Transcriptome analyses unveiled differential regulation of AGO and DCL genes by pepino mosaic virus strains.

Pepino mosaic virus (PepMV) is a single-stranded (ss), positive-sense (+) RNA potexvirus that affects tomato crops worldwide. We have described an in planta antagonistic interaction between PepMV isolates of two strains in which the EU isolate represses the accumulation of the CH2 isolate during mixed infections. Reports describing transcriptomic responses to mixed infections are scant. We carried out transcriptomic analyses of tomato plants singly and mixed-infected with two PepMV isolates of both strains. Comparison of the transcriptomes of singly infected plants showed that deeper transcriptomic alterations occurred at early infection times, and also that each of the viral strains modulated the host transcriptome differentially. Mixed infections caused transcriptomic alterations similar to those for the sum of single infections at early infection times, but clearly differing at later times postinfection. We next tested the hypothesis that PepMV-EU, in either single or mixed infections, deregulates host gene expression differentially so that virus accumulation of both strains gets repressed. That seemed to be the case for the genes AGO1a, DCL2d, AGO2a, and DCL2b, which are involved in the antiviral silencing pathway and were upregulated by PepMV-EU but not by PepMV-CH2 at early times postinfection. The pattern of AGO2a expression was validated by reverse transcription-quantitative PCR in tomato and Nicotiana benthamiana plants. Using an N. benthamiana ago2 mutant line, we showed that AGO2 indeed plays an important role in the antiviral defence against PepMV, but it is not the primary determinant of the outcome of the antagonistic interaction between the two PepMV strains.

The gibberellic acid derived from the plastidial MEP pathway is involved in the accumulation of Bamboo mosaic virus.

A gene upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) infection was revealed as 1-deoxy-d-xylulose-5-phosphate reductoisomerase (NbDXR). DXR is the key enzyme in the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway that catalyzes the conversion of 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol-4-phosphate. Knockdown and overexpression of NbDXR followed by BaMV inoculation revealed that NbDXR is involved in BaMV accumulation. Treating leaves with fosmidomycin, an inhibitor of DXR function, reduced BaMV accumulation. Subcellular localization confirmed that DXR is a chloroplast-localized protein by confocal microscopy. Furthermore, knockdown of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, one of the enzymes in the MEP pathway, also reduced BaMV accumulation. The accumulation of BaMV increased significantly in protoplasts treated with isopentenyl pyrophosphate. Thus, the metabolites of the MEP pathway could be involved in BaMV infection. To identify the critical components involved in BaMV accumulation, we knocked down the crucial enzyme of isoprenoid synthesis, NbGGPPS11 or NbGGPPS2. Only NbGGPPS2 was involved in BaMV infection. The geranylgeranyl pyrophosphate (GGPP) synthesized by NbGGPPS2 is known for gibberellin synthesis. We confirmed this result by supplying gibberellic acid exogenously on leaves, which increased BaMV accumulation. The de novo synthesis of gibberellic acid could assist BaMV accumulation.

Effects of Poty-Potexvirus Synergism on Growth, Photosynthesis and Metabolite Status of Nicotiana benthamiana.

Mixed virus infections threaten crop production because interactions between the host and the pathogen mix may lead to viral synergism. While individual infections by potato virus A (PVA), a potyvirus, and potato virus X (PVX), a potexvirus, can be mild, co-infection leads to synergistic enhancement of PVX and severe symptoms. We combined image-based phenotyping with metabolite analysis of single and mixed PVA and PVX infections and compared their effects on growth, photosynthesis, and metabolites in Nicotiana benthamiana. Viral synergism was evident in symptom severity and impaired growth in the plants. Indicative of stress, the co-infection increased leaf temperature and decreased photosynthetic parameters. In contrast, singly infected plants sustained photosynthetic activity. The host's metabolic response differed significantly between single and mixed infections. Over 200 metabolites were differentially regulated in the mixed infection: especially defense-related metabolites and aromatic and branched-chain amino acids increased compared to the control. Changes in the levels of methionine cycle intermediates and a low S-adenosylmethionine/S-adenosylhomocysteine ratio suggested a decline in the methylation potential in co-infected plants. The decreased ratio between reduced glutathione, an important scavenger of reactive oxygen species, and its oxidized form, indicated that severe oxidative stress developed during co-infection. Based on the results, infection-associated oxidative stress is successfully controlled in the single infections but not in the synergistic infection, where activated defense pathways are not sufficient to counter the impact of the infections on plant growth.

