Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of tobacco etch virus.

Restriction of long-distance movement of tobacco etch virus (TEV) in Arabidopsis ecotype Col-0 plants requires the function of at least three genes: RTM1 (restricted TEV movement 1), RTM2, and RTM3. The mechanism of TEV movement restriction remains poorly understood, although it does not involve a hypersensitive response or systemic acquired resistance. A functional characterization of RTM1 and RTM2 was done. The RTM1 protein was found to be soluble with the potential to form self-interacting complexes. The regulatory regions of both the RTM1 and RTM2 genes were analyzed using reporter constructs. The regulatory sequences from both genes directed expression of beta-glucuronidase exclusively in phloem-associated cells. Translational fusion proteins containing the green fluorescent protein and RTM1 or RTM2 localized to sieve elements when expressed from their native regulatory sequences. Thus, components of the RTM system may function within phloem, and sieve elements in particular, to restrict TEV long-distance movement.

Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system.

The Agrobacterium-mediated transient expression assay in intact tissues has emerged as a rapid and useful method to analyze genes and gene products in plants. In many cases, high levels of active protein can be produced without the need to produce transgenic plants. In this study, a series of tools were developed to enable strong or weak induction of RNA silencing and to suppress RNA silencing in the absence of stable transgenes. Transient delivery of a gene directing production of a double-stranded green fluorescent protein (GFP) transcript rapidly induced RNA silencing of a codelivered GFP reporter gene, effectively preventing accumulation of GFP protein and mRNA. RNA silencing triggered by the strong dsGFP inducer was partially inhibited by the tobacco etch virus silencing suppressor, P1/HC-Pro. In the absence of the strong double-stranded GFP inducer, the functional GFP gene served as a weak RNA silencing inducer in the transient assay, severely limiting accumulation of the GFP mRNA over time. The weak silencing induced by the GFP gene was suppressed by P1/HC-Pro. These results indicate RNA silencing can be triggered by a variety of inducers and analyzed entirely using transient gene delivery systems. They also indicate that RNA silencing may be a significant limitation to expression of genes in the Agrobacterium-mediated transient assay but that this limitation can be overcome by using RNA silencing suppressors.

Long-distance movement and replication maintenance functions correlate with silencing suppression activity of potyviral HC-Pro.

The tobacco etch potyviral protein, HC-Pro, is a multifunctional proteinase required for long-distance movement in plants and maintenance of genome replication at the single-cell level. It also functions in a counterdefensive capacity as a suppressor of posttranscriptional gene silencing (PTGS). To determine whether the requirements for HC-Pro during long distance movement and replication maintenance are due to the silencing suppressor function of the protein, a series of HC-Pro alanine scanning and other site-directed mutants were analyzed. Using a transient silencing suppression assay in Agrobacterium-injected leaf tissue, several suppression-defective mutants were identified. Each of six HC-Pro mutations, which were shown previously to confer long-distance movement and replication maintenance defects, conferred PTGS suppression defects. Interestingly, the genes encoding these defective HC-Pro derivatives were themselves susceptible targets of PTGS, resulting in low levels of mRNA and protein accumulation. Mutations that inactivated the proteinase domain active site had no effect on PTGS suppression function. The results are consistent with the hypothesis that the role of HC-Pro in long-distance movement and genome replication depends on PTGS suppression function and that this function is independent of HC-Pro proteolytic activity.

Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway.

Certain plant viruses encode suppressors of posttranscriptional gene silencing (PTGS), an adaptive antiviral defense response that limits virus replication and spread. The tobacco etch potyvirus protein, helper component-proteinase (HC-Pro), suppresses PTGS of silenced transgenes. The effect of HC-Pro on different steps of the silencing pathway was analyzed by using both transient Agrobacterium tumefaciens-based delivery and transgenic systems. HC-Pro inactivated PTGS in plants containing a preexisting silenced beta-glucuronidase (GUS) transgene. PTGS in this system was associated with both small RNA molecules (21-26 nt) corresponding to the 3' proximal region of the transcribed GUS sequence and cytosine methylation of specific sites near the 3' end of the GUS transgene. Introduction of HC-Pro into these plants resulted in loss of PTGS, loss of small RNAs, and partial loss of methylation. These results suggest that HC-Pro targets a PTGS maintenance (as opposed to an initiation or signaling) component at a point that affects accumulation of small RNAs and methylation of genomic DNA.

Strain-specific interaction of the tobacco etch virus NIa protein with the translation initiation factor eIF4E in the yeast two-hybrid system.

The NIa protein of potyviruses provides VPg and proteolytic functions during virus replication. It has also been shown to confer host genotype-specific movement functions in plants. Specifically, NIa from tobacco etch virus (TEV)-Oxnard, but not from most other strains, confers the ability to move long distances in Nicotiana tabacum cultivar "V-20." This led to the hypothesis that all or part of NIa may interact with one or more cellular factors. To identify cellular proteins that interact with NIa in a host- or strain-specific manner, a yeast two-hybrid search of a tomato cDNA library was done. Ten proteins that interacted with NIa were recovered, with translation initiation factor eIF4E being by far the most common protein identified. Interaction of eIF4E with NIa was shown to be TEV strain-specific. eIF4E from both tomato and tobacco interacted well with NIa from the HAT strain, but not from the Oxnard strain. However, using chimeric NIa proteins, the determinant for systemic infection of V20 plants was found to be genetically distinct from the determinant controlling eIF4E interaction. In TEV-eIF4E coexpression experiments, evidence suggesting that eIF4E provides a positive effect on genome amplification was obtained.

Arabidopsis RTM2 gene is necessary for specific restriction of tobacco etch virus and encodes an unusual small heat shock-like protein.

Arabidopsis plants have a system to specifically restrict the long-distance movement of tobacco etch potyvirus (TEV) without involving either hypersensitive cell death or systemic acquired resistance. At least two dominant genes, RTM1 and RTM2, are necessary for this restriction. Through a series of coinfection experiments with heterologous viruses, the RTM1/RTM2-mediated restriction was shown to be highly specific for TEV. The RTM2 gene was isolated by a map-based cloning strategy. Isolation of RTM2 was confirmed by transgenic complementation and sequence analysis of wild-type and mutant alleles. The RTM2 gene product is a multidomain protein containing an N-terminal region with high similarity to plant small heat shock proteins (HSPs). Phylogenetic analysis revealed that the RTM2 small HSP-like domain is evolutionarily distinct from each of the five known classes of plant small HSPs. Unlike most other plant genes encoding small HSPs, expression of the RTM2 gene was not induced by high temperature and did not contribute to thermotolerance of seedlings. The RTM2 gene product was also shown to contain a large C-terminal region with multiple repeating sequences.

Cloning of the Arabidopsis RTM1 gene, which controls restriction of long-distance movement of tobacco etch virus.

The locus RTM1 is necessary for restriction of long-distance movement of tobacco etch virus in Arabidopsis thaliana without causing a hypersensitive response or inducing systemic acquired resistance. The RTM1 gene was isolated by map-based cloning. The deduced gene product is similar to the alpha-chain of the Artocarpus integrifolia lectin, jacalin, and to several proteins that contain multiple repeats of a jacalin-like sequence. These proteins comprise a family with members containing modular organizations of one or more jacalin repeat units and are implicated in defense against viruses, fungi, and insects.

Functional analysis of the interaction between VPg-proteinase (NIa) and RNA polymerase (NIb) of tobacco etch potyvirus, using conditional and suppressor mutants.

The tobacco etch potyