Structural characterization of a new subclass of panicum mosaic virus-like 3' cap-independent translation enhancer.

Canonical eukaryotic mRNA translation requires 5'cap recognition by initiation factor 4E (eIF4E). In contrast, many positive-strand RNA virus genomes lack a 5'cap and promote translation by non-canonical mechanisms. Among plant viruses, PTEs are a major class of cap-independent translation enhancers located in/near the 3'UTR that recruit eIF4E to greatly enhance viral translation. Previous work proposed a single form of PTE characterized by a Y-shaped secondary structure with two terminal stem-loops (SL1 and SL2) atop a supporting stem containing a large, G-rich asymmetric loop that forms an essential pseudoknot (PK) involving C/U residues located between SL1 and SL2. We found that PTEs with less than three consecutive cytidylates available for PK formation have an upstream stem-loop that forms a kissing loop interaction with the apical loop of SL2, important for formation/stabilization of PK. PKs found in both subclasses of PTE assume a specific conformation with a hyperreactive guanylate (G*) in SHAPE structure probing, previously found critical for binding eIF4E. While PTE PKs were proposed to be formed by Watson-Crick base-pairing, alternative chemical probing and 3D modeling indicate that the Watson-Crick faces of G* and an adjacent guanylate have high solvent accessibilities. Thus, PTE PKs are likely composed primarily of non-canonical interactions.

Subcellular dynamics of red clover necrotic mosaic virus double-stranded RNAs in infected plant cells.

New evidences are emerging to support the importance of viral replication complexes (VRCs) in not only viral replication, but also viral cell-to-cell movement. Currently, how VRCs grow in size and colocalize with viral movement proteins (MPs) remains unclear. Herein, we performed live-cell imaging of red clover necrotic mosaic virus (RCNMV) dsRNA by using reporter B2-GFP plants. Tiny granules of dsRNA were formed along the endoplasmic reticulum (ER) at an early stage of infection. Importantly, the colocalization of the dsRNA granules with the virus-encoded p27 replication protein showed that these structures are components of VRCs. These granules moved throughout the cytoplasm, driven by the acto-myosin system, and coalesced with each other to form larger aggregates; the MPs were not associated with these processes. Notably, the MPs colocalized preferentially with large dsRNA aggregates, rather than with tiny dsRNA granules, suggesting that the increase in the size of VRCs promotes their colocalization with MPs.

Effects of the Noncoding Subgenomic RNA of Red Clover Necrotic Mosaic Virus in Virus Infection.

In recent years, a new class of viral noncoding subgenomic RNA (ncsgRNA) has been identified. This RNA is generated as a stable degradation product via an exoribonuclease-resistant RNA (xrRNA) structure, which blocks the progression of 5'→3' exoribonuclease on viral RNAs in infected cells. Here, we assess the effects of the ncsgRNA of red clover necrotic mosaic virus (RCNMV), called SR1f, in infected plants. We demonstrate the following: (i) the absence of SR1f reduces symptoms and decreases viral RNA accumulation in Nicotiana benthamiana and Arabidopsis thaliana plants; (ii) SR1f has an essential function other than suppression of RNA silencing; and (iii) the cytoplasmic exoribonuclease involved in mRNA turnover, XRN4, is not required for SR1f production or virus infection. A comparative transcriptomic analysis in N. benthamiana infected with wild-type RCNMV or an SR1f-deficient mutant RCNMV revealed that wild-type RCNMV infection, which produces SR1f and much higher levels of virus, has a greater and more significant impact on cellular gene expression than the SR1f-deficient mutant. Upregulated pathways include plant hormone signaling, plant-pathogen interaction, MAPK signaling, and several metabolic pathways, while photosynthesis-related genes were downregulated. We compare this to host genes known to participate in infection by other tombusvirids. Viral reads revealed a 10- to 100-fold ratio of positive to negative strand, and the abundance of reads of both strands mapping to the 3' region of RCNMV RNA1 support the premature transcription termination mechanism of synthesis for the coding sgRNA. These results provide a framework for future studies of the interactions and functions of noncoding RNAs of plant viruses. IMPORTANCE Knowledge of how RNA viruses manipulate host and viral gene expression is crucial to our understanding of infection and disease. Unlike viral protein-host interactions, little is known about the control of gene expression by viral RNA. Here, we begin to address this question by investigating the noncoding subgenomic RNA (ncsgRNA) of red clover necrotic mosaic virus (RCNMV), called SR1f. Similar exoribonuclease-resistant RNAs of flaviviruses are well studied, but the roles of plant viral ncsgRNAs, and how they arise, are poorly understood. Surprisingly, we find the likely exonuclease candidate, XRN4, is not required to generate SR1f, and we assess the effects of SR1f on virus accumulation and symptom development. Finally, we compare the effects of infection by wild-type RCNMV versus an SR1f-deficient mutant on host gene expression in Nicotiana benthamiana, which reveals that ncsgRNAs such as SR1f are key players in virus-host interactions to facilitate productive infection.

