Potato virus X-delivered CRISPR activation programs lead to strong endogenous gene induction and transient metabolic reprogramming in Nicotiana benthamiana.

Programmable transcriptional regulators based on CRISPR architecture are promising tools for the induction of plant gene expression. In plants, CRISPR gene activation is effective with respect to modulating development processes, such as the flowering time or customizing biochemical composition. The most widely used method for delivering CRISPR components into the plant is Agrobacterium tumefaciens-mediated genetic transformation, either transient or stable. However, as a result of their versatility and their ability to move, virus-derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular guide RNA (gRNA), which represents the variable component in CRISPR strategies. In the present study, we describe a Potato virus X-derived vector that, upon agroinfection in Nicotiana benthamiana, serves as a vehicle for delivery of gRNAs, producing highly specific virus-induced gene activation. The system works in combination with a N. benthamiana transgenic line carrying the remaining complementary CRISPR gene activation components, specifically the dCasEV2.1 cassette, which has been shown previously to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non-invasive spraying method, we show that gRNA-mediated activation programs move locally and systemically, generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this Potato virus X-based virus-induced gene reprogramming strategy results in program-specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid-enriched metabolite profiles.

Engineering aphid transmission of foxtail mosaic virus in the presence of potyvirus helper component proteinase through coat protein modifications.

Biotechnologies that use plant viruses as plant enhancement tools have shown great potential to flexibly engineer crop traits, but field applications of these technologies are still limited by efficient dissemination methods. Potyviruses can be rapidly inoculated into plants by aphid vectors due to the presence of the potyviral helper component proteinase (HC-Pro), which binds to the DAG motif of the coat protein (CP) of the virion. Previously it was determined that a naturally occurring DAG motif in the non-aphid-transmissible potexvirus, potato aucuba mosaic virus (PAMV), is functional when a potyviral HC-Pro is provided to aphids in plants. The DAG motif of PAMV was successfully transferred to the CP of another non-aphid-transmissible potexvirus, potato virus X, to convey aphid transmission capabilities in the presence of HC-Pro. Here, we demonstrate that DAG-containing segments of the CP from two different potyviruses (sugarcane mosaic virus and turnip mosaic virus), and from the previously used potexvirus, PAMV, can make the potexvirus, foxtail mosaic virus (FoMV), aphid-transmissible when fused with the FoMV CP. We show that DAG-containing FoMVs are transmissible by aphids that have prior access to HC-Pro through potyvirus-infected plants or ectopic expression of HC-Pro. The transmission efficiency of the DAG-containing FoMVs varied from less than 10 % to over 70 % depending on the length and composition of the surrounding amino acid sequences of the DAG-containing segment, as well as due to the recipient plant species. Finally, we show that the engineered aphid-transmissible FoMV is still functional as a plant enhancement resource, as endogenous host target genes were silenced in FoMV-infected plants after aphid transmission. These results suggest that aphid transmission could be engineered into non-aphid-transmissible plant enhancement viral resources to facilitate their field applications.

The tomato calcium-permeable channel 4.1 (SlOSCA4.1) is a susceptibility factor for pepino mosaic virus.

The hyperosmolality-gated calcium permeable channel 4.1 (OSCA4.1) belongs to an evolutionarily conserved small family of mechano-sensitive channels. OSCA members may represent key players in plant resistance to drought and to pathogen infection but are scarcely studied. After screening for resistance to pepino mosaic virus (PepMV) a collection of 1000 mutagenized tomato families, we identified a mutant showing no symptoms and reduced virus accumulation. Resistance was mapped to chromosome 2 between positions 46 309 531 to 47 044 163, where a missense mutation caused the putative truncation of the OSCA4.1 protein. A CRISPR/Cas9 slosca4.1 mutant was resistant to PepMV, but not to tobacco mosaic virus or potato virus X. Inoculation of mutant and wild type tomato protoplasts showed that resistance was expressed in single cells, suggesting a role for SlOSCA4.1 in early viral function(s); congruently, SlOSCA4.1 re-localized to structures reminiscent of viral replication complexes. We propose that SlOSCA4.1 contributes to the correct regulation of the Ca2+ homeostasis necessary for optimal PepMV infection. PepMV is a pandemic virus that causes significant losses in tomato crops worldwide. In spite of its importance, no tomato-resistant varieties have been deployed yet; the mutant identified here has great potential to breed tomato varieties resistant to PepMV.

