Turnip Crinkle Virus Coat Protein Research Articles

Molecular modeling and interaction between Arabidopsis sulfite oxidase and the GW motif of Turnip crinkle virus coat protein

Previous study has shown that Hibiscus sulfite oxidase (SO) interacts with Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) and triggers sulfur enhanced defense (SED). In this study, we show the interaction of Arabidopsis SO (AtSO) and Turnip crinkle virus (TCV) CP in Arabidopsis thaliana plants. We identified the binding sites of TCV CP (W274) and AtSO (D223) using bioinformatics and confirmed it experimentally. Mutation of binding site W274 to A274 in TCV CP resulted in failure of TCV infection. TCV accumulation in SO over-expression (SO_OE) plants was lower than that in wild-type (WT) and SO knock-out (SO_KO) plants at 7 dpi but reached a level similar to that of WT and SO_KO plants at 10 dpi. AtSO competed with Argonaute 1 (AGO1) for TCV CP binding in vitro. AtSO may serve as an anti-viral factor through sequestering TCV CP for binding with AGO1 and confers virus resistance.

Co-expressing Turnip Crinkle Virus-coat protein with the serine protease α-thrombin precursor (pFIIa) in Nicotiana benthamiana Domin

The serine protease α-thrombin (FIIa) plays a fundamental role in blood clotting. In the present report, a FIIa precursor (pFIIa) was expressed in Nicotiana benthamiana Domin. The expression construct featured the Kozak consensus sequence and the 2S2 Arabidopsis thaliana (L.) Heynh. signal peptide to direct the protein into the secretory pathway (sec-pFIIa). A version carrying the KDEL endoplasmic reticulum (ER) retention signal (pFIIa-ER) was also constructed. Transient expression of pFIIa in N. benthamiana leaves was achieved by Agrobacterium tumefaciens infiltration. The influence of post-transcriptional gene silencing (PTGS) was analyzed by co-infiltrating with an A. tumefaciens strain carrying the construct for the Turnip Crinkle Virus-coat protein (TCV-CP) known for interfering with PTGS. Reverse transcription polymerase chain reaction and Western blot analyses confirmed the presence of the corresponding messenger RNA and the recombinant pFIIa protein in plant extracts. A positive effect of the addition of the PTGS inhibitor was demonstrated. The accumulation of sec-pFIIa and pFIIa-ER was estimated to be 6 μg g−1 fresh weight (FW) (0.07% (w/w) total protein concentration; TPC) and 17 μg g−1 FW (0.21% (w/w) TPC), respectively. Furthermore, stably transformed callus and suspension cultures were obtained. The recombinant protein was detected only in the biomass of the pFIIa-ER cell suspension line at a concentration of 0.25 μg mL−1 (0.017% (w/w) of total soluble protein). This appears to be the first report describing the expression of a precursor of FIIa in plants.

Production of Mosaic Turnip Crinkle Virus-Like Particles Derived by Coinfiltration of Wild-Type and Modified Forms of Virus Coat Protein in Plants.

When the coat protein reading frame of turnip crinkle virus (TCV) is transiently expressed in leaves, virus-like particles (VLPs) are readily formed. However, after introducing genetic modifications to the full-length coat protein sequence, such as the introduction of an epitope-specific sequence within the coat protein sequence or the in-frame carboxyl terminal fusion of GFP, the formation of such modified VLPs is poor. However, by coexpression of one of these modified forms with wild-type TCV coat protein by the coinfiltration of appropriate Agrobacterium suspensions, VLP generation is enhanced through the formation of "mosaics," that is, individual VLPs consisting of both modified and wild-type subunits (also known as phenotypically mixed VLPs). Here we describe methods for the introduction of genetic modifications into the TCV coat protein sequence, the production of mosaic TCV VLPs and their characterization.

A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus.

To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher-order RNA structure, an unstructured sequence of about 84 nucleotides (nt) immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) open reading frame (ORF) has been found to promote internal expression of the CP from the genomic RNA (gRNA) both in vitro and in vivo An absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing. Analysis of the IRES region in vitro by use of both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that an overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulated in vivo in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA invivo Since the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation, suggesting that this viral IRES and cellular IRES may have similar strategies for internal translation initiation.IMPORTANCE Cap-independent translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. Viral IRES, in general, contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary structure has IRES activity and produces low levels of viral coat protein in vitro and in vivo Our findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common.

