Resistance to turnip crinkle virus in Arabidopsis is regulated by two host genes and is salicylic acid dependent but NPR1, ethylene, and jasmonate independent.
(9/132)Inoculation of turnip crinkle virus (TCV) on the resistant Arabidopsis ecotype Dijon (Di-17) results in the development of a hypersensitive response (HR) on the inoculated leaves. To assess the role of the recently cloned HRT gene in conferring resistance, we monitored both HR and resistance (lack of viral spread to systemic tissues) in the progeny of a cross between resistant Di-17 and susceptible Columbia plants. As expected, HR development segregated as a dominant trait that corresponded with the presence of HRT. However, all of the F(1) plants and three-fourths of HR(+) F(2) plants were susceptible to the virus. These results suggest the presence of a second gene, termed RRT, that regulates resistance to TCV. The allele present in Di-17 appears to be recessive to the allele or alleles present in TCV-susceptible ecotypes. We also demonstrate that HR formation and TCV resistance are dependent on salicylic acid but not on ethylene or jasmonic acid. Furthermore, these phenomena are unaffected by mutations in NPR1. Thus, TCV resistance requires a yet undefined salicylic acid-dependent, NPR1-independent signaling pathway. (+info)
CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases.
(10/132)It has previously been observed that the only specific requirement for transcriptional initiation on viral RNA in vitro by the RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus is the CCA at the 3' end of the genome. We now compare the abilities of this RdRp, turnip crinkle virus RdRp, and Qbeta replicase, an enzyme capable of supporting the complete viral replication cycle in vitro, to transcribe RNA templates containing multiple CCA boxes but lacking specific viral sequences. Each enzyme is able to initiate transcription from several CCA boxes within these RNAs, and no special reaction conditions are required for these activities. The transcriptional yields produced from templates comprised of multiple CCA or CCCA repeats relative to templates derived from native viral RNA sequences vary between 2:1 and 0.1:1 for the different RdRps. Control of initiation by such redundant sequences presents a challenge to the specificity of viral transcription and replication. We identify 3'-preferential initiation and sensitivity to structural presentation as two specificity mechanisms that can limit initiation among potential CCA initiation sites. These two specificity mechanisms are used to different degrees by the three RdRps. The finding that three viral RdRps representing two of the three supergroups within the positive-strand RNA viral RdRp phylogeny support substantial transcription in the absence of unique promoters suggests that this phenomenon may be common among positive-strand viruses. A framework is presented arguing that replication of viral RNA in the absence of unique promoter elements is feasible. (+info)
3'-End stem-loops of the subviral RNAs associated with turnip crinkle virus are involved in symptom modulation and coat protein binding.
(11/132)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. (+info)
Cell-to-cell and systemic movement of recombinant green fluorescent protein-tagged turnip crinkle viruses.
(12/132)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 DeltaCP) 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 DeltaCP 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. (+info)
Nuclear localization of turnip crinkle virus movement protein p8.
(13/132)Turnip crinkle virus (TCV) is a single-stranded positive-sense RNA virus of the Carmovirus genus. Two of its five open reading frames (ORFs), encoding proteins of 8 and 9 kDa, are required for cell-to-cell movement of the virus. These movement proteins (MPs) were fused to green fluorescent protein (GFP) to determine their cellular localization. In protoplasts, p9-GFP, like GFP itself, is found throughout the cytoplasm, as well as in cell nuclei. In contrast, p8-GFP was confined to the cell nucleus. Similar localization patterns were observed when specific small peptide epitopes were fused to p8 and p9 proteins instead of GFP. The cytoplasmic localization of p9-GFP and nuclear localization of p8-GFP were also detected in leaves after particle bombardment of DNA encoding these fusion proteins or after overexpression of p8-GFP in transgenic Arabidopsis seedlings. The expression of the GFP fusion proteins by recombinant TCV viruses in infected protoplasts or on inoculated Arabidopsis leaves produced similar patterns. Unlike TMV-MP and other MPs studied to date, no obvious punctuate expression in the cell wall or association with the cytoskeleton was detected. The sequence analysis of p8 revealed two unique nuclear localization signals (NLSs), which were not conserved within p8 homologues of other viruses in the genus Carmovirus. Mutation in either of these NLSs did not disrupt the nuclear localization of p8-GFP. However, when both NLSs were mutated, p8-GFP expression was no longer restricted to cell nuclei. The NLSs are not required for cell-to-cell movement; TCV recombinant viruses mutated in one or both NLSs could still facilitate cell-to-cell movement of the virus. The nuclear localization of p8 suggests a novel function for this protein in the cell nucleus. (+info)
Partial purification and characterization of Cucumber necrosis virus and Tomato bushy stunt virus RNA-dependent RNA polymerases: similarities and differences in template usage between tombusvirus and carmovirus RNA-dependent RNA polymerases.