Genetic evidence for the involvement of Dicer-like 2 and 4 as well as Argonaute 2 in the Nicotiana benthamiana response against Pelargonium line pattern virus.

In plants, RNA silencing functions as a potent antiviral mechanism. Virus-derived double-stranded RNAs (dsRNAs) trigger this mechanism, being cleaved by Dicer-like (DCL) enzymes into virus small RNAs (vsRNAs). These vsRNAs guide sequence-specific RNA degradation upon their incorporation into an RNA-induced silencing complex (RISC) that contains a slicer of the Argonaute (AGO) family. Host RNA dependent-RNA polymerases, particularly RDR6, strengthen antiviral silencing by generating more dsRNA templates from RISC-cleavage products that, in turn, are converted into secondary vsRNAs by DCLs. Previous work showed that Pelargonium line pattern virus (PLPV) is a very efficient inducer and target of RNA silencing as PLPV-infected Nicotiana benthamiana plants accumulate extraordinarily high amounts of vsRNAs that, strikingly, are independent of RDR6 activity. Several scenarios may explain these observations including a major contribution of dicing versus slicing for defence against PLPV, as the dicing step would not be affected by the RNA silencing suppressor encoded by the virus, a protein that acts via vsRNA sequestration. Taking advantage of the availability of lines of N. benthamiana with DCL or AGO2 functions impaired, here we have tried to get further insights into the components of the silencing machinery that are involved in anti-PLPV-silencing. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection. Interestingly, AGO2 apparently makes even a higher contribution in the defence against PLPV, extending the number of viruses that are affected by this particular slicer. The data support that both dicing and slicing activities participate in the host race against PLPV.

The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism.

Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes [viral replication factories or complex (VRC)], in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle.

Three-dimensional reconstruction and comparison of vacuolar membranes in response to viral infection.

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV-AC ), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.

A trans-activator-like structure in RCNMV RNA1 evokes the origin of the trans-activator in RNA2.

The Red clover necrotic mosaic virus (RCNMV) genome consists of two plus-strand RNA genome segments, RNA1 and RNA2. RNA2 contains a multifunctional RNA structure known as the trans-activator (TA) that (i) promotes subgenomic mRNA transcription from RNA1, (ii) facilitates replication of RNA2, and (iii) mediates particle assembly and copackaging of genome segments. The TA has long been considered a unique RNA element in RCNMV. However, by examining results from RCNMV genome analyses in the ViRAD virus (re-)annotation database, a putative functional RNA element in the polymerase-coding region of RNA1 was identified. Structural and functional analyses revealed that the novel RNA element adopts a TA-like structure (TALS) and, similar to the requirement of the TA for RNA2 replication, the TALS is necessary for the replication of RNA1. Both the TA and TALS possess near-identical asymmetrical internal loops that are critical for efficient replication of their corresponding genome segments, and these structural motifs were found to be functionally interchangeable. Moreover, replacement of the TA in RNA2 with a stabilized form of the TALS directed both RNA2 replication and packaging of both genome segments. Based on their comparable properties and considering evolutionary factors, we propose that the TALS appeared de novo in RNA1 first and, subsequently, the TA arose de novo in RNA2 as a functional mimic of the TALS. This and other related information were used to formulate a plausible evolutionary pathway to describe the genesis of the bi-segmented RCNMV genome. The resulting scenario provides an evolutionary framework to further explore and test possible origins of this segmented RNA plant virus.