Application of Plant Viruses in Biotechnology, Medicine, and Human Health.

Plant-based nanotechnology programs using virus-like particles (VLPs) and virus nanoparticles (VNPs) are emerging platforms that are increasingly used for a variety of applications in biotechnology and medicine. Tobacco mosaic virus (TMV) and potato virus X (PVX), by virtue of having high aspect ratios, make ideal platforms for drug delivery. TMV and PVX both possess rod-shaped structures and single-stranded RNA genomes encapsidated by their respective capsid proteins and have shown great promise as drug delivery systems. Cowpea mosaic virus (CPMV) has an icosahedral structure, and thus brings unique benefits as a nanoparticle. The uses of these three plant viruses as either nanostructures or expression vectors for high value pharmaceutical proteins such as vaccines and antibodies are discussed extensively in the following review. In addition, the potential uses of geminiviruses in medical biotechnology are explored. The uses of these expression vectors in plant biotechnology applications are also discussed. Finally, in this review, we project future prospects for plant viruses in the fields of medicine, human health, prophylaxis, and therapy of human diseases.

Thermal remodelling of Alternanthera mosaic virus virions and virus-like particles into protein spherical particles.

The present work addresses the thermal remodelling of flexible plant viruses with a helical structure and virus-like particles (VLPs). Here, for the first time, the possibility of filamentous Alternanthera mosaic virus (AltMV) virions' thermal transition into structurally modified spherical particles (SP) has been demonstrated. The work has established differences in formation conditions of SP from virions (SPV) and VLPs (SPVLP) that are in accordance with structural data (on AltMV virions and VLPs). SP originate from AltMV virions through an intermediate stage. However, the same intermediate stage was not detected during AltMV VLPs' structural remodelling. According to the biochemical analysis, AltMV SPV consist of protein and do not include RNA. The structural characterisation of AltMV SPV/VLP by circular dichroism, intrinsic fluorescence spectroscopy and thioflavin T fluorescence assay has been performed. AltMV SPV/VLP adsorption properties and the availability of chemically reactive surface amino acids have been analysed. The revealed characteristics of AltMV SPV/VLP indicate that they could be applied as protein platforms for target molecules presentation and for the design of functionally active complexes.

The Lipid Transfer Protein 1 from Nicotiana benthamiana Assists Bamboo mosaic virus Accumulation.

Host factors play a pivotal role in regulating virus infection. Uncovering the mechanism of how host factors are involved in virus infection could pave the way to defeat viral disease. In this study, we characterized a lipid transfer protein, designated NbLTP1 in Nicotiana benthamiana, which was downregulated after Bamboo mosaic virus (BaMV) inoculation. BaMV accumulation significantly decreased in NbLTP1-knockdown leaves and protoplasts compared with the controls. The subcellular localization of the NbLTP1-orange fluorescent protein (OFP) was mainly the extracellular matrix. However, when we removed the signal peptide (NbLTP1/ΔSP-OFP), most of the expressed protein targeted chloroplasts. Both NbLTP1-OFP and NbLTP1/ΔSP-OFP were localized in chloroplasts when we removed the cell wall. These results suggest that NbLTP1 may have a secondary targeting signal. Transient overexpression of NbLTP1 had no effect on BaMV accumulation, but that of NbLTP1/ΔSP significantly increased BaMV expression. NbLTP1 may be a positive regulator of BaMV accumulation especially when its expression is associated with chloroplasts, where BaMV replicates. The mutation was introduced to the predicted phosphorylation site to simulate the phosphorylated status, NbLTP/ΔSP/P(+), which could still assist BaMV accumulation. By contrast, a mutant lacking calmodulin-binding or simulates the phosphorylation-negative status could not support BaMV accumulation. The lipid-binding activity of LTP1 was reported to be associated with calmodulin-binding and phosphorylation, by which the C-terminus functional domain of NbLTP1 may play a critical role in BaMV accumulation.

