Harnessing RNA interference for the control of Fusarium species: A critical review.

Fusarium fungi are a pervasive threat to global agricultural productivity. They cause a spectrum of plant diseases that result in significant yield losses and threaten food safety by producing mycotoxins that are harmful to human and animal health. In recent years, the exploitation of the RNA interference (RNAi) mechanism has emerged as a promising avenue for the control of Fusarium-induced diseases, providing both a mechanistic understanding of Fusarium gene function and a potential strategy for environmentally sustainable disease management. However, despite significant progress in elucidating the presence and function of the RNAi pathway in different Fusarium species, a comprehensive understanding of its individual protein components and underlying silencing mechanisms remains elusive. Accordingly, while a considerable number of RNAi-based approaches to Fusarium control have been developed and many reports of RNAi applications in Fusarium control under laboratory conditions have been published, the applicability of this knowledge in agronomic settings remains an open question, and few convincing data on RNAi-based disease control under field conditions have been published. This review aims to consolidate the current knowledge on the role of RNAi in Fusarium disease control by evaluating current research and highlighting important avenues for future investigation.

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.

Molecular approaches for the management of papaya ringspot virus infecting papaya: a comprehensive review.

Papaya ringspot virus (PRSV) is a catastrophic disease that causes huge yield losses in papaya cultivation around the world. Yield losses in severely infected plants can be upto 100%. Because of this disease, papaya cultivation has been shifted to other crops in some areas of the world. Many conventional methods and breeding approaches are used against this disease, which turns out to be less effective. Considering the yield loss caused by PRSV in papaya, it is high time to focus on alternative control methods. To implement effective management strategies, molecular approaches such as Marker Assisted Breeding (MAS) or transgenic methods involving post-transcriptional gene silencing targeting the genome viz., coat protein, replicase gene, or HC Pro can be pursued. However, the public's reluctance to widely accept the transgenic approach due to health and environmental concerns necessitates a consideration of non-transgenic alternatives. Prioritizing safety and ensuring efficient virus control, non-transgenic approaches which encompass cross-protection, genome editing, and topical applications of dsRNA to induce gene silencing within the host, can be adopted. This review aims to provide comprehensive insights of various molecular tools used in managing PRSV which in turn will help in sustainable agriculture.

Remodeling of perturbed chromatin can initiate de novo transcriptional and post-transcriptional silencing.

In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa, de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6, fungal ortholog of the SWI/SNF chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin.

Challenges and Opportunities Arising from Host-Botrytis cinerea Interactions to Outline Novel and Sustainable Control Strategies: The Key Role of RNA Interference.

The necrotrophic plant pathogenic fungus Botrytis cinerea (Pers., 1794), the causative agent of gray mold disease, causes significant losses in agricultural production. Control of this fungal pathogen is quite difficult due to its wide host range and environmental persistence. Currently, the management of the disease is still mainly based on chemicals, which can have harmful effects not only on the environment and on human health but also because they favor the development of strains resistant to fungicides. The flexibility and plasticity of B. cinerea in challenging plant defense mechanisms and its ability to evolve strategies to escape chemicals require the development of new control strategies for successful disease management. In this review, some aspects of the host-pathogen interactions from which novel and sustainable control strategies could be developed (e.g., signaling pathways, molecules involved in plant immune mechanisms, hormones, post-transcriptional gene silencing) were analyzed. New biotechnological tools based on the use of RNA interference (RNAi) are emerging in the crop protection scenario as versatile, sustainable, effective, and environmentally friendly alternatives to the use of chemicals. RNAi-based fungicides are expected to be approved soon, although they will face several challenges before reaching the market.

Emerging roles of plant transcriptional gene silencing under heat.

Plants continuously endure unpredictable environmental fluctuations that upset their physiology, with stressful conditions negatively impacting yield and survival. As a contemporary threat of rapid progression, global warming has become one of the most menacing ecological challenges. Thus, understanding how plants integrate and respond to elevated temperatures is crucial for ensuring future crop productivity and furthering our knowledge of historical environmental acclimation and adaptation. While the canonical heat-shock response and thermomorphogenesis have been extensively studied, evidence increasingly highlights the critical role of regulatory epigenetic mechanisms. Among these, the involvement under heat of heterochromatic suppression mediated by transcriptional gene silencing (TGS) remains the least understood. TGS refers to a multilayered metabolic machinery largely responsible for the epigenetic silencing of invasive parasitic nucleic acids and the maintenance of parental imprints. Its molecular effectors include DNA methylation, histone variants and their post-translational modifications, and chromatin packing and remodeling. This work focuses on both established and emerging insights into the contribution of TGS to the physiology of plants under stressful high temperatures. We summarized potential roles of constitutive and facultative heterochromatin as well as the most impactful regulatory genes, highlighting events where the loss of epigenetic suppression has not yet been associated with corresponding changes in epigenetic marks.

