Plant Virus Replication Research Articles

A novel pH-responsive multi-component nanodelivery system with siRNA for plant disease management

At the present time, the control of plant viral diseases is primarily achieved through the use of traditional pesticides. However, the tendency of RNA viruses to mutate and develop resistance to pesticides represents a significant challenge. The development of nanoscale strategies based on RNA interference (RNAi) represents a rapidly evolving approach to sustainable plant disease control. In this study, an siRNA was screened that effectively targets the 70-kDa heat shock protein (HSP70), which is closely associated with plant virus infection and replication. Additionally, a novel nanosystem for hierarchical delivery of siRNA was developed. This consisted of the cellular shuttle peptide (CPP)-modified tetrahedral DNA nanoparticles (TDN) as the core and polydopamine-hybridized mesoporous silica nanoparticles (PDA-MSN) as the shell. This was named TDN-siR099@CPP@PDA-MSNs. The results demonstrated that upon entering the leaf tissue through the stomata, TDN-siR099@CPP@PDA-MSNs encountered a weak acidic environment, prompting the PDA-MSN to release TDN-siR099@CPP. This resulted in the intercellular shuttling of TDN-siR099@CPP. The nanoscale dimensions of TDN-siR099@CPP facilitate facile displacement, while the intrinsic properties of CPP enhance the efficacy of long-distance siR099 intercellular shuttling. In this nanosystem, we also demonstrated that PDA-MSN retarded the susceptible degradation of siRNA in vitro and responded to the acidic release of the drug, which improve the utilization efficiency of siRNA in terms of both improving the delivery efficiency and delaying the degradation. It effectively down-regulated 56.2% of HSP70 protein and inhibited the infection of tobacco mosaic virus (TMV) and potato Y virus (PVY) by 70.2% and 41.0%, respectively. By skillfully constructing a framework for siRNA inhibition of target genes, this technology broadens the application of RNA silencing in plant disease management.

Small RNA and Degradome Deep Sequencing Reveal Regulatory Roles of MicroRNAs in Response to Sugarcane Mosaic Virus Infection on Two Contrasting Sugarcane Cultivars.

MicroRNAs (miRNAs) play an essential regulatory role in plant-virus interaction. However, few studies have focused on the roles of miRNAs and their targets after sugarcane mosaic virus (SCMV) infection in sugarcane. To address this issue, we conducted small RNA (sRNA) and degradome sequencing on two contrasting sugarcanes (SCMV-resistant 'Fuoguo1' [FG1] and susceptible 'Badila') infected by SCMV at five time points. A total of 1,578 miRNAs were profiled from 30 sRNA libraries, comprising 660 known miRNAs and 380 novel miRNAs. Differential expression analysis of miRNAs revealed that most were highly expressed during the SCMV exponential phase in Badila at 18 h postinfection, with expression profiles positively correlated with virus replication dynamics as observed through clustering. Analysis of degradome data indicated a higher number of differential miRNA targets in Badila compared to FG1 at 18 h postinfection. Gene ontology (GO) enrichment analysis significantly enriched the stimulus-response pathway, suggesting negative regulatory roles to SCMV resistance. Specifically, miR160 upregulated expression patterns and validated in Badila through quantitative real-time PCR (qRT-PCR) in the early stages of SCMV multiplication. Our research provides new insights into the dynamic response of plant miRNA and virus replication and contributes valuable information on the intricate interplay between miRNAs and SCMV infection dynamics. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Replication Assay of Begomovirus in Arabidopsis Protoplasts.

Protoplasts are isolated plant cells from which the cell walls have been removed by treatment with fungal cellulase and macerozyme enzymes, which degrade the primary components of the cell wall. The protoplasts are totipotent, sensitive, and versatile; thereby, they have been extensively used to study signal transduction pathways, cell-autonomous responses, and replication of plant viruses. This system has several advantages over the use of whole plants for viral replication, including a high percentage of infected cells and uncoupling virus movement from replication assays. Here, we describe a simple and efficient protocol for begomovirus transfection and replication in Arabidopsis protoplasts. The method consists of four steps, (i) protoplast isolation, (ii) PEG-calcium transfection of begomovirus infectious clones, (iii) elimination of input plasmid DNA by DpnI digestion, and (iv) quantification of the viral newly synthesized DNA by qPCR. The protoplasts can be transformed efficiently with begomovirus infectious clones, and virus replication can be monitored by the accumulation of nascent viral DNA in the infected protoplasts.