Effect of the Coat Protein N-Terminal Domain Structure on the Structure and Physicochemical Properties of Virions of Potato Virus X and Alternanthera Mosaic Virus.

The amino acid sequences of the coat proteins (CPs) of the potexviruses potato virus X (PVX) and alternanthera mosaic virus (AltMV) share ~40% identity. The N-terminal domains of these proteins differ in the amino acid sequence and the presence of the N-terminal fragment of 28 residues (ΔN peptide) in the PVX CP. Here, we determined the effect of the N-terminal domain on the structure and physicochemical properties of PVX and AltMV virions. The circular dichroism spectra of these viruses differed significantly, and the melting point of PVX virions was 10-12°C higher than that of AltMV virions. Alignment of the existing high-resolution 3D structures of the potexviral CPs showed that the RMSD value between the Cα-atoms was the largest for the N-terminal domains of the two compared models. Based on the computer modeling, the ΔN peptide of the PVX CP is fully disordered. According to the synchrotron small-angle X-ray scattering (SAXS) data, the structure of CPs from the PVX and AltMV virions differ; in particular, the PVX CP has a larger portion of crystalline regions and, therefore, is more ordered. Based on the SAXS data, the diameters of the PVX and AltMV virions and helix parameters in solution were calculated. The influence of the conformation of the PVX CP N-terminal domain and its position relative to the virion surface on the virion structure was investigated. Presumably, an increased thermal stability of PVX virions vs. AltMV is provided by the extended N-terminal domain (ΔN peptide, 28 amino acid residues), which forms additional contacts between the adjacent CP subunits in the PVX virion.

Development of an efficient expression system with large cargo capacity for interrogation of gene function in bamboo based on bamboo mosaic virus.

Bamboo is one of the fastest growing plants among monocotyledonous species and is grown extensively in subtropical regions. Although bamboo has high economic value and produces much biomass quickly, gene functional research is hindered by the low efficiency of genetic transformation in this species. We therefore explored the potential of a bamboo mosaic virus (BaMV)-mediated expression system to investigate genotype-phenotype associations. We determined that the sites between the triple gene block proteins (TGBps) and the coat protein (CP) of BaMV are the most efficient insertion sites for the expression of exogenous genes in both monopodial and sympodial bamboo species. Moreover, we validated this system by individually overexpressing the two endogenous genes ACE1 and DEC1, which resulted in the promotion and suppression of internode elongation, respectively. In particular, this system was able to drive the expression of three 2A-linked betalain biosynthesis genes (more than 4 kb in length) to produce betalain, indicating that it has high cargo capacity and may provide the prerequisite basis for the development of a DNA-free bamboo genome editing platform in the future. Since BaMV can infect multiple bamboo species, we anticipate that the system described in this study will greatly contribute to gene function research and further promote the molecular breeding of bamboo.

Perturbation of nuclear-cytosolic shuttling of Rx1 compromises extreme resistance and translational arrest of potato virus X transcripts.