Panicum Mosaic Virus and Its Satellites Acquire RNA Modifications Associated with Host-Mediated Antiviral Degradation.

Positive-sense RNA viruses in the Tombusviridae family have genomes lacking a 5' cap structure and prototypical 3' polyadenylation sequence. Instead, these viruses utilize an extensive network of intramolecular RNA-RNA interactions to direct viral replication and gene expression. Here we demonstrate that the genomic RNAs of Panicum mosaic virus (PMV) and its satellites undergo sequence modifications at their 3' ends upon infection of host cells. Changes to the viral and subviral genomes arise de novo within Brachypodium distachyon (herein called Brachypodium) and proso millet, two alternative hosts of PMV, and exist in the infections of a native host, St. Augustinegrass. These modifications are defined by polyadenylation [poly(A)] events and significant truncations of the helper virus 3' untranslated region-a region containing satellite RNA recombination motifs and conserved viral translational enhancer elements. The genomes of PMV and its satellite virus (SPMV) were reconstructed from multiple poly(A)-selected Brachypodium transcriptome data sets. Moreover, the polyadenylated forms of PMV and SPMV RNAs copurify with their respective mature icosahedral virions. The changes to viral and subviral genomes upon infection are discussed in the context of a previously understudied poly(A)-mediated antiviral RNA degradation pathway and the potential impact on virus evolution.IMPORTANCE The genomes of positive-sense RNA viruses have an intrinsic capacity to serve directly as mRNAs upon viral entry into a host cell. These RNAs often lack a 5' cap structure and 3' polyadenylation sequence, requiring unconventional strategies for cap-independent translation and subversion of the cellular RNA degradation machinery. For tombusviruses, critical translational regulatory elements are encoded within the 3' untranslated region of the viral genomes. Here we describe RNA modifications occurring within the genomes of Panicum mosaic virus (PMV), a prototypical tombusvirus, and its satellite agents (i.e., satellite virus and noncoding satellite RNAs), all of which depend on the PMV-encoded RNA polymerase for replication. The atypical RNAs are defined by terminal polyadenylation and truncation events within the 3' untranslated region of the PMV genome. These modifications are reminiscent of host-mediated RNA degradation strategies and likely represent a previously underappreciated defense mechanism against invasive nucleic acids.

A polysaccharide found in Dendrobium nobile Lindl stimulates calcium signaling pathway and enhances tobacco defense against TMV.

A neutral polysaccharide separated from Dendrobium nobile Lindl was designated as DNPE6(4). It was structurally characterized using a combination of spectral and chemical analysis. Its average molecule weight was 99.2 kDa. The monosaccharide composition was Araf, Glcp, Galp, and Manp in a molar ratio of 2.5:0.9:0.3:0.8. Their linkage types were →1)-L-Araf-(3→, →1)-D-Glcp-(4→, →1)-D-Galp-(3→, →1)-D-Galp-(6→, →1)-D-Manp-(3, 6→, and T-D-Manp. The polysaccharide was found to have anti-TMV and anti-CMV activities for the first time in vivo. Notably, DNPE6(4) exhibited excellent protective activity against TMV. Furthermore, several proteins related to calcium signaling pathway and pathogen related proteins were up-regulated, and we also found expression levels of EDS1, ICS1, and PR1 involved in SA pathway up-regulated after DNPE6(4) treatment. In addition, some defense enzymes increased in the same condition. All these findings revealed DNPE6(4) was an elicitor to stimulate calcium signaling pathway to enhance the tobacco defense against TMV. This study therefore revealed that DNPE6(4) was a promising antiviral agent for future study.