A DNA-Binding Bromodomain-Containing Protein Interacts with and Reduces Rx1-Mediated Immune Response to Potato Virus X.

Plant NLR proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming. Some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato binds and distorts double-stranded DNA. However, the components of the chromatin-localized Rx1 complex are largely unknown. Here, we report a physical and functional interaction between Rx1 and NbDBCP, a bromodomain-containing chromatin-interacting protein. NbDBCP accumulates in the nucleoplasm and nucleolus, interacts with chromatin, and redistributes Rx1 to the nucleolus in a subpopulation of imaged cells. Rx1 overexpression reduces the interaction between NbDBCP and chromatin. NbDBCP is a negative regulator of Rx1-mediated immune responses to potato virus X (PVX), and this activity requires an intact bromodomain. Previously, Rx1 has been shown to regulate the DNA-binding activity of a Golden2-like transcription factor, NbGlk1. Rx1 and NbDBCP act synergistically to reduce NbGlk1 DNA binding, suggesting a mode of action for NbDBCP's inhibitory effect on immunity. This study provides new mechanistic insight into the mechanism by which a chromatin-localized NLR complex co-ordinates immune signaling after pathogen perception.

The Barley stripe mosaic virus γb protein promotes viral cell-to-cell movement by enhancing ATPase-mediated assembly of ribonucleoprotein movement complexes.

Nine genera of viruses in five different families use triple gene block (TGB) proteins for virus movement. The TGB modules fall into two classes: hordei-like and potex-like. Although TGB-mediated viral movement has been extensively studied, determination of the constituents of the viral ribonucleoprotein (vRNP) movement complexes and the mechanisms underlying their involvement in vRNP-mediated movement are far from complete. In the current study, immunoprecipitation of TGB1 protein complexes formed during Barley stripe mosaic virus (BSMV) infection revealed the presence of the γb protein in the products. Further experiments demonstrated that TGB1 interacts with γb in vitro and in vivo, and that γb-TGB1 localizes at the periphery of chloroplasts and plasmodesmata (PD). Subcellular localization analyses of the γb protein in Nicotiana benthamiana epidermal cells indicated that in addition to chloroplast localization, γb also targets the ER, actin filaments and PD at different stages of viral infection. By tracking γb localization during BSMV infection, we demonstrated that γb is required for efficient cell-to-cell movement. The N-terminus of γb interacts with the TGB1 ATPase/helicase domain and enhances ATPase activity of the domain. Inactivation of the TGB1 ATPase activity also significantly impaired PD targeting. In vitro translation together with co-immunoprecipitation (co-IP) analyses revealed that TGB1-TGB3-TGB2 complex formation is enhanced by ATP hydrolysis. The γb protein positively regulates complex formation in the presence of ATP, suggesting that γb has a novel role in BSMV cell-to-cell movement by directly promoting TGB1 ATPase-mediated vRNP movement complex assembly. We further demonstrated that elimination of ATPase activity abrogates PD and actin targeting of Potato virus X (PVX) and Beet necrotic yellow vein virus (BNYVV) TGB1 proteins. These results expand our understanding of the multifunctional roles of γb and provide new insight into the functions of TGB1 ATPase domains in the movement of TGB-encoding viruses.

Interplay between potato virus X and RNA granules in Nicotiana benthamiana.

Cytoplasmic RNA granules consist of microscopic agglomerates of mRNAs and proteins and occur when the translation is reversibly and temporally halted (stress granules, SGs) or mRNAs are targeted for decapping (processing bodies, PBs). The induction of RNA granules formation by virus infection is a common feature of mammalian cells. However, plant-virus systems still remain poorly characterized. In this work, the SG marker AtUBP1b was expressed in Nicotiana benthamiana plants to decipher how the virus infection of plant cells affects SG dynamics. We found that the hypoxia-induced SG assembly was substantially inhibited in Potato virus X (PVX)-infected cells. Furthermore, we determined that the expression of PVX movement protein TGBp1 by itself, mimics the inhibitory effect of PVX on SG formation under hypoxia. Importantly, overexpression of AtUBP1b showed inhibition of the PVX spreading, whereas the overexpression of the dominant negative AtUBP1brrm enhanced PVX spreding, indicating that AtUBP1b negatively affects PVX infection. Notably, PVX infection did not inhibit the formation of processing bodies (PBs), indicating PVX has distinct effects depending on the type of RNA granule. Our results suggest that SG inhibition could be part of the virus strategy to infect the plant.