RETICULON-LIKE PROTEIN B2 is a proviral factor co-opted for the biogenesis of viral replication organelles in plants.

Endomembrane remodeling to form a viral replication complex (VRC) is crucial for a virus to establish infection in a host. Although the composition and function of VRCs have been intensively studied, host factors involved in the assembly of VRCs for plant RNA viruses have not been fully explored. TurboID-based proximity labeling (PL) has emerged as a robust tool for probing molecular interactions in planta. However, few studies have employed the TurboID-based PL technique for investigating plant virus replication. Here, we used Beet black scorch virus (BBSV), an endoplasmic reticulum (ER)-replicating virus, as a model and systematically investigated the composition of BBSV VRCs in Nicotiana benthamiana by fusing the TurboID enzyme to viral replication protein p23. Among the 185 identified p23-proximal proteins, the reticulon family of proteins showed high reproducibility in the mass spectrometry data sets. We focused on RETICULON-LIKE PROTEIN B2 (RTNLB2) and demonstrated its proviral functions in BBSV replication. We showed that RTNLB2 binds to p23, induces ER membrane curvature, and constricts ER tubules to facilitate the assembly of BBSV VRCs. Our comprehensive proximal interactome analysis of BBSV VRCs provides a resource for understanding plant viral replication and offers additional insights into the formation of membrane scaffolds for viral RNA synthesis.

Topical application of coat protein specific dsRNA molecules confers resistance against cognate Potato virus Y (PVY) infecting potato of North-East India

Sustainable agriculture relies on techniques and technology that combine efficiency with the smallest possible ecological and environmental impact. RNA interference (RNAi), a post-transcriptional eukaryotic pathway in eukaryotes can be co-opted for the control of plant pathogens in the topical application system. RNAi is the biological mechanism of mRNA degradation triggered by endogenous or exogenous double-stranded small interfering RNAs (dsRNA/siRNA) and regulates the target gene expression. Topically applied double-stranded RNA (dsRNA) molecules on plants imitate the viral RNA intermediate which is involved in plant virus replication and thereby triggers post-transcriptional gene silencing via., RNAi pathway and reducing viral replication. In this study, a non-transgenic-based RNAi approach was adopted to induce resistance against PVY in potato crop. dsRNA was successfully produced through in vitro transcription and hybridization. To test the effect of dsRNA molecules, in vitro synthesized dsRNA were applied on potato leaves along with PVY. dsRNA-treated plants showed higher resistance to viral disease and exhibited normal growth compared with PVY-treated plants. Therefore, the topical application of dsRNA molecules from the PVY CP gene could serve as a promising method for suppressing PVY infection and ultimately enhancing potato yield.

Priming crop plants with rosemary (Salvia rosmarinus Spenn, syn Rosmarinus officinalis L.) extract triggers protective defense response against pathogens

Plant bioactive compounds provide novel straightforward approaches to control plant diseases. Rosemary (Salvia rosmarinus)-derived extracts carry many prominent pharmacological activities, including antimicrobial and antioxidant, mainly due to its phenolic compounds, rosmarinic acid (RA), carnosic acid and carnosol. However, the effects of these extracts on plant diseases are still unknown, which constrains its potential application as bioprotectant in the agricultural production. In this study we demonstrate the antiviral effect of the aqueous rosemary extract (ARE) against tobacco necrosis virus strain A (TNVA) in ARE-treated tobacco (Nicotiana tabacum) plants. Our results show that ARE-treatment enhances plant defense response, contributing to reduce virus replication and systemic movement in tobacco plants. RA, the main phenolic compound detected in this extract, is one of the main inducers of TNVA control. The ARE-induced protection in TNVA-infected plants was characterized by the expression of H2O2 scavengers and defense-related genes, involving salicylic acid- and jasmonic acid-regulated pathways. Furthermore, treatment with ARE in lemon (Citrus limon) and soybean (Glycine max) leaves protects the plants against Xanthomonas citri subsp. citri and Diaporthe phaseolorum var. meridionalis, respectively. Additionally, ARE treatment also promotes growth and development, suggesting a biostimulant activity in soybean. These results open the way for the potential use of ARE as a bioprotective agent in disease management.