Many plant intracellular immune receptors mount a hypersensitive response (HR) upon pathogen perception. The concomitant localized cell death is proposed to trap pathogens, such as viruses, inside infected cells, thereby preventing their spread. Notably, extreme resistance (ER) conferred by the potato immune receptor Rx1 to potato virus X (PVX) does not involve the death of infected cells. It is unknown what defines ER and how it differs from HR-based resistance. Interestingly, Rx1 can trigger an HR, but only upon artificial (over)expression of PVX or its avirulence coat protein (CP). Rx1 has a nucleocytoplasmic distribution and both pools are required for HR upon transient expression of a PVX-GFP amplicon. It is unknown whether mislocalized Rx1 variants can induce ER upon natural PVX infection. Here, we generated transgenic Nicotiana benthamiana producing nuclear- or cytosol-restricted Rx1 variants. We found that these variants can still mount an HR. However, nuclear- or cytosol-restricted Rx1 variants can no longer trigger ER or restricts viral infection. Interestingly, unlike the mislocalized Rx1 variants, wild-type Rx1 was found to compromise CP protein accumulation. We show that the lack of CP accumulation does not result from its degradation but is likely to be linked with translational arrest of its mRNA. Together, our findings suggest that translational arrest of viral genes is a major component of ER and, unlike the HR, is required for resistance to PVX.

NbPsbO1 Interacts Specifically with the Bamboo Mosaic Virus (BaMV) Subgenomic RNA (sgRNA) Promoter and Is Required for Efficient BaMV sgRNA Transcription.

Many positive-strand (+) RNA viruses produce subgenomic RNAs (sgRNAs) in the infection cycle through the combined activities of viral replicase and host proteins. However, knowledge about host proteins involved in direct sgRNA promoter recognition is limited. Here, in the partially purified replicase complexes from Bamboo mosaic virus (BaMV)-infected tissue, we have identified the Nicotiana benthamiana photosystem II oxygen-evolving complex protein, NbPsbO1, which specifically interacted with the promoter of sgRNA but not that of genomic RNA (gRNA). Silencing of NbPsbO1 expression suppressed BaMV accumulation in N. benthamiana protoplasts without affecting viral gRNA replication. Overexpression of wild-type NbPsbO1 stimulated BaMV sgRNA accumulation. Fluorescent microscopy examination revealed that the fluorescence associated with NbPsbO1 was redistributed from chloroplast granal thylakoids to stroma in BaMV-infected cells. Overexpression of a mislocalized mutant of NbPsbO1, dTPPsbO1-T7, inhibited BaMV RNA accumulation in N. benthamiana, whereas overexpression of an NbPsbO1 derivative, sPsbO1-T7, designed to be targeted to chloroplast stroma, upregulated the sgRNA level. Furthermore, depletion of NbPsbO1 in BaMV RdRp preparation significantly inhibited sgRNA synthesis in vitro but exerted no effect on (+) or (-) gRNA synthesis, which indicates that NbPsbO1 is required for efficient sgRNA synthesis. These results reveal a novel role for NbPsbO1 in the selective enhancement of BaMV sgRNA transcription, most likely via direct interaction with the sgRNA promoter. IMPORTANCE Production of subgenomic RNAs (sgRNAs) for efficient translation of downstream viral proteins is one of the major strategies adapted for viruses that contain a multicistronic RNA genome. Both viral genomic RNA (gRNA) replication and sgRNA transcription rely on the combined activities of viral replicase and host proteins, which recognize promoter regions for the initiation of RNA synthesis. However, compared to the cis-acting elements involved in the regulation of sgRNA synthesis, the host factors involved in sgRNA promoter recognition mostly remain to be elucidated. Here, we found a chloroplast protein, NbPsbO1, which specifically interacts with Bamboo mosaic virus (BaMV) sgRNA promoter. We showed that NbPsbO1 is relocated to the BaMV replication site in BaMV-infected cells and demonstrated that NbPsbO1 is required for efficient BaMV sgRNA transcription but exerts no effect on gRNA replication. This study provides a new insight into the regulating mechanism of viral gRNA and sgRNA synthesis.

Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation.

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

Multiple ER-to-nucleus stress signaling pathways are activated during Plantago asiatica mosaic virus and Turnip mosaic virus infection in Arabidopsis thaliana.