A Two-Amino Acid Difference in the Coat Protein of Satellite panicum mosaic virus Isolates Is Responsible for Differential Synergistic Interactions with Panicum mosaic virus.

Panicum mosaic virus (PMV) (genus Panicovirus, family Tombusviridae) and its molecular parasite, Satellite panicum mosaic virus (SPMV), synergistically interact in coinfected proso and pearl millet (Panicum miliaceum L.) plants resulting in a severe symptom phenotype. In this study, we examined synergistic interactions between the isolates of PMV and SPMV by using PMV-NE, PMV85, SPMV-KS, and SPMV-Type as interacting partner viruses in different combinations. Coinfection of proso millet plants by PMV-NE and SPMV-KS elicited severe mosaic, chlorosis, stunting, and eventual plant death compared with moderate mosaic, chlorotic streaks, and stunting by PMV85 and SPMV-Type. In reciprocal combinations, coinfection of proso millet by either isolate of PMV with SPMV-KS but not with SPMV-Type elicited severe disease synergism, suggesting that SPMV-KS was the main contributor for efficient synergistic interaction with PMV isolates. Coinfection of proso millet plants by either isolate of PMV and SPMV-KS or SPMV-Type caused increased accumulation of coat protein (CP) and genomic RNA copies of PMV, compared with infections by individual PMV isolates. Additionally, CP and genomic RNA copies of SPMV-KS accumulated at substantially higher levels, compared with SMPV-Type in coinfected proso millet plants with either isolate of PMV. Hybrid viruses between SPMV-KS and SPMV-Type revealed that SPMV isolates harboring a CP fragment with four differing amino acids at positions 18, 35, 59, and 98 were responsible for differential synergistic interactions with PMV in proso millet plants. Mutation of amino acid residues at these positions in different combinations in SPMV-KS, similar to those as in SPMV-Type or vice-versa, revealed that A35 and R98 in SPMV-KS CP play critical roles in enhanced synergistic interactions with PMV isolates. Taken together, these data suggest that the two distinct amino acids at positions 35 and 98 in the CP of SPMV-KS and SPMV-Type are involved in the differential synergistic interactions with the helper viruses.

Hijacking a host scaffold protein, RACK1, for replication of a plant RNA virus.

Receptor for activated C kinase 1 (RACK1) is strictly conserved across eukaryotes and acts as a versatile scaffold protein involved in various signaling pathways. Plant RACK1 is known to exert important functions in innate immunity against fungal and bacterial pathogens. However, the role of the RACK1 in plant-virus interactions remains unknown. Here, we addressed the role of RACK1 of Nicotiana benthamiana during infection by red clover necrotic mosaic virus (RCNMV), a plant positive-stranded RNA virus. NbRACK1 was shown to be recruited by the p27 viral replication protein into endoplasmic reticulum-derived aggregated structures (possible replication sites). Downregulation of NbRACK1 by virus-induced gene silencing inhibited viral cap-independent translation and p27-mediated reactive oxygen species (ROS) accumulation, which are prerequisite for RCNMV replication. We also found that NbRACK1 interacted with a host calcium-dependent protein kinase (NbCDPKiso2) that activated a ROS-generating enzyme. Interestingly, NbRACK1 was required for the interaction of p27 with NbCDPKiso2, suggesting that NbRACK1 acts as a bridge between the p27 viral replication protein and NbCDPKiso2. Collectively, our findings provide an example of a viral strategy in which a host multifaceted scaffold protein RACK1 is highjacked for promoting viral protein-triggered ROS production necessary for robust viral replication.