Autophagy is involved in assisting the replication of Bamboo mosaic virus in Nicotiana benthamiana.

Autophagy plays a critical role in plants under biotic stress, including the response to pathogen infection. We investigated whether autophagy-related genes (ATGs) are involved in infection with Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. Initially, we observed that BaMV infection in Nicotiana benthamiana leaves upregulated the expression of ATGs but did not trigger cell death. The induction of ATGs, which possibly triggers autophagy, increased rather than diminished BaMV accumulation in the leaves, as revealed by gene knockdown and transient expression experiments. Furthermore, the inhibitor 3-methyladenine blocked autophagosome formation and the autophagy inducer rapamycin, which negatively and positively affected BaMV accumulation, respectively. Pull-down experiments with an antibody against orange fluorescent protein (OFP)-NbATG8f, an autophagosome marker protein, showed that both plus- and minus-sense BaMV RNAs could associate with NbATG8f. Confocal microscopy revealed that ATG8f-enriched vesicles possibly derived from chloroplasts contained both the BaMV viral RNA and its replicase. Thus, BaMV infection may induce the expression of ATGs possibly via autophagy to selectively engulf a portion of viral RNA-containing chloroplast. Virus-induced vesicles enriched with ATG8f could provide an alternative site for viral RNA replication or a shelter from the host silencing mechanism.

Acibenzolar-S-Methyl Restricts Infection of Nicotiana benthamiana by Plantago Asiatica Mosaic Virus at Two Distinct Stages.

Plant activators, including acibenzolar-S-methyl (ASM), are chemical compounds that stimulate plant defense responses to pathogens. ASM treatment inhibits infection by a variety of plant viruses, however, the mechanisms of this broad-spectrum and strong effect remain poorly understood. We employed green fluorescent protein (GFP)-expressing viruses and Nicotiana benthamiana plants to identify the infection stages that are restricted by ASM. ASM suppressed infection by three viral species, plantago asiatica mosaic virus (PlAMV), potato virus X (PVX), and turnip mosaic virus (TuMV), in inoculated cells. Furthermore, ASM delayed the long-distance movement of PlAMV and PVX, and the cell-to-cell (short range) movement of TuMV. The ASM-mediated delay of long-distance movement of PlAMV was not due to the suppression of viral accumulation in the inoculated leaves, indicating that ASM restricts PlAMV infection in at least two independent steps. We used Arabidopsis thaliana mutants to show that the ASM-mediated restriction of PlAMV infection requires the NPR1 gene but was independent of the dicer-like genes essential for RNA silencing. Furthermore, experiments using protoplasts showed that ASM treatment inhibited PlAMV replication without cell death. Our approach, using GFP-expressing viruses, will be useful for the analysis of mechanisms underlying plant activator-mediated virus restriction.

Functional conservation of EXA1 among diverse plant species for the infection by a family of plant viruses.

Since the propagation of plant viruses depends on various host susceptibility factors, deficiency in them can prevent viral infection in cultivated and model plants. Recently, we identified the susceptibility factor Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana, and revealed that EXA1-mediated resistance was effective against three potexviruses. Although EXA1 homolog genes are found in tomato and rice, little is known about which viruses depend on EXA1 for their infection capability and whether the function of EXA1 homologs in viral infection is conserved across multiple plant species, including crops. To address these questions, we generated knockdown mutants using virus-induced gene silencing in two Solanaceae species, Nicotiana benthamiana and tomato. In N. benthamiana, silencing of an EXA1 homolog significantly compromised the accumulation of potexviruses and a lolavirus, a close relative of potexviruses, whereas transient expression of EXA1 homologs from tomato and rice complemented viral infection. EXA1 dependency for potexviral infection was also conserved in tomato. These results indicate that EXA1 is necessary for effective accumulation of potexviruses and a lolavirus, and that the function of EXA1 in viral infection is conserved among diverse plant species.