Endoplasmic reticulum stress responses function in the HRT-mediated hypersensitive response in Nicotiana benthamiana.

HRT is a plant coiled-coil, nucleotide-binding and leucine-rich repeat (CC-NB-LRR) disease resistance protein that triggers the hypersensitive response (HR) on recognition of Turnip crinkle virus (TCV) coat protein (CP). The molecular mechanism and significance of HR-mediated cell death for TCV resistance have not been fully elucidated. To identify the genes involved in HRT/TCV CP-mediated HR in Nicotiana benthamiana, we performed virus-induced gene silencing (VIGS) of 459 expressed sequence tags (ESTs) of pathogen-responsive Capsicum annuum genes. VIGS of CaBLP5, which encodes an endoplasmic reticulum (ER)-associated immunoglobulin-binding protein (BiP), silenced NbBiP4 and NbBiP5 and significantly reduced HRT-mediated HR. The induction of ER stress-responsive genes and the accumulation of ER-targeted BiPs in response to HRT-mediated HR suggest that ER is involved in HR in N. benthamiana. BiP4/5 silencing significantly down-regulated HRT at the mRNA and protein levels, and affected SGT1 and HSP90 expression. Co-expression of TCV CP in BiP4/5-silenced plants completely abolished HRT induction. Transient expression of TCV CP alone induced selected ER stress-responsive gene transcripts only in Tobacco rattle virus (TRV)-infected plants, and most of these genes were induced by HRT/TCV CP, except for bZIP60, which was induced specifically in response to HRT/TCV CP. TCV CP-mediated induction of ER stress-responsive genes still occurred in BiP4/5-silenced plants, but HRT/TCV CP-mediated induction of these genes was defective. Tunicamycin, a chemical that inhibits protein N-glycosylation, inhibited HRT-mediated HR, suggesting that ER has a role in HR regulation. These results indicate that BiP and ER, which modulate pattern recognition receptors in innate immunity, also regulate R protein-mediated resistance.

The Generation of Turnip Crinkle Virus-Like Particles in Plants by the Transient Expression of Wild-Type and Modified Forms of Its Coat Protein.

Turnip crinkle virus (TCV), a member of the genus carmovirus of the Tombusviridae family, has a genome consisting of a single positive-sense RNA molecule that is encapsidated in an icosahedral particle composed of 180 copies of a single type of coat protein. We have employed the CPMV-HT transient expression system to investigate the formation of TCV-like particles following the expression of the wild-type coat protein or modified forms of it that contain either deletions and/or additions. Transient expression of the coat protein in plants results in the formation of capsid structures that morphologically resemble TCV virions (T = 3 structure) but encapsidate heterogeneous cellular RNAs, rather than the specific TCV coat protein messenger RNA. Expression of an amino-terminal deleted form of the coat protein resulted in the formation of smaller T = 1 structures that are free of RNA. The possibility of utilizing TCV as a carrier for the presentation of foreign proteins on the particle surface was also explored by fusing the sequence of GFP to the C-terminus of the coat protein. The expression of coat protein-GFP hybrids permitted the formation of VLPs but the yield of particles is diminished compared to the yield obtained with unmodified coat protein. Our results confirm the importance of the N-terminus of the coat protein for the encapsidation of RNA and show that the coat protein's exterior P domain plays a key role in particle formation.

Turnip crinkle virus coat protein inhibits the basal immune response to virus invasion in Arabidopsis by binding to the NAC transcription factor TIP

Turnip crinkle virus (TCV) has been shown to interact with a NAC transcription factor, TIP, of Arabidopsis thaliana, via its coat protein (CP). This interaction correlates with the resistance response manifested in TCV-resistant Arabidopsis ecotype Di-17. We report that failure of a mutated CP to interact with TIP triggered the corresponding TCV mutant (R6A) to cause more severe symptoms in the TCV-susceptible ecotype Col-0. We hypothesized that TCV regulates antiviral basal immunity through TIP-CP interaction. Consistent with this hypothesis, we found that the rate of accumulation of R6A was measurably slower than wild-type TCV over the course of an infection. Notably, R6A was able to accumulate at similar rates as wild-type TCV in mutant plants with defects in salicylic acid (SA) signaling. Finally, plants with altered TIP expression provided evidence R6A's inability to evade the basal resistance response was likely associated with loss of ability for CP to bind TIP.