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.

RNAi-Based Therapy: Combating Shrimp Viral Diseases.

Shrimp aquaculture has become a vital industry, meeting the growing global demand for seafood. Shrimp viral diseases have posed significant challenges to the aquaculture industry, causing major economic losses worldwide. Conventional treatment methods have proven to be ineffective in controlling these diseases. However, recent advances in RNA interference (RNAi) technology have opened new possibilities for combating shrimp viral diseases. This cutting-edge technology uses cellular machinery to silence specific viral genes, preventing viral replication and spread. Numerous studies have shown the effectiveness of RNAi-based therapies in various model organisms, paving the way for their use in shrimp health. By precisely targeting viral pathogens, RNAi has the potential to provide a sustainable and environmentally friendly solution to combat viral diseases in shrimp aquaculture. This review paper provides an overview of RNAi-based therapy and its potential as a game-changer for shrimp viral diseases. We discuss the principles of RNAi, its application in combating viral infections, and the current progress made in RNAi-based therapy for shrimp viral diseases. We also address the challenges and prospects of this innovative approach.

Regulation of plant immunity via small RNA-mediated control of NLR expression.

Plants use different receptors to detect potential pathogens: membrane-anchored pattern recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI); and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) activated by detection of pathogen-derived effectors, activating effector-triggered immunity (ETI). The interconnections between PTI and ETI responses have been increasingly reported. Elevated NLR levels may cause autoimmunity, with symptoms ranging from fitness cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide miRNAs with the unique ability to trigger secondary siRNA production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22 nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses.

Knockdown of Bmp1 and Pls1 Virulence Genes by Exogenous Application of RNAi-Inducing dsRNA in Botrytis cinerea.

Botrytis cinerea is a pathogen of wide agronomic and scientific importance partly due to its tendency to develop fungicide resistance. Recently, there has been great interest in the use of RNA interference as a control strategy against B. cinerea. In order to reduce the possible effects on non-target species, the sequence-dependent nature of RNAi can be used as an advantage to customize the design of dsRNA molecules. We selected two genes related to virulence: BcBmp1 (a MAP kinase essential for fungal pathogenesis) and BcPls1 (a tetraspanin related to appressorium penetration). After performing a prediction analysis of small interfering RNAs, dsRNAs of 344 (BcBmp1) and 413 (BcPls1) nucleotides were synthesized in vitro. We tested the effect of topical applications of dsRNAs, both in vitro by a fungal growth assay in microtiter plates and in vivo on artificially inoculated detached lettuce leaves. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination for BcBmp1, an evident growth retardation for BcPls1, and a strong reduction in necrotic lesions on lettuce leaves for both genes. Furthermore, a strongly reduced expression of the BcBmp1 and BcPls1 genes was observed in both in vitro and in vivo experiments, suggesting that these genes could be promising targets for the development of RNAi-based fungicides against B. cinerea.

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.

Enzymatic reactions of AGO4 in RNA-directed DNA methylation: siRNA duplex loading, passenger strand elimination, target RNA slicing, and sliced target retention.

RNA-directed DNA methylation in plants is guided by 24-nt siRNAs generated in parallel with 23-nt RNAs of unknown function. We show that 23-nt RNAs function as passenger strands during 24-nt siRNA incorporation into AGO4. The 23-nt RNAs are then sliced into 11- and 12-nt fragments, with 12-nt fragments remaining associated with AGO4. Slicing recapitulated with recombinant AGO4 and synthetic RNAs reveals that siRNAs of 21-24 nt, with any 5'-terminal nucleotide, can guide slicing, with sliced RNAs then retained by AGO4. In vivo, RdDM target locus RNAs that copurify with AGO4 also display a sequence signature of slicing. Comparing plants expressing slicing-competent versus slicing-defective AGO4 shows that slicing elevates cytosine methylation levels at virtually all RdDM loci. We propose that siRNA passenger strand elimination and AGO4 tethering to sliced target RNAs are distinct modes by which AGO4 slicing enhances RNA-directed DNA methylation.