Evaluation of Potato virus X mild mutants for cross protection against severe infection in China.

BACKGROUND: Cross protection is a promising alternative to control plant viral diseases. One critical factor limiting the application of cross protection is the availability of attenuated mutants or mild strains. Potato virus X (PVX) infects many crops and induces huge economic losses to agricultural production. However, researches on the variability and mechanism of PVX virulence are scarce. METHODS: The mutants were obtained by introducing mutations into the RNA dependent RNA polymerase (RdRp) gene of PVX via site-directed mutagenesis. Attenuated mutants were screen according to their symptoms in Nicotiana benthamiana plants. The protection efficacy against severe infection were evaluated with interval of 5, 10 and 15 days. RESULTS: Among the 40 mutants obtained, four mutants carrying substitutions of either Glu46, Asn863, Asn968 or Glu1001 to Ala in PVX RdRp showed drastically attenuated symptom, accompanying with reduced accumulation levels of coat protein, plus- and minus-sense RNAs. When the interval between protective and challenging inoculations was 15 days, mutant E1001A (with substitution of Glu1001 to Ala in RdRp) provided complete protection against severe infection in both Nicotiana benthamiana and tomato, while E46A (Glu46 mutated to Ala) provided incomplete protection. To reduce the risk of reverse mutation, we constructed mutant dM which carries double mutations of both Glu46 and Glu1001 to Ala in RdRp. The mutant dM could provide effective protection against severe PVX infection. CONCLUSION: Mutations of Glu46, Asn863, Asn968 or Glu1001 to Ala in PVX RdRp significantly reduced the viral symptoms. Mutants E1001A and E46A could provide effective protection against wild type PVX in both Nicotiana benthamiana and tomato. These results provide theoretical and practical bases for the control of PVX via cross protection.

An E3 ubiquitin ligase from Nicotiana benthamiana targets the replicase of Bamboo mosaic virus and restricts its replication.

One up-regulated host gene identified previously was found involved in the infection process of Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. The full length cDNA of this gene was cloned by 5' and 3'-rapid amplification of cDNA ends and found to encode a polypeptide containing a conserved really interesting new gene (RING) domain and a transmembrane domain. The gene might function as an ubiquitin E3 ligase. We designated this protein in Nicotiana benthamiana as ubiquitin E3 ligase containing RING domain 1 (NbUbE3R1). Further characterization by using Tobacco rattle virus-based virus-induced gene silencing (loss-of-function) revealed that increased BaMV accumulation was in both knockdown plants and protoplasts. The gene might have a defensive role in the replication step of BaMV infection. To further inspect the functional role of NbUbE3R1 in BaMV accumulation, NbUbE3R1 was expressed in N. benthamiana plants. The wild-type NbUbE3R1-orange fluorescent protein (NbUbE3R1-OFP), NbUbE3R1/△TM-OFP (removal of the transmembrane domain) and NbUbE3R1/mRING-OFP (mutation at the RING domain, the E2 interaction site) were transiently expressed in plants. NbUbE3R1 and its derivatives all functioned in restricting the accumulation of BaMV. The common feature of these constructs was the intact substrate-interacting domain. Yeast two-hybrid and co-immunoprecipitation experiments used to determine the possible viral-encoded substrate of NbUbE3R1 revealed the replicase of BaMV as the possible substrate. In conclusion, we identified an up-regulated gene, NbUbE3R1 that plays a role in BaMV replication.

The hypersensitive induced reaction 3 (HIR3) gene contributes to plant basal resistance via an EDS1 and salicylic acid-dependent pathway.