Manipulation of the Cellular Membrane-Cytoskeleton Network for RNA Virus Replication and Movement in Plants.

Viruses infect all cellular life forms and cause various diseases and significant economic losses worldwide. The majority of viruses are positive-sense RNA viruses. A common feature of infection by diverse RNA viruses is to induce the formation of altered membrane structures in infected host cells. Indeed, upon entry into host cells, plant-infecting RNA viruses target preferred organelles of the cellular endomembrane system and remodel organellar membranes to form organelle-like structures for virus genome replication, termed as the viral replication organelle (VRO) or the viral replication complex (VRC). Different viruses may recruit different host factors for membrane modifications. These membrane-enclosed virus-induced replication factories provide an optimum, protective microenvironment to concentrate viral and host components for robust viral replication. Although different viruses prefer specific organelles to build VROs, at least some of them have the ability to exploit alternative organellar membranes for replication. Besides being responsible for viral replication, VROs of some viruses can be mobile to reach plasmodesmata (PD) via the endomembrane system, as well as the cytoskeleton machinery. Viral movement protein (MP) and/or MP-associated viral movement complexes also exploit the endomembrane-cytoskeleton network for trafficking to PD where progeny viruses pass through the cell-wall barrier to enter neighboring cells.

The Crucial Role of Chloroplast-Related Proteins in Viral Genome Replication and Host Defense against Positive-Sense Single-Stranded RNA Viruses.

Plant viruses are responsible for worldwide production losses of numerous economically important crops. The most common plant RNA viruses are positivesense single-stranded RNA viruses [(+)ss RNA viruses]. These viruses have small genomes that encode a limited number of proteins. The viruses depend on their host's machinery for the replication of their RNA genome, assembly, movement, and attraction to the vectors for dispersal. Recently researchers have reported that chloroplast proteins are crucial for replicating (+)ss plant RNA viruses. Some chloroplast proteins, including translation initiation factor [eIF(iso)4E] and 75 DEAD-box RNA helicase RH8, help viruses fulfill their infection cycle in plants. In contrast, other chloroplast proteins such as PAP2.1, PSaC, and ATPsyn-α play active roles in plant defense against viruses. This is also consistent with the idea that reactive oxygen species, salicylic acid, jasmonic acid, and abscisic acid are produced in chloroplast. However, knowledge of molecular mechanisms and functions underlying these chloroplast host factors during the virus infection is still scarce and remains largely unknown. Our review briefly summarizes the latest knowledge regarding the possible role of chloroplast in plant virus replication, emphasizing chloroplast-related proteins. We have highlighted current advances regarding chloroplast-related proteins' role in replicating plant (+)ss RNA viruses.

Role of an RNA pseudoknot involving the polyA tail in replication of Pepino mosaic potexvirus and related plant viruses

Pepino mosaic virus (PepMV) is a potexvirus of the family Alphaflexiviridae within the order of Tymovirales that threatens tomato production worldwide. PepMV possesses a positive-strand RNA genome with a 5′-methylguanosine cap and a 3′-polyA tail. Previously, using partially-purified viral RNA polymerase important secondary structures within the 3′-untranslated region (UTR) of PepMV RNA were identified. Here we show that an RNA pseudoknot can be formed in the 3′-UTR that includes part of the polyA tail. Using protoplasts, we demonstrate that the pseudoknot is required for replication of PepMV RNA. Mutational analysis and native gel electrophoresis further show that the pseudoknot is stabilized by UAU base triples, as is the human telomerase RNA pseudoknot. The presence of a pseudoknot in several other members of the Alpha- and Betaflexiviridae is supported by covariance analysis and native gel electrophoresis of other potexvirus, capillovirus and trichovirus RNAs. The ubiquitous presence of the pseudoknot in viruses of the Betaflexiviridae, suggests that the pseudoknot is a typical trait of the Betaflexiviridae that may have been adopted by many potexviruses during evolution.