Interaction between Cucumber mosaic virus and Turnip crinkle virus in Arabidopsis thaliana

AbstractThe interaction between Cucumber mosaic virus (CMV) and Turnip crinkle virus (TCV) in Arabidopsis thaliana is reported. Although TCV coat protein accumulates to a similar level in singly or doubly infected plants, CMV coat protein is significantly decreased in doubly infected plants, which develop symptoms similar to those of TCV infection alone. TCV thus strongly interfers with infection by CMV. A significant reduction in CMV 2b gene expression also occurs in co‐infected plants. A defence signalling initiator, salicylic acid (SA), further inhibits CMV accumulation during CMV–TCV co‐infection. This interference is correlated with both enhanced virus gene silencing and defence signalling.

Characterization of resistance mechanism in transgenic Nicotiana benthamiana containing Turnip crinkle virus coat protein

Two transgenic lines, of Nicotiana benthamiana expressing Turnip crinkle virus (TCV)-coat protein (CP) gene with contrasting phenotype, the highest (#3) and the lowest (#18) CP expressers, were selected and challenged with the homologous TCV. The former, the highest expresser, showed nearly five times more CP expression than the latter. Progenies of #3 and #18 lines showed 30 and 100% infection rates, respectively. The infected progenies of #3 line showed mild and delayed symptom with TCV. This is a coat protein-mediated resistance (CP-MR), and its resistance level is directly proportional to CP transgene expression. However, CP-MR of the transgenic plants was specific only for TCV but not for heterologous viruses. Newly growing leaves of those infected progenies of #3 line did not show any visible symptoms at 4-week post-inoculation (wpi) with TCV, suggesting a reversal from infection. This was confirmed by RT-PCR analysis with the disappearance of the target at 4 wpi. This is a case of RNA-mediated resistance, and a threshold level of transgene expression may be needed to achieve the silent state. To confirm the RNA silencing, we infiltrated Agrobacterium carrying TCV-CP into leaves of progenies of #3 and performed RT-PCR analysis. The results indicate that TCV-CP's suppressor activity against RNA silencing itself can be silenced by the homologous expression of TCV-CP in the transgenic plants. The transgenic plants containing TCV-CP seem to be a model system to study viral protection mediated by a combination of protein and RNA silencing.

Importance of coat protein and RNA silencing in satellite RNA/virus interactions

RNA silencing is a major defense mechanism plants use to fight an invading virus. The silencing suppressor of Turnip crinkle virus (TCV) is the viral coat protein (CP), which obstructs the DCL2/DCL4 silencing pathway. TCV is associated with a virulent satellite RNA (satC) that represses the accumulation of TCV genomic RNA and whose accumulation is repressed by the TCV CP. To investigate if reduced TCV accumulation due to satC involves RNA silencing and/or the suppressor activity of the CP, TCV was altered to contain a mutation reported to target CP silencing suppressor activity (Deleris et al., Science 313, 68, 2006). However, the mutation did not cause an exclusive defect in silencing suppression, but rather produced a generally non-functional protein. We demonstrate that a functional CP, but not DCL2/DCL4, is required for satC-mediated repression of TCV. In addition, enhancement of satC accumulation in the absence of a functional CP requires DCL2/DCL4.

A Versatile Assay for the Identification of RNA Silencing Suppressors Based on Complementation of Viral Movement

The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed.

Host-induced avirulence of hibiscus chlorotic ringspot virus mutants correlates with reduced gene-silencing suppression activity

Post-transcriptional gene silencing (PTGS) and virus-encoded gene-silencing suppressors are defence and counterdefence strategies developed by host and pathogens during evolution. Using a green fluorescence protein-based transient suppression system, the coat protein (CP) of Hibiscus chlorotic ringspot virus (HCRSV) was identified as a strong gene-silencing suppressor. CP suppressed sense RNA-induced but not dsRNA-induced local and systemic PTGS. This is different from another virus in the genus Carmovirus, Turnip crinkle virus (TCV), the CP of which strongly suppresses dsRNA-induced PTGS. HCRSV CP domain deletion mutants lost their suppression function, indicating that the complete CP is essential for suppression of PTGS. When CP was expressed from a Potato virus X (PVX) vector, it was able to enhance the symptom severity and to increase the accumulation of PVX RNA. Here, it is proposed that HCRSV CP suppresses PTGS at the initiation step, which is different from TCV CP. In addition, a previous study demonstrated that CP mutants resulting from serial passage of HCRSV in its local lesion host also showed a significantly reduced suppression function, indicating that host-induced mutations that lead to avirulence of HCRSV in kenaf correlate with its reduced ability to suppress PTGS.