New Insights into the Nucleolar Localization of a Plant RNA Virus-Encoded Protein That Acts in Both RNA Packaging and RNA Silencing Suppression: Involvement of Importins Alpha and Relevance for Viral Infection.

Despite the fact that replication of plus-strand RNA viruses takes place in the cytoplasm of host cells, different proteins encoded by these infectious agents have been shown to localize in the nucleus, with high accumulation at the nucleolus. In most cases, the molecular determinants or biological significance of such subcellular localization remains elusive. Recently, we reported that protein p37 encoded by Pelargonium line pattern virus (family Tombusviridae) acts in both RNA packaging and RNA silencing suppression. Consistently with these functions, p37 was detected in the cytoplasm of plant cells, although it was also present in the nucleus and, particularly, in the nucleolus. Here, we searched for further insights into factors influencing p37 nucleolar localization and into its potential relevance for viral infection. Besides mapping the protein region containing the nucleolar localization signal, we have found that p37 interacts with distinct members of the importin alpha family-main cellular transporters for nucleo-cytoplasmic traffic of proteins-and that these interactions are crucial for nucleolar targeting of p37. Impairment of p37 nucleolar localization through downregulation of importin alpha expression resulted in a reduction of viral accumulation, suggesting that sorting of the protein to the major subnuclear compartment is advantageous for the infection process.

Hsc70-2 is required for Beet black scorch virus infection through interaction with replication and capsid proteins.

Dissecting the complex molecular interplay between the host plant and invading virus improves our understanding of the mechanisms underlying viral pathogenesis. In this study, immunoprecipitation together with the mass spectrometry analysis revealed that the heat shock protein 70 (Hsp70) family homolog, Hsc70-2, was co-purified with beet black scorch virus (BBSV) replication protein p23 and coat protein (CP), respectively. Further experiments demonstrated that Hsc70-2 interacts directly with both p23 and CP, whereas there is no interaction between p23 and CP. Hsc70-2 expression is induced slightly during BBSV infection of Nicotiana benthamiana, and overexpression of Hsc70-2 promotes BBSV accumulation, while knockdown of Hsc70-2 in N. benthamiana leads to drastic reduction of BBSV accumulation. Infection experiments revealed that CP negatively regulates BBSV replication, which can be mitigated by overexpression of Hsc70-2. Further experiments indicate that CP impairs the interaction between Hsc70-2 and p23 in a dose-dependent manner. Altogether, we provide evidence that besides specific functions of Hsp70 family proteins in certain aspects of viral infection, they can serve as a mediator for the orchestration of virus infection by interacting with different viral components. Our results provide new insight into the role of Hsp70 family proteins in virus infection.

De novo generation of helper virus-satellite chimera RNAs results in disease attenuation and satellite sequence acquisition in a host-dependent manner.

Panicum mosaic virus (PMV) is a helper RNA virus for satellite RNAs (satRNAs) and a satellite virus (SPMV). Here, we describe modifications that occur at the 3'-end of a satRNA of PMV, satS. Co-infections of PMV+satS result in attenuation of the disease symptoms induced by PMV alone in Brachypodium distachyon and proso millet. The 375 nt satS acquires ~100-200 nts from the 3'-end of PMV during infection and is associated with decreased abundance of the PMV RNA and capsid protein in millet. PMV-satS chimera RNAs were isolated from native infections of St. Augustinegrass and switchgrass. Phylogenetic analyses revealed that the chimeric RNAs clustered according to the host species from which they were isolated. Additionally, the chimera satRNAs acquired non-viral "linker" sequences in a host-specific manner. These results highlight the dynamic regulation of viral pathogenicity by satellites, and the selective host-dependent, sequence-based pressures for driving satRNA generation and genome compositions.

Genomic Approaches to Analyze Alternative Splicing, A Key Regulator of Transcriptome and Proteome Diversity in Brachypodium distachyon.