A spotlight on non-host resistance to plant viruses.

Plant viruses encounter a range of host defenses including non-host resistance (NHR), leading to the arrest of virus replication and movement in plants. Viruses have limited host ranges, and adaptation to a new host is an atypical phenomenon. The entire genotypes of plant species which are imperceptive to every single isolate of a genetically variable virus species are described as non-hosts. NHR is the non-specific resistance manifested by an innately immune non-host due to pre-existing and inducible defense responses, which cannot be evaded by yet-to-be adapted plant viruses. NHR-to-plant viruses are widespread, but the phenotypic variation is often not detectable within plant species. Therefore, molecular and genetic mechanisms of NHR need to be systematically studied to enable exploitation in crop protection. This article comprehensively describes the possible mechanisms of NHR against plant viruses. Also, the previous definition of NHR to plant viruses is insufficient, and the main aim of this article is to sensitize plant pathologists to the existence of NHR to plant viruses and to highlight the need for immediate and elaborate research in this area.

Targeting conserved co-opted host factors to block virus replication: Using allosteric inhibitors of the cytosolic Hsp70s to interfere with tomato bushy stunt virus replication

To further our understanding of the pro-viral roles of the host cytosolic heat shock protein 70 (Hsp70) family, we chose the conserved Arabidopsis thaliana Hsp70-2 and the unique Erd2 (early response to dehydration 2), which contain Hsp70 domains. Based on in vitro studies with purified components, we show that AtHsp70-2 and AtErd2 perform pro-viral functions equivalent to that of the yeast Ssa1 Hsp70. These functions include activation of the tombusvirus RdRp, and stimulation of replicase assembly. Yeast-based complementation studies demonstrate that AtHsp70-2 or AtErd2 are present in the purified tombusvirus replicase. RNA silencing and over-expression studies in Nicotiana benthamiana suggest that both Hsp70-2 and Erd2 are co-opted by tomato bushy stunt virus (TBSV). Moreover, we used allosteric inhibitors of Hsp70s to inhibit replication of TBSV and related plant viruses in plants. Altogether, interfering with the functions of the co-opted Hsp70s could be an effective antiviral approach against tombusviruses in plants.

High overexpression of CERES, a plant regulator of translation, induces different phenotypical defence responses during TuMV infection.

Mutations in the eukaryotic translation initiation factors eIF4E and eIF(iso)4E confer potyvirus resistance in a range of plant hosts. This supports the notion that, in addition to their role in translation of cellular mRNAs, eIF4E isoforms are also essential for the potyvirus cycle. CERES is a plant eIF4E- and eIF(iso)4E-binding protein that, through its binding to the eIF4Es, modulates translation initiation; however, its possible role in potyvirus resistance is unknown. In this article, we analyse if the ectopic expression of AtCERES is able to interfere with turnip mosaic virus replication in plants. Our results demonstrate that, during infection, the ectopic expression of CERES in Nicotiana benthamiana promotes the development of a mosaic phenotype when it is accumulated to moderate levels, but induces veinal necrosis when it is accumulated to higher levels. This necrotic process resembles a hypersensitive response (HR)-like response that occurs with different HR hallmarks. Remarkably, Arabidopsis plants inoculated with a virus clone that promotes high expression of CERES do not show signs of infection. These final phenotypical outcomes are independent of the capacity of CERES to bind to eIF4E. All these data suggest that CERES, most likely due to its leucine-rich repeat nature, could act as a resistance protein, able to promote a range of different defence responses when it is highly overexpressed from viral constructs.

Imaging Techniques to Study Plant Virus Replication and Vertical Transmission.

Plant viruses are obligate parasites that need to usurp plant cell metabolism in order to infect their hosts. Imaging techniques have been used for quite a long time to study plant virus–host interactions, making it possible to have major advances in the knowledge of plant virus infection cycles. The imaging techniques used to study plant–virus interactions have included light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopies. Here, we review the use of these techniques in plant virology, illustrating recent advances in the area with examples from plant virus replication and virus plant-to-plant vertical transmission processes.

Viral subversion of nonsense-mediated mRNA decay.