Pathogens and other adverse environmental conditions can trigger endoplasmic reticulum (ER) stress. ER stress signaling increases the expression of cytoprotective ER-chaperones. The inositol-requiring enzyme (IRE1) is one ER stress sensor that is activated to splice the bZIP60 mRNA that produces a truncated transcription factor that activates gene expression in the nucleus. The IRE1/bZIP60 pathway is associated with restricting potyvirus and potexvirus infection. This study shows that the Plantago asiatica mosaic virus (PlAMV) triple gene block 3 (TGB3) and the Turnip mosaic virus (TuMV) 6K2 proteins activate alternative transcription pathways involving the bZIP17, bZIP28, BAG7, NAC089 and NAC103 factors in Arabidopsis thaliana. Using the corresponding knockout mutant lines, we show that bZIP17, bZIP60, BAG7 and NAC089 are factors in reducing PlAMV infection, whereas bZIP28 and bZIP60 are factors in reducing TuMV infection. We propose a model in which bZIP60 and bZIP17 synergistically induce genes restricting PlAMV infection, while bZIP60 and bZIP28 independently induce genes supporting PlAMV infection. Regarding TuMV-green fluorescent protein (GFP) infection, bZIP60 and bZIP28 serve to repress local and systemic infection. Finally, tauroursodeoxycholic acid treatments were used to demonstrate that the protein folding capacity significantly influences PlAMV accumulation.

Potato virus X-mediated constitutive expression of Plutella xylostella PxSDF2L1 gene in Nicotiana benthamiana confers resistance to Phytophthora parasitica var. nicotianae.

BACKGROUND: The Plutella xylostella PxSDF2L1 gene was previously reported to enhance insect resistance to pathogen at high basal transcription rate. PxSDF2L1 shows similitude with the stromal cell-derived factor 2 (SDF2), an ER stress-induced chaperon protein that is highly conserved throughout animals and plants. The precise biological function of SDF2 is not clear, but its expression is required for innate immunity in plants. Here, we investigate whether a continuous expression of PxSDF2L1 in Nicotiana benthamiana can similarly confer resistance to plant pathogen, particularly, the black shank Phytophthora parasitica var. nicotianae. RESULTS: The N. benthamiana plants were inoculated with agrobacteria transformed with a PVX-based binary vector carrying the PxSDF2L1 gene; similar agroinoculation experiments with a PVX vector carrying the GFP gene were used for controls. In pot trials, agroinfected N. benthamiana plants constitutively expressing PxSDF2L1 showed a significant reduction of stem disease symptoms caused by the inoculation with P. parasitica, compared with controls. CONCLUSIONS: We confirm a role of PxSDF2L1 in resistance to black shank, with a potential application to engineering active resistance against this oomycete in the commercial N. tabacum species and propose its evaluation in other crop families and plant pathogens.

Potato Virus X Vector-Mediated DNA-Free Genome Editing in Plants.

Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ≤18% of shoots contained T-DNA. The PVX vector was also used to express a base editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.

The Potato Virus X TGBp2 Protein Plays Dual Functional Roles in Viral Replication and Movement.

Plant viruses usually encode one or more movement proteins (MP) to accomplish their intercellular movement. A group of positive-strand RNA plant viruses requires three viral proteins (TGBp1, TGBp2, and TGBp3) that are encoded by an evolutionarily conserved genetic module of three partially overlapping open reading frames (ORFs), termed the triple gene block (TGB). However, how these three viral movement proteins function cooperatively in viral intercellular movement is still elusive. Using a novel in vivo double-stranded RNA (dsRNA) labeling system, we showed that the dsRNAs generated by potato virus X (PVX) RNA-dependent RNA polymerase (RdRp) are colocalized with viral RdRp, which are further tightly covered by "chain mail"-like TGBp2 aggregates and localizes alongside TGBp3 aggregates. We also discovered that TGBp2 interacts with the C-terminal domain of PVX RdRp, and this interaction is required for the localization of TGBp3 and itself to the RdRp/dsRNA bodies. Moreover, we reveal that the central and C-terminal hydrophilic domains of TGBp2 are required to interact with viral RdRp. Finally, we demonstrate that knockout of the entire TGBp2 or the domain involved in interacting with viral RdRp attenuates both PVX replication and movement. Collectively, these findings suggest that TGBp2 plays dual functional roles in PVX replication and intercellular movement.IMPORTANCE Many plant viruses contain three partially overlapping open reading frames (ORFs), termed the triple gene block (TGB), for intercellular movement. However, how the corresponding three proteins coordinate their functions remains obscure. In the present study, we provided multiple lines of evidence supporting the notion that PVX TGBp2 functions as the molecular adaptor bridging the interaction between the RdRp/dsRNA body and TGBp3 by forming "chain mail"-like structures in the RdRp/dsRNA body, which can also enhance viral replication. Taken together, our results provide new insights into the replication and movement of PVX and possibly also other TGB-containing plant viruses.