Alternative splicing (AS) promotes transcriptome and proteome diversity in plants, which influences growth and development, and host responses to stress. Advancements in next-generation sequencing, bioinformatics, and computational biology tools have allowed biologists to investigate AS landscapes on a genome-wide scale in several plant species. Furthermore, the development of Brachypodium distachyon (Brachypodium) as a model system for grasses has facilitated comparative studies of AS within the Poaceae. These analyses revealed a plethora of genes in several biological processes that are alternatively spliced and identified conserved AS patterns among monocot and dicot plants. In this chapter, using a Brachypodium-virus pathosystem as a research template, we provide an overview of genomic and bioinformatic tools that can be used to investigate constitutive and alternative splicing in plants.

Dual function of a cis-acting RNA element that acts as a replication enhancer and a translation repressor in a plant positive-stranded RNA virus.

The genome of red clover necrotic mosaic virus is divided into two positive-stranded RNA molecules of RNA1 and RNA2, which have no 5' cap structure and no 3' poly(A) tail. Previously, we showed that any mutations in the cis-acting RNA replication elements of RNA2 abolished its cap-independent translational activity, suggesting a strong link between RNA replication and translation. Here, we investigated the functions of the 5' untranslated region (UTR) of RNA2 and revealed that the basal stem-structure (5'BS) predicted in the 5' UTR is essential for robust RNA replication. Interestingly, RNA2 mutants with substitution or deletion in the right side of the 5'BS showed strong translational activity, despite their impaired replication competency. Furthermore, nucleotide sequences other than the 5'BS of the 5' UTR were essential to facilitate the replication-associated translation. Overall, these cis-acting RNA elements seem to coordinately regulate the balance between RNA replication and replication-associated translation.

Differential use of 3'CITEs by the subgenomic RNA of Pea enation mosaic virus 2.

The genomic RNA (gRNA) of Pea enation mosaic virus 2 (PEMV2) is the template for p33 and -1 frameshift product p94. The PEMV2 subgenomic RNA (sgRNA) encodes two overlapping ORFs, p26 and p27, which are required for movement and stability of the gRNA. Efficient translation of p33 requires two of three 3' proximal cap-independent translation enhancers (3'CITEs): the kl-TSS, which binds ribosomes and engages in a long-distance interaction with the 5'end; and the adjacent eIF4E-binding PTE. Unlike the gRNA, all three 3'CITEs were required for efficient translation of the sgRNA, which included the ribosome-binding 3'TSS. A hairpin in the 5' proximal coding region of p26/p27 supported translation by the 3'CITEs by engaging in a long-distance RNA:RNA interaction with the kl-TSS. These results strongly suggest that the 5' ends of PEMV2 gRNA and sgRNA connect with the 3'UTR through similar long-distance interactions while having different requirements for 3'CITEs.

Requirement for eukaryotic translation initiation factors in cap-independent translation differs between bipartite genomic RNAs of red clover necrotic mosaic virus.

The bipartite genomic RNAs of red clover necrotic mosaic virus (RCNMV) lack a 5' cap and a 3' poly(A) tail. RNA1 encodes viral replication proteins, and RNA2 encodes a movement protein (MP). These proteins are translated in a cap-independent manner. We previously identified two cis-acting RNA elements that cooperatively recruit eukaryotic translation initiation factor (eIF) complex eIF4F or eIFiso4F to RNA1. Such cis-acting RNA elements and host factors have not been identified in RNA2. Here we found that translation of RNA1 was significantly compromised in Arabidopsis thaliana carrying eif4f mutation. RNA1 replicated efficiently in eifiso4f1 mutants, suggesting vigorous translation of the replication proteins from RNA1 in the plants. In contrast, MP accumulation was decreased in eifiso4f1 mutants but not in eif4f mutants. Collectively, these results suggest that RCNMV uses different eIF complexes for translation of its bipartite genomic RNAs, which may contribute to fine-tuning viral gene expression during infection.

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus.

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

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 ß-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.