Viruses have evolved in tandem with the organisms that they infect. Afflictions of the plant and animal kingdoms with viral infections have forced the host organism to evolve new or exploit existing systems to develop the countermeasures needed to offset viral insults. As one example, nonsense-mediated mRNA decay, a cellular quality-control mechanism ensuring the translational fidelity of mRNA transcripts, has been used to restrict virus replication in both plants and animals. In response, viruses have developed a slew of means to disrupt or become insensitive to NMD, providing researchers with potential new reagents that can be used to more fully understand the NMD mechanism.

Co-opted Cellular Sac1 Lipid Phosphatase and PI(4)P Phosphoinositide Are Key Host Factors during the Biogenesis of the Tombusvirus Replication Compartment.

Positive-strand RNA [(+)RNA] viruses assemble numerous membrane-bound viral replicase complexes (VRCs) with the help of viral replication proteins and co-opted host proteins within large viral replication compartments in the cytosol of infected cells. In this study, we found that deletion or depletion of Sac1 phosphatidylinositol 4-phosphate [PI(4)P] phosphatase reduced tomato bushy stunt virus (TBSV) replication in yeast (Saccharomyces cerevisiae) and plants. We demonstrate a critical role for Sac1 in TBSV replicase assembly in a cell-free replicase reconstitution assay. The effect of Sac1 seems to be direct, based on its interaction with the TBSV p33 replication protein, its copurification with the tombusvirus replicase, and its presence in the virus-induced membrane contact sites and within the TBSV replication compartment. The proviral functions of Sac1 include manipulation of lipid composition, sterol enrichment within the VRCs, and recruitment of additional host factors into VRCs. Depletion of Sac1 inhibited the recruitment of Rab5 GTPase-positive endosomes and enrichment of phosphatidylethanolamine in the viral replication compartment. We propose that Sac1 might be a component of the assembly hub for VRCs, likely in collaboration with the co-opted the syntaxin18-like Ufe1 SNARE protein within the TBSV replication compartments. This work also led to demonstration of the enrichment of PI(4)P phosphoinositide within the replication compartment. Reduction in the PI(4)P level due to chemical inhibition in plant protoplasts; depletion of two PI(4)P kinases, Stt4p and Pik1p; or sequestration of free PI(4)P via expression of a PI(4)P-binding protein in yeast strongly inhibited TBSV replication. Altogether, Sac1 and PI(4)P play important proviral roles during TBSV replication.IMPORTANCE Replication of positive-strand RNA viruses depends on recruitment of host components into viral replication compartments or organelles. Using TBSV, we uncovered the critical roles of Sac1 PI(4)P phosphatase and its substrate, PI(4)P phosphoinositide, in promoting viral replication. Both Sac1 and PI(4)P are recruited to the site of viral replication to facilitate the assembly of the viral replicase complexes, which perform viral RNA replication. We found that Sac1 affects the recruitment of other host factors and enrichment of phosphatidylethanolamine and sterol lipids within the subverted host membranes to promote optimal viral replication. In summary, this work demonstrates the novel functions of Sac1 and PI(4)P in TBSV replication in the model host yeast and in plants.

Whitefly HES1 binds to the intergenic region of Tomato yellow leaf curl China virus and promotes viral gene transcription

Intergenic region of begomovirus genome is vital to virus replication and viral gene transcription in plants. Previous studies have reported that Tomato yellow leaf curl China virus (TYLCCNV), a begomovirus, is able to accumulate and transcribe in its whitefly vector. However, the viral and host components that participate in begomovirus transcription in whiteflies are hitherto unknown. Using a yeast one-hybrid system, we identified >50 whitefly proteins that interacted with TYLCCNV intergenic region. Dual luciferase analysis revealed that one of the identified proteins, the hairy and enhancer of split homolog-1 (HES1), specifically bound to CACGTG motif in TYLCCNV intergenic region. Silencing HES1 decreased viral transcription, accumulation and transmission. These results demonstrate that the interactions between whitefly proteins and the intergenic region of TYLCCNV may contribute to viral transcription in the whitefly vector. Our findings offer valuable clues for the research and development of novel strategies to interfere with begomovirus transmission.