Broken up but still living together: how ARGONAUTE's retention of cleaved fragments explains its role during chromatin modification.

Throughout the eukaryotic kingdoms, small RNAs direct chromatin modification. ARGONAUTE proteins sit at the nexus of this process, linking the small RNA information to the programming of chromatin. ARGONAUTE proteins physically incorporate the small RNAs as guides to target specific regions of the genome. In this issue of Genes & Development, Wang and colleagues (pp. 103-118) add substantial new detail to the processes of ARGONAUTE RNA loading, preference, cleavage, and retention, which together accomplish RNA-directed chromatin modification. They show that after catalytic cleavage by the plant ARGONAUTE protein AGO4, the cleaved fragment remains bound. This happens during two distinct RNA cleavage reactions performed by AGO4: first for a passenger RNA strand of the siRNA duplex, and second for a nascent transcript at the target DNA locus. Cleaved fragment retention of the nascent transcript explains how the protein complex accumulates to high levels at the target locus, amplifying chromatin modification.

Multiple integrations of a sense transgene, including a tandem inverted repeat confer stable RNA-silencing mediated virus resistance under different abiotic and biotic conditions.

In a previous study, tobacco plants, transformed with a sense construct of the 57K domain of the replicase gene of tobacco rattle virus (TRV), provided resistance against genetically distant isolates of the virus. In this work, 57K-specific siRNAs were detected with RT-qPCR solely in the resistant line verifying the RNA-silencing base of the resistance. The integration sites of the transgene into the plant genome were identified with inverse-PCR. Moreover, the resistance against TRV was practically unaffected by low temperature conditions and the presence of heterologous viruses. The mechanism of the resistance was further examined by a gene expression analysis that showed increased transcript levels of genes with a key-role in the RNA silencing pathway and the basal antiviral defence. This work provides a comprehensive characterization of the robust virus resistance obtained by a sense transgene and underlines the usefulness of transgenic plants obtained by such a strategy.

Establishment of Transcriptional Gene Silencing Targeting the Promoter Regions of GFP, PDS, and PSY Genes in Cotton using Virus-Induced Gene Silencing.

Virus-induced gene silencing (VIGS) by deploying viral-based vectors such as tobacco rattle virus (TRV) is a homology-based gene silencing technique in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) to validate the function of particular genes. The study presented here showed the induction of DNA methylation in the promoter regions of three phenotypic marker genes in different cotton accessions, including two endogenous genes such as phytoene desaturase (PDS) and phytoene synthase (PSY), and an exogenous gene, such as green fluorescent protein (GFP). First, DNA methylation was established in transgenic GFP cotton where methylation persisted up to S3 generation. Afterward, the promoter of PSY was targeted following the same conditions. Significant silencing of PSY was observed and methylation of the promoter was found up to S2 generation in red leaf cotton as detected in GFP cotton. Silencing of PDS resulted in a photobleaching phenotype; interestingly, the strength of this phenotype was diverse within the plants and was not observed in the next generation. Bisulfite sequencing results showed methylation percentage of the cytosine residues was high at CG and CHG sites of the targeted promoter sequences in the silenced plants. The findings of this paper suggest that TRV-based vector system can be used to monitor DNA methylation for both exogenous and endogenous gene levels in cotton and offer a very useful tool for plant epigenetic modification.

Plant terminators: the unsung heroes of gene expression.

To be properly expressed, genes need to be accompanied by a terminator, a region downstream of the coding sequence that contains the information necessary for the maturation of the mRNA 3' end. The main event in this process is the addition of a poly(A) tail at the 3' end of the new transcript, a critical step in mRNA biology that has important consequences for the expression of genes. Here, we review the mechanism leading to cleavage and polyadenylation of newly transcribed mRNAs and how this process can affect the final levels of gene expression. We give special attention to an aspect often overlooked, the effect that different terminators can have on the expression of genes. We also discuss some exciting findings connecting the choice of terminator to the biogenesis of small RNAs, which are a central part of one of the most important mechanisms of regulation of gene expression in plants.

Rearranged Endogenized Plant Pararetroviruses as Evidence of Heritable RNA-based Immunity.