The hypersensitive-induced reaction (HIR) gene family is associated with the hypersensitive response (HR) that is a part of the plant defense system against bacterial and fungal pathogens. The involvement of HIR genes in response to viral pathogens has not yet been studied. We now report that the HIR3 genes of Nicotiana benthamiana and Oryza sativa (rice) were upregulated following rice stripe virus (RSV) infection. Silencing of HIR3s in N. benthamiana resulted in an increased accumulation of RSV RNAs, whereas overexpression of HIR3s in N. benthamiana or rice reduced the expression of RSV RNAs and decreased symptom severity, while also conferring resistance to Turnip mosaic virus, Potato virus X, and the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. Silencing of HIR3 genes in N. benthamiana reduced the content of salicylic acid (SA) and was accompanied by the downregulated expression of genes in the SA pathway. Transient expression of the two HIR3 gene homologs from N. benthamiana or the rice HIR3 gene in N. benthamiana leaves caused cell death and an accumulation of SA, but did not do so in EDS1-silenced plants or in plants expressing NahG. The results indicate that HIR3 contributes to plant basal resistance via an EDS1- and SA-dependent pathway.

The Plant Noncanonical Antiviral Resistance Protein JAX1 Inhibits Potexviral Replication by Targeting the Viral RNA-Dependent RNA Polymerase.

Understanding the innate immune mechanisms of plants is necessary for the breeding of disease-resistant lines. Previously, we identified the antiviral resistance gene JAX1 from Arabidopsis thaliana, which inhibits infection by potexviruses. JAX1 encodes a unique jacalin-type lectin protein. In this study, we analyzed the molecular mechanisms of JAX1-mediated resistance. JAX1 restricted the multiplication of a potexviral replicon lacking movement-associated proteins, suggesting inhibition of viral replication. Therefore, we developed an in vitro potato virus X (PVX) translation/replication system using vacuole- and nucleus-free lysates from tobacco protoplasts, and we revealed that JAX1 inhibits viral RNA synthesis but not the translation of the viral RNA-dependent RNA polymerase (RdRp). JAX1 did not affect the replication of a resistance-breaking mutant of PVX. Blue native polyacrylamide gel electrophoresis of fractions separated by sucrose gradient sedimentation showed that PVX RdRp constituted the high-molecular-weight complex that seems to be crucial for viral replication. JAX1 was detected in this complex of the wild-type PVX replicon but not in that of the resistance-breaking mutant. In addition, JAX1 interacted with the RdRp of the wild-type virus but not with that of a virus with a point mutation at the resistance-breaking residue. These results suggest that JAX1 targets RdRp to inhibit potexviral replication.IMPORTANCE Resistance genes play a crucial role in plant antiviral innate immunity. The roles of conventional nucleotide-binding leucine-rich repeat (NLR) proteins and the associated defense pathways have long been studied. In contrast, recently discovered resistance genes that do not encode NLR proteins (non-NLR resistance genes) have not been investigated extensively. Here we report that the non-NLR resistance factor JAX1, a unique jacalin-type lectin protein, inhibits de novo potexviral RNA synthesis by targeting the huge complex of viral replicase. This is unlike other known antiviral resistance mechanisms. Molecular elucidation of the target in lectin-type protein-mediated antiviral immunity will enhance our understanding of the non-NLR-mediated plant resistance system.

Suppression of nbe-miR166h-p5 attenuates leaf yellowing symptoms of potato virus X on Nicotiana benthamiana and reduces virus accumulation.

MicroRNAs (miRNAs) play essential roles in plant development. There is increasing evidence that changed expression of miRNAs in virus-infected plants contributes to the development of viral symptoms. Here, we analysed the altered expression of miRNAs of Nicotiana benthamiana in response to Potato virus X (PVX) by Illumina Solexa sequencing. One of the 21 miRNAs significantly affected, nbe-miR166h-p5, was closely associated with viral symptoms. Using the Tobacco rattle virus-based miRNA suppression (VbMS) system, we found that the suppression of nbe-miR166h-p5 in plants caused leaves to turn dark green with increased chlorophyll. When PVX was inoculated on nbe-miR166h-p5-suppressed plants, the leaf yellowing symptom of PVX was largely attenuated with less reduction in chlorophyll content, and the accumulation of PVX was decreased. nbe-miR166h-p5 was also up-regulated in plants infected by Turnip mosaic virus (TuMV), and its suppression attenuated the leaf yellowing symptom of TuMV and decreased viral accumulation. Three potential targets of nbe-miR166h-p5 were identified. The results indicate the association of nbe-miR166h-p5 with symptoms of PVX and also with those of TuMV, providing useful information on the relationship between miRNA and viral infection.