The nuclear localization of the Arabidopsis transcription factor TIP is blocked by its interaction with the coat protein of Turnip crinkle virus

We have previously reported that TIP, an Arabidopsis protein, interacts with the coat protein (CP) of Turnip crinkle virus (TCV) in yeast cells and that this interaction correlated with the resistance response in the TCV-resistant Arabidopsis ecotype Dijon-17. TIP was also able to activate transcription of reporter genes in yeast cells, suggesting that it is likely a transcription factor. We have now verified the physical interaction between TIP and TCV CP in vitro and showed that CP mutants unable to interact with TIP in yeast cells bind TIP with much lower affinity in vitro. Secondly, we have performed gel shift experiments demonstrating that TIP does not bind to DNA in a sequence-specific manner. The subcellular localization of TIP was also investigated by transiently expressing green fluorescence protein (GFP)-tagged TIP in Nicotiana benthamiana plant cells, which showed that GFP-tagged TIP localizes primarily to nuclei. Significantly, co-expression of TCVCP and GFP-TIP prevented the nuclear localization of TIP. Together, these results suggest that TIP might be a transcription factor involved in regulating the defense response of Arabidopsis to TCV and that its normal role is compromised by interaction with the invading viral CP.

Enhanced viral pathogenesis associated with a virulent mutant virus or a virulent satellite RNA correlates with reduced virion accumulation and abundance of free coat protein

SatC, a chimeric satellite RNA associated with Turnip crinkle virus (TCV), intensifies the symptoms of TCV on all symptomatic hosts yet attenuates the symptoms of a TCV mutant that expresses low levels of a defective coat protein (CP). We now report that TCV virion levels were substantially reduced by the presence of satC or when two amino acids were inserted at the N-terminus of the CP, resulting in similarly enhanced symptoms. Since the TCV CP is a suppressor of RNA silencing, increased levels of resultant free CP could augment silencing suppression, resulting in enhanced colonization of the plant.

Turnip crinkle virus coat protein mediates suppression of RNA silencing in nicotiana benthamiana

All of the protein products of Turnip crinkle virus (TCV; Tombusviridae, Carmovirus) were tested for their ability to suppress RNA silencing of a reporter gene after transient expression in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Only the capsid protein, P38, showed suppression activity, although this was not obvious when P38 was expressed as part of a TCV infection of the same tissues. When P38 was expressed from a PVX vector, symptoms with enhanced severity that correlated with increased PVX RNA accumulation were observed. This contradiction between ectopic expression of P38 and TCV infection could be accounted for if the active determinant of suppressor activity within P38 was sequestered within the capsid protein structure. The N-terminal 25 amino acids were shown to be important for this activity. This region forms part of the unexposed R-domain that interacts with the RNA within the virus particle. This observation throws light on some of the complex biology exhibited by TCV.

Efficient infection of Nicotiana benthamiana by Tomato bushy stunt virus is facilitated by the coat protein and maintained by p19 through suppression of gene silencing.

Tomato bushy stunt virus (TBSV) is one of few RNA plant viruses capable of moving systemically in some hosts in the absence of coat protein (CP). TBSV also encodes another protein (p19) that is not required for systemic movement but functions as a symptom determinant in Nicotiana benthamiana. Here, the role of both CP and p19 in the systemic spread has been reevaluated by utilizing transgenic N. benthamiana plants expressing the movement protein (MP) of Red clover necrotic mosaic virus and chimeric TBSV mutants that express CP of Turnip crinkle virus. Through careful examination of the infection phenotype of a series of mutants with changes in the CP and p19 genes, we demonstrate that both of these genes are required for efficient systemic invasion of TBSV in N. benthamiana. The CP likely enables efficient viral unloading from the vascular system in the form of assembled virions, whereas p19 enhances systemic infection by suppressing the virus-induced gene silencing.

The amino terminus of the coat protein of Turnip crinkle virus is the AVR factor recognized by resistant arabidopsis.