(14/132)Tombusviruses are small, plus-sense, single-stranded RNA viruses of plants. RNA-dependent RNA polymerases (RdRp) of two tombusviruses, Tomato bushy stunt virus (TBSV) and Cucumber necrosis virus (CNV), have been partially purified from infected Nicotiana benthamiana plants. The obtained RdRp complexes are capable of de novo initiation of complementary RNA synthesis using either plus- or minus-strand templates derived from tombusvirus defective interfering (DI) RNAs. In addition to template-sized products, shorter than full-length products were also generated efficiently apparently because of internal initiation of RNA synthesis by the tombusvirus RdRp. This property could be important for the formation of DI RNAs that are observed in tombusvirus infections. The tombusvirus RdRp is also able to use heterologous RNAs derived from satellite RNAs associated with Turnip crinkle virus (TCV) as templates. Generation of full-length, complementary RNA by the tombusvirus RdRp suggests that it can correctly and efficiently recognize the heterologous TCV-specific promoters. Reduced generation of a 3'-terminal extension product in the preceding assay suggests that the previously characterized replication enhancer present in sat-RNA C (Nagy et al., 1999, EMBO J. 18, 5653-5665) does not stimulate tombusvirus RdRp activity. Taken together, these results suggest that template usage by the tombusvirus and carmovirus RdRps are similar, but not identical. (+info)
HRT gene function requires interaction between a NAC protein and viral capsid protein to confer resistance to turnip crinkle virus.
(15/132)An Arabidopsis protein was found to interact specifically with the capsid protein (CP) of turnip crinkle virus (TCV) through yeast two-hybrid screening. This protein, designated TIP (for TCV-interacting protein), was found to be a member of the recently recognized NAC family of proteins. NAC proteins have been implicated in the regulation of development of plant embryos and flowers. TIP alone was able to activate expression of reporter genes in yeast if fused to a DNA binding domain, suggesting that it may be a transcriptional activator. The TIP binding region in the TCV CP has been mapped to the N-terminal 25 amino acids. Site-directed mutagenesis within this region revealed that loss of the TIP-CP interaction in the yeast two-hybrid assay correlated with loss of the ability of TCV to induce the hypersensitive response and resistance in the TCV-resistant Arabidopsis ecotype Dijon (Di-0 and its inbred line Di-17). These data suggest that TIP is an essential component in the TCV resistance response pathway. (+info)
Polymerization of nontemplate bases before transcription initiation at the 3' ends of templates by an RNA-dependent RNA polymerase: an activity involved in 3' end repair of viral RNAs.
(16/132)The 3' ends of RNAs associated with turnip crinkle virus (TCV), including subviral satellite (sat)C, terminate with the motif CCUGCCC-3'. Transcripts of satC with a deletion of the motif are repaired to wild type (wt) in vivo by RNA-dependent RNA polymerase (RdRp)-mediated extension of abortively synthesized oligoribonucleotide primers complementary to the 3' end of the TCV genomic RNA. Repair of shorter deletions, however, are repaired by other mechanisms. SatC transcripts with the 3' terminal CCC replaced by eight nonviral bases were repaired in plants by homologous recombination between the similar 3' ends of satC and TCV. Transcripts with deletions of four or five 3' terminal bases, in the presence or absence of nonviral bases, generated progeny with a mixture of wt and non-wt 3' ends in vivo. In vitro, RdRp-containing extracts were able to polymerize nucleotides in a template-independent fashion before using these primers to initiate transcription at or near the 3' end of truncated satC templates. The nontemplate additions at the 5' ends of the nascent complementary strands were not random, with a preference for consecutive identical nucleotides. The RdRp was also able to initiate transcription opposite cytidylate, uridylate, guanylate, and possibly adenylate residues without exhibiting an obvious preference, flexibility previously unreported for viral RdRp. The unexpected existence of three different repair mechanisms for TCV suggests that 3' end reconstruction is critical to virus survival. (+info)