Systemic Infection of Nicotiana benthamiana with Potato virus X Nanoparticles Presenting a Fluorescent iLOV Polypeptide Fused Directly to the Coat Protein.

Plant virus-based nanoparticles can be produced in plants on a large scale and are easily modified to introduce new functions, making them suitable for applications such as vaccination and drug delivery, tissue engineering, and in vivo imaging. The latter is often achieved using green fluorescent protein and its derivatives, but the monovalent fluorescent protein iLOV is smaller and more robust. Here, we fused the iLOV polypeptide to the N-terminus of the Potato virus X (PVX) coat protein, directly or via the Foot-and-mouth disease virus 2A sequence, for expression in Nicotiana benthamiana. Direct fusion of the iLOV polypeptide did not prevent the assembly or systemic spread of the virus and we verified the presence of fusion proteins and iLOV hybrid virus particles in leaf extracts. Compared to wild-type PVX virions, the PVX particles displaying the iLOV peptide showed an atypical, intertwined morphology. Our results confirm that a direct fusion of the iLOV fluorescent protein to filamentous PVX nanoparticles offers a promising tool for imaging applications.

Plasma membrane-associated cation-binding protein 1-like protein negatively regulates intercellular movement of BaMV.

To establish a successful infection, a virus needs to replicate and move cell-to-cell efficiently. We investigated whether one of the genes upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) inoculation was involved in regulating virus movement. We revealed the gene to be a plasma membrane-associated cation-binding protein 1-like protein, designated NbPCaP1L. The expression of NbPCaP1L in N. benthamiana was knocked down using Tobacco rattle virus-based gene silencing and consequently the accumulation of BaMV increased significantly to that of control plants. Further analysis indicated no significant difference in the accumulation of BaMV in NbPCaP1L knockdown and control protoplasts, suggesting NbPCaP1L may affect cell-to-cell movement of BaMV. Using a viral vector expressing green fluorescent protein in the knockdown plants, the mean area of viral focus, as determined by fluorescence, was found to be larger in NbPCaP1L knockdown plants. Orange fluorescence protein (OFP)-fused NbPCaP1L, NbPCaP1L-OFP, was expressed in N. benthamiana and reduced the accumulation of BaMV to 46%. To reveal the possible interaction of viral protein with NbPCaP1L, we performed yeast two-hybrid and co-immunoprecipitation experiments. The results indicated that NbPCaP1L interacted with BaMV replicase. The results also suggested that NbPCaP1L could trap the BaMV movement RNP complex via interaction with the viral replicase in the complex and so restricted viral cell-to-cell movement.

Virulence determines beneficial trade-offs in the response of virus-infected plants to drought via induction of salicylic acid.

It has been hypothesized that plants can get beneficial trade-offs from viral infections when grown under drought conditions. However, experimental support for a positive correlation between virus-induced drought tolerance and increased host fitness is scarce. We investigated whether increased virulence exhibited by the synergistic interaction involving Potato virus X (PVX) and Plum pox virus (PPV) improves tolerance to drought and host fitness in Nicotiana benthamiana and Arabidopsis thaliana. Infection by the pair PPV/PVX and by PPV expressing the virulence protein P25 of PVX conferred an enhanced drought-tolerant phenotype compared with single infections with either PPV or PVX. Decreased transpiration rates in virus-infected plants were correlated with drought tolerance in N. benthamiana but not in Arabidopsis. Metabolite and hormonal profiles of Arabidopsis plants infected with the different viruses showed a range of changes that positively correlated with a greater impact on drought tolerance. Virus infection enhanced drought tolerance in both species by increasing salicylic acid accumulation in an abscisic acid-independent manner. Viable offspring derived from Arabidopsis plants infected with PPV increased relative to non-infected plants, when exposed to drought. By contrast, the detrimental effect caused by the more virulent viruses overcame potential benefits associated with increased drought tolerance on host fitness.