Eukaryotic genomics frequently revealed historical spontaneous endogenization events of external invading nucleic acids, such as viral elements. In plants, an extensive occurrence of endogenous plant pararetroviruses (EPRVs) is usually believed to endow hosts with an additional layer of internal suppressive weaponry. However, an actual demonstration of this activity remains speculative. We analyzed the EPRV component and accompanying silencing effectors of Solanum lycopersicum, documenting that intronic/intergenic pararetroviral integrations bearing inverted-repeats fuel the plant's RNA-based immune system with suitable transcripts capable of evoking a silencing response. A surprisingly small set of rearrangements explained a substantial fraction of pararetroviral-derived endogenous small-interfering (si)RNAs, enriched in 22-nt forms typically associated with anti-viral post-transcriptional gene silencing. We provide preliminary evidence that such genetic and immunological signals may be found in other species outside the genus Solanum. Based on molecular dating, bioinformatics, and empirical explorations, we propose that homology-dependent silencing emerging from particular immuno-competent rearranged chromosomal areas that constitute an adaptive heritable trans-acting record of past infections, with potential impact against the unlocking of plant latent EPRVs and cognate-free pararetroviruses.

Small interfering RNA: Discovery, pharmacology and clinical development-An introductory review.

Post-transcriptional gene silencing targets and degrades mRNA transcripts, silencing the expression of specific genes. RNA interference technology, using synthetic structurally well-defined short double-stranded RNA (small interfering RNA [siRNA]), has advanced rapidly in recent years. This introductory review describes the utility of siRNA, by exploring the underpinning biology, pharmacology, recent advances and clinical developments, alongside potential limitations and ongoing challenges. Mediated by the RNA-induced silencing complex, siRNAs bind to specific complementary mRNAs, which are subsequently degraded. siRNA therapy offers advantages over other therapeutic approaches, including ability of specifically designed siRNAs to potentially target any mRNA and improved patient adherence through infrequent administration associated with a very long duration of action. Key pharmacokinetic and pharmacodynamic challenges include targeted administration, poor tissue penetration, nuclease inactivation, rapid renal elimination, immune activation and off-target effects. These have been overcome by chemical modification of siRNA and/or by utilising a range of delivery systems, increasing bioavailability and stability to allow successful clinical translation. Patisiran (hereditary transthyretin-mediated amyloidosis) was the first licensed siRNA, followed by givosiran (acute hepatic porphyria), lumasiran (primary hyperoxaluria type 1) and inclisiran (familial hypercholesterolaemia), which all use N-acetylgalactosamine (GalNAc) linkage for effective liver-directed delivery. Others are currently under development for indications varying from rare genetic diseases to common chronic non-communicable diseases (hypertension, cancer). Technological advances are paving the way for broader clinical use. Ongoing challenges remain in targeting organs beyond the liver and reaching special sites (e.g., brain). By overcoming these barriers, siRNA therapy has the potential to substantially widen its therapeutic impact.

Structural features of T-DNA that induce transcriptional gene silencing during agroinfiltration.

Agrobacterium tumefaciens (Rhizobium radiobacter) is used for the transient expression of foreign genes by the agroinfiltration method, but the introduction of foreign genes often induces transcriptional and/or post-transcriptional gene silencing (TGS and/or PTGS). In this study, we characterized the structural features of T-DNA that induce TGS during agroinfiltration. When A. tumefaciens cells harboring an empty T-DNA plasmid containing the cauliflower mosaic virus (CaMV) 35S promoter were infiltrated into the leaves of Nicotiana benthamiana line 16c with a GFP gene over-expressed under the control of the same promoter, no small interfering RNAs (siRNAs) were derived from the GFP sequence. However, siRNAs derived from the CaMV 35S promoter were detected, indicating that TGS against the GFP gene was induced. When the GFP gene was inserted into the T-DNA plasmid, PTGS against the GFP gene was induced whereas TGS against the CaMV 35S promoter was suppressed. We also showed the importance of terminator sequences in T-DNA for gene silencing. Therefore, depending on the combination of promoter, terminator and coding sequences on T-DNA and the host nuclear genome, either or both TGS and/or PTGS could be induced by agroinfiltration. Furthermore, we showed the possible involvement of three siRNA-producing Dicers (DCL2, DCL3 and DCL4) in the induction of TGS by the co-agroinfiltration method. Especially, DCL2 was probably the most important among them in the initial step of TGS induction. These results are valuable for controlling gene expression by agroinfiltration.