Proliferating Cell Nuclear Antigen Suppresses RNA Replication of Bamboo Mosaic Virus through an Interaction with the Viral Genome.

Bamboo mosaic virus (BaMV), a member of the Potexvirus genus, has a monopartite positive-strand RNA genome on which five open reading frames (ORFs) are organized. ORF1 encodes a 155-kDa nonstructural protein (REPBaMV) that plays a core function in replication/transcription of the viral genome. To find out cellular factors modulating the replication efficiency of BaMV, a putative REPBaMV-associated protein complex from Nicotiana benthamiana leaf was isolated on an SDS-PAGE gel, and a few proteins preferentially associated with REPBaMV were identified by tandem mass spectrometry. Among them, proliferating cell nuclear antigen (PCNA) was particularly noted. Overexpression of PCNA strongly suppressed the accumulation of BaMV coat protein and RNAs in leaf protoplasts. In addition, PCNA exhibited an inhibitory effect on BaMV polymerase activity. A pulldown assay confirmed a binding capability of PCNA toward BaMV genomic RNA. Mutations at D41 or F114 residues, which are critical for PCNA to function in nuclear DNA replication and repair, disabled PCNA from binding BaMV genomic RNA as well as suppressing BaMV replication. This suggests that PCNA bound to the viral RNA may interfere with the formation of a potent replication complex or block the replication process. Interestingly, BaMV is almost invisible in the newly emerging leaves where PCNA is actively expressed. Accordingly, PCNA is probably one of the factors restricting the proliferation of BaMV in young leaves. Foxtail mosaic virus and Potato virus X were also suppressed by PCNA in the protoplast experiment, suggesting a general inhibitory effect of PCNA on the replication of potexviruses.IMPORTANCE Knowing the dynamic interplay between plant RNA viruses and their host is a basic step toward first understanding how the viruses survive the plant defense mechanisms and second gaining knowledge of pathogenic control in the field. This study found that plant proliferating cell nuclear antigen (PCNA) imposes a strong inhibition on the replication of several potexviruses, including Bamboo mosaic virus, Foxtail mosaic virus, and Potato virus X Based on the tests on Bamboo mosaic virus, PCNA is able to bind the viral genomic RNA, and this binding is a prerequisite for the protein to suppress the virus replication. This study also suggests that PCNA plays an important role in restricting the proliferation of potexviruses in the rapidly dividing tissues of plants.

Allexiviruses may have acquired inserted sequences between the CP and CRP genes to change the translation reinitiation strategy of CRP.

Allexiviruses are economically important garlic viruses that are involved in garlic mosaic diseases. In this study, we characterized the allexivirus cysteine-rich protein (CRP) gene located just downstream of the coat protein (CP) gene in the viral genome. We determined the nucleotide sequences of the CP and CRP genes from numerous allexivirus isolates and performed a phylogenetic analysis. According to the resulting phylogenetic tree, we found that allexiviruses were clearly divided into two major groups (group I and group II) based on the sequences of the CP and CRP genes. In addition, the allexiviruses in group II had distinct sequences just before the CRP gene, while group I isolates did not. The inserted sequence between the CP and CRP genes was partially complementary to garlic 18S rRNA. Using a potato virus X vector, we showed that the CRPs affected viral accumulation and symptom induction in Nicotiana benthamiana, suggesting that the allexivirus CRP is a pathogenicity determinant. We assume that the inserted sequences before the CRP gene may have been generated during viral evolution to alter the termination-reinitiation mechanism for coupled translation of CP and CRP.