Plant trichomes as microbial habitats and infection sites

Trichomes, also simply referred to as hairs, are fine outgrowths of epidermal cells in many organisms including plants and bacteria. Plant trichomes have long been known for their multiple beneficial roles, ranging from protection against insect herbivores and ultraviolet light to the reduction of transpiration. However, there is increasing evidence that the presence of trichomes may have detrimental consequences for plants. For example, plant pathogenic bacteria can enter hosts through the open bases or broken stalks of damaged trichomes. Similarly, trichomes are considered a preferred site for fungal infection, and in this regard, the colonization and penetration of trichomes by fungi and oomycetes have been visualized using light, fluorescence, and scanning electron microscopy in a variety of plants from grasses to shrubs and trees. In addition to parasitic interactions, trichomes also form a host site for endophytic relationships with fungi, thereby serving as an unusual fungal niche. The replication and presence of plant viruses in trichomes have also been confirmed after inoculation. In contrast, the well-known beneficial Azolla–Anabaena symbiosis is facilitated through epidermal trichomes of the seedless vascular plant Azolla. These observations indicate that plant trichomes are involved in multiple interactions in terms of providing microbial habitats and infection sites as well as functioning as protective structures. Trichome-related microbial parasitism and endophytism can, in many ways, be considered comparable to those associated with root hairs.

Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Plant Virus Replication.

Positive-sense (+) RNA viruses represent the most abundant group of viruses and are dependent on the host cell machinery to replicate. One remarkable feature that occurs after (+) RNA virus entry into cells is the remodeling of host endomembranes, leading to the formation of viral replication factories. Recently, rapid progress in three-dimensional (3D) imaging technologies, such as electron tomography (ET) and focused ion beam-scanning electron microscopy (FIB-SEM), has enabled researchers to visualize the novel membrane structures induced by viruses at high resolution. These 3D imaging technologies provide new mechanistic insights into the viral infection cycle. In this review, we summarize the latest reports on the cellular remodeling that occurs during plant virus infection; in particular, we focus on studies that provide 3D architectural information on viral replication factories. We also outline the mechanisms underlying the formation of these membranous structures and discuss possible future research directions.

The roles of membranes and associated cytoskeleton in plant virus replication and cell-to-cell movement

The infection of plants by viruses depends on cellular mechanisms that support the replication of the viral genomes, and the cell-to-cell and systemic movement of the virus via plasmodesmata (PD) and the connected phloem. While the propagation of some viruses requires the conventional endoplasmic reticulum (ER)-Golgi pathway, others replicate and spread between cells in association with the ER and are independent of this pathway. Using selected viruses as examples, this review re-examines the involvement of membranes and the cytoskeleton during virus infection and proposes potential roles of class VIII myosins and membrane-tethering proteins in controlling viral functions at specific ER subdomains, such as cortical microtubule-associated ER sites, ER-plasma membrane contact sites, and PD.

Quantification of Pea enation mosaic virus 1 and 2 during infection of Pisum sativum by one step real-time RT-PCR

Pea enation mosaic virus 1 (PEMV1) and Pea enation mosaic virus 2 (PEMV2) are two viruses in an obligate symbiosis that cause pea enation mosaic disease mainly in plants in the Fabaceae family. This virus system is a valuable model to investigate plant virus replication, movement and vector transmission. Thus, here we describe growth conditions, virus detection methods, and virus accumulation behavior. To measure the accumulation and movement of PEMV1 and PEMV2 in plants during the course of infection, we developed a quantitative real-time one-step reverse transcription PCR procedure using the SYBR-green® technology. Viral primers were designed that anneal to conserved but distinct regions in the RNA-dependent RNA polymerase gene of each virus. Moreover, the normalization of viral accumulation was performed to correct for sample-to-sample variation by designing primers to two different Pisum sativum housekeeping genes: actin and β-tubulin. Transcript levels for these housekeeping genes did not change significantly in response to PEMV infection. Conditions were established for maximum PCR efficiency for each gene, and quantification using QuBit® technology. Both viruses reached maximum accumulation around 21days post-inoculation of pea plants. These results provide valuable tools and knowledge to allow reproducible studies of this emerging model virus system virus complex.