in a complex transgene locus in rice

Despite the presence in nature of many functional gene families that contain several to many highly similar sequences, the presence of identical DNA sequence repeats is widely thought to predispose transgene inserts to homology dependent gene silencing (HDGS). The induction of transcriptional gene silencing (TGS) by RNAs homologous to promoter sequences has been reported recently in Arabidopsis and humans. However, mechanisms for TGS have not been studied in detail for rice, the most widely cultivated crop plant. Taking advantage of a well-characterized homozygous silenced transgenic rice line (siJKA), supertransformation was performed with a binary vector bearing mUbi1 and 35S promoter sequences identical to those in the resident transgenes. Analysis of the incoming and resident transgenes in the supertransformants revealed that the incoming mUbi1 transgene promoter was not silenced whereas the incoming 35S transgene promoter was silenced. That the resident silenced mUbi1-bar was not reactivated in these experiments as a result of passage through tissue culture and regeneration was established by the finding that regenerants from siJKA immature embryos were all silenced for mUbi1-bar. In a parallel experiment, when wild type rice calli were transformed with the same binary vector, neither of the incoming transgene promoters was silenced. Following 5-azacytidine (5-azaC) treatment of siJKA, aberrant RNA species corresponding to the 35S promoter, but not to the mUbi1 promoter, were detected. Nevertheless, no 21-25 nt RNAs corresponding to the 35S promoter sequence were detected. These results, together with detailed analyses of the progenies from the primary transformants and supertransformants, revealed that HDGS of the resident silenced locus was caused not by simple transgene duplication, but by aberrant transcripts derived from rearranged promoters present in siJKA. Practical consequences of this study include a justification for the use of multiple copies of a given promoter for transformation without inducing silencing, provided that their genomic integration does not result in aberrant transcription of the promoters.

The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 Gene Codes for an S-Adenosyl-l-Homocysteine Hydrolase Required for DNA Methylation-Dependent Gene Silencing

Genes introduced into higher plant genomes can become silent (gene silencing) and/or cause silencing of homologous genes at unlinked sites (homology-dependent gene silencing or HDG silencing). Mutations of the HOMOLOGY-DEPENDENT GENE SILENCING1 (HOG1) locus relieve transcriptional gene silencing and methylation-dependent HDG silencing and result in genome-wide demethylation. The hog1 mutant plants also grow slowly and have low fertility and reduced seed germination. Three independent mutants of HOG1 were each found to have point mutations at the 3' end of a gene coding for S-adenosyl-l-homocysteine (SAH) hydrolase, and hog1-1 plants show reduced SAH hydrolase activity. A transposon (hog1-4) and a T-DNA tag (hog1-5) in the HOG1 gene each behaved as zygotic embryo lethal mutants and could not be made homozygous. The results suggest that the homozygous hog1 point mutants are leaky and result in genome demethylation and poor growth and that homozygous insertion mutations result in zygotic lethality. Complementation of the hog1-1 point mutation with a T-DNA containing the gene coding for SAH hydrolase restored gene silencing, HDG silencing, DNA methylation, fast growth, and normal seed viability. The same T-DNA also complemented the zygotic embryo lethal phenotype of the hog1-4 tagged mutant. A model relating the HOG1 gene, DNA methylation, and methylation-dependent HDG silencing is presented.

Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa.

RNA interference (RNAi) in animals, cosuppression in plants, and quelling in fungi are homology-dependent gene silencing mechanisms in which the introduction of either double-stranded RNA (dsRNA) or transgenes induces sequence-specific mRNA degradation. These phenomena share a common genetic and mechanistic basis. The accumulation of short interfering RNA (siRNA) molecules that guide sequence-specific mRNA degradation is a common feature in both silencing mechanisms, as is the component of the RNase complex involved in mRNA cleavage. During RNAi in animal cells, dsRNA is processed into siRNA by an RNase III enzyme called Dicer. Here we show that elimination of the activity of two Dicer-like genes by mutation in the fungus Neurospora crassa eliminates transgene-induced gene silencing (quelling) and the processing of dsRNA to an siRNA form. The two Dicer-like genes appear redundant because single mutants are quelling proficient. This first demonstration of the involvement of Dicer in gene silencing induced by transgenes supports a model by which a dsRNA produced by the activity of cellular RNA-dependent RNA polymerases on transgenic transcripts is an essential intermediate of silencing.