A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1.

Pepino mosaic virus (PepMV) is a mechanically-transmitted tomato pathogen of importance worldwide. Interactions between the PepMV coat protein and triple gene block protein (TGBp1) with the host heat shock cognate protein 70 and catalase 1 (CAT1), respectively, have been previously reported by our lab. In this study, a novel tomato interactor (SlTXND9) was shown to bind the PepMV TGBp1 in yeast-two-hybrid screening, in vitro pull-down and bimolecular fluorescent complementation (BiFC) assays. SlTXND9 possesses part of the conserved thioredoxin (TRX) active site sequence (W__PC vs. WCXPC), and TXND9 orthologues cluster within the TRX phylogenetic superfamily closest to phosducin-like protein-3. In PepMV-infected and healthy Nicotiana benthamiana plants, NbTXND9 mRNA levels were comparable, and expression levels remained stable in both local and systemic leaves for 10 days post inoculation (dpi), as was also the case for catalase 1 (CAT1). To localize the TXND9 in plant cells, a polyclonal antiserum was produced. Purified α-SlTXND9 immunoglobulin (IgG) consistently detected a set of three protein bands in the range of 27⁻35 kDa, in the 1000 and 30,000 g pellets, and the soluble fraction of extracts of healthy and PepMV-infected N. benthamiana leaves, but not in the cell wall. These bands likely consist of the homologous protein NbTXND9 and its post-translationally modified derivatives. On electron microscopy, immuno-gold labelling of ultrathin sections of PepMV-infected N. benthamiana leaves using α-SlTXND9 IgG revealed particle accumulation close to plasmodesmata, suggesting a role in virus movement. Taken together, this study highlights a novel tomato-PepMV protein interaction and provides data on its localization in planta. Currently, studies focusing on the biological function of this interaction during PepMV infection are in progress.

Engineering Potato Virus X Particles for a Covalent Protein Based Attachment of Enzymes.

Plant virus nanoparticles are often used to display functional amino acids or small peptides, thus serving as building blocks in application areas as diverse as nanoelectronics, bioimaging, vaccination, drug delivery, and bone differentiation. This is most easily achieved by expressing coat protein fusions, but the assembly of the corresponding virus particles can be hampered by factors such as the fusion protein size, amino acid composition, and post-translational modifications. Size constraints can be overcome by using the Foot and mouth disease virus 2A sequence, but the compositional limitations cannot be avoided without the introduction of time-consuming chemical modifications. SpyTag/SpyCatcher technology is used in the present study to covalently attach the Trichoderma reesei endoglucanase Cel12A to Potato virus X (PVX) nanoparticles. The formation of PVX particles is confirmed by western blot, and the ability of the particles to display Cel12A is demonstrated by enzyme-linked immunosorbent assays and transmission electron microscopy. Enzymatic assays show optimal reaction conditions of 50 °C and pH 6.5, and an increased substrate conversion rate compared to free enzymes. It is concluded that PVX displaying the SpyTag can serve as new scaffold for protein display, most notably for proteins with post-translational modifications.

Three gene products of a begomovirus-betasatellite complex restore expression of a transcriptionally silenced green fluorescent protein transgene in Nicotiana benthamiana.