We have isolated three naturally occurring strains of Turnip crinkle virus (TCV) that break resistance in Di-17 Arabidopsis. Two mutations in the N terminus of the TCV coat protein, D4N and P5S, were shown to confer this phenotype. Thus, this region of the coat protein is involved in eliciting resistance responses in Arabidopsis.

Cell-to-Cell and Systemic Movement of Recombinant Green Fluorescent Protein-Tagged Turnip Crinkle Viruses

To facilitate analyses of turnip crinkle virus (TCV) cell-to-cell and systemic movement, we created a series of recombinant viruses expressing green fluorescent protein (GFP) either as substitutions of coat protein (CP) sequences or as fusions to movement proteins (MPs). Constructs were used to inoculate leaves of Arabidopsis seedlings. TCV carrying its two native MPs and GFP fused near the start of CP translation (GFP ΔCP) resulted in cell-to-cell movement manifested by the expansion of fluorescent foci on inoculated leaves. GFP fusions to either MP were inactive for movement. However, TCV carrying the p9–GFP fusion, which expresses a functional p8 gene, could be complemented for cell-to-cell movement by coinoculation with virus carrying native p9 but mutant for p8. This same coinoculation combination also lead to systemic spread of GFP fluorescence to noninoculated leaves, as the complementing virus carries native CP. Complementation for systemic movement of virus carrying GFP ΔCP constructs was achieved by inoculation onto transgenic plants expressing TCV CP. GFP-tagged TCV movement was detected throughout the plant, including the inflorescence stem, cauline leaves, flowers, siliques, and substructures such as organ primordia and meristematic regions. The recombinant viruses described herein provide (1) genetic information relevant to define regions of TCV that can, or cannot, be manipulated by insertion of foreign coding sequences and (2) a set of tools to allow the study of viral cell-to-cell and long-distance movement in the model plant system Arabidopsis.

3'-End stem-loops of the subviral RNAs associated with turnip crinkle virus are involved in symptom modulation and coat protein binding.

Many plant RNA viruses are associated with one or more subviral RNAs. Two subviral RNAs, satellite RNA C (satC) and defective interfering RNA G (diG) intensify the symptoms of their helper, turnip crinkle virus (TCV). However, when the coat protein (CP) of TCV was replaced with that of the related Cardamine chlorotic fleck virus (CCFV), both subviral RNAs attenuated symptoms of the hybrid virus TCV-CP(CCFV). In contrast, when the translation initiation codon of the TCV CP was altered to ACG and reduced levels of CP were synthesized, satC attenuated symptoms while diG neither intensified nor attenuated symptoms. The determinants for this differential symptom modulation were previously localized to the 3'-terminal 100 bases of the subviral RNAs, which contain six positional differences (Q. Kong, J.-W. Oh, C. D. Carpenter, and A. E. Simon, Virology 238:478-485, 1997). In the current study, we have determined that certain sequences within the 3'-terminal stem-loop structures of satC and diG, which also serve as promoters for complementary strand synthesis, are critical for symptom modulation. Furthermore, the ability to attenuate symptoms was correlated with weakened binding of TCV CP to the hairpin structure.

Members of the Arabidopsis HRT/RPP8 family of resistance genes confer resistance to both viral and oomycete pathogens.

Turnip crinkle virus (TCV) inoculation onto TCV-resistant Arabidopsis leads to a hypersensitive response (HR) controlled by the dominant gene HRT. HRT is a member of the class of resistance (R) genes that contain a leucine zipper, a nucleotide binding site, and leucine-rich repeats. The chromosomal position of HRT and its homology to resistance gene RPP8 and two RPP8 homologs indicate that unequal crossing over and gene conversion may have contributed to HRT evolution. RPP8 confers resistance to an oomycete pathogen, Peronospora parasitica. Despite very strong similarities within the HRT/RPP8 family, HRT and RPP8 are specific for the respective pathogens they detect. Hence, the HRT/RPP8 family provides molecular evidence that sequence changes between closely related members of multigene families can generate novel specificities for radically different pathogens. Transgenic plants expressing HRT developed an HR but generally remained susceptible to TCV because of a second gene, RRT, that regulates resistance to TCV. However, several transgenic plants that overexpressed HRT produced micro-HRs or no HR when inoculated with TCV and were resistant to infection. Expression of the TCV coat protein gene in seedlings containing HRT resulted in massive necrosis and death, indicating that the avirulence factor detected by the HRT-encoded protein is the TCV coat protein.