Single-stranded DNA geminiviruses replicate via double-stranded DNA intermediates forming mini-chromosomes that are targets for transcriptional gene silencing (TGS) in plants. The ability of the cotton leaf curl Kokhran virus (CLCuKoV)-cotton leaf curl Multan betasatellite (CLCuMuB) proteins, replication-associated protein (Rep), transcriptional activator protein (TrAP), C4, V2 and ßC1, to suppress TGS was investigated by using the Nicotiana benthamiana line 16-TGS (16-TGS) harbouring a transcriptionally silenced green fluorescent protein (GFP) transgene. Inoculation of 16-TGS plants with a recombinant potato virus X vector carrying Rep, TrAP or ßC1 resulted in re-expression of GFP. Northern blot analysis confirmed that the observed GFP fluorescence was associated with GFP mRNA accumulation. These results indicated that Rep, TrAP and ßC1 proteins of CLCuKoV-CLCuMuB can re-activate the expression of a transcriptionally silenced GFP transgene in N. benthamiana. Although Rep, TrAP, or ßC1 proteins have, for other begomoviruses or begomoviruses-betasatellites, been previously shown to have TGS suppressor activity, this is the first report demonstrating that a single begomovirus-betasatellite complex encodes three suppressors of TGS.

Multifaceted capsid proteins: multiple interactions suggest multiple roles for Pepino mosaic virus capsid protein.

Pepino mosaic virus (PepMV) (family Alphaflexiviridae, genus Potexvirus) is a mechanically transmitted tomato pathogen that, over the last decade, has evolved from emerging to endemic worldwide. Here, two heat-shock cognate (Hsc70) isoforms were identified as part of the coat protein (CP)/Hsc70 complex in vivo, following full-length PepMV and CP agroinoculation. PepMV accumulation was severely reduced in Hsp70 virus-induced gene silenced and in quercetin-treated Nicotiana benthamiana plants. Similarly, in vitro-transcribed as well as virion RNA input levels were reduced in quercetin-treated protoplasts, suggesting an essential role for Hsp70 in PepMV replication. As for Potato virus X, the PepMV CP and triple gene-block protein 1 (TGBp1) self-associate and interact with each other in vitro but, unlike in the prototype, both PepMV proteins represent suppressors of transgene-induced RNA silencing with different modes of action; CP is a more efficient suppressor of RNA silencing, sequesters the silencing signal by preventing its spread to neighboring cells and its systemic movement. Here, we provide evidence for additional roles of the PepMV CP and host-encoded Hsp70 in viral infection, the first as a truly multifunctional protein able to specifically bind to a host chaperone and to counterattack an RNA-based defense mechanism, and the latter as an essential factor for PepMV infection.

Structural determinants at the interface of the ARC2 and leucine-rich repeat domains control the activation of the plant immune receptors Rx1 and Gpa2.

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins.

Influence of host chloroplast proteins on Tobacco mosaic virus accumulation and intercellular movement.

Tobacco mosaic virus (TMV) forms dense cytoplasmic bodies containing replication-associated proteins (virus replication complexes [VRCs]) upon infection. To identify host proteins that interact with individual viral components of VRCs or VRCs in toto, we isolated viral replicase- and VRC-enriched fractions from TMV-infected Nicotiana tabacum plants. Two host proteins in enriched fractions, ATP-synthase γ-subunit (AtpC) and Rubisco activase (RCA) were identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry. Through pull-down analysis, RCA bound predominantly to the region between the methyltransferase and helicase domains of the TMV replicase. Tobamovirus, but not Cucumber mosaic virus or Potato virus X, infection of N. tabacum plants resulted in 50% reductions in Rca and AtpC messenger RNA levels. To investigate the role of these host proteins in TMV accumulation and plant defense, we used a Tobacco rattle virus vector to silence these genes in Nicotiana benthamiana plants prior to challenge with TMV expressing green fluorescent protein. TMV-induced fluorescent lesions on Rca- or AtpC-silenced leaves were, respectively, similar or twice the size of those on leaves expressing these genes. Silencing Rca and AtpC did not influence the spread of Tomato bushy stunt virus and Potato virus X. In AtpC- and Rca-silenced leaves TMV accumulation and pathogenicity were greatly enhanced, suggesting a role of both host-encoded proteins in a defense response against TMV. In addition, silencing these host genes altered the phenotype of the TMV infection foci and VRCs, yielding foci with concentric fluorescent rings and dramatically more but smaller VRCs. The concentric rings occurred through renewed virus accumulation internal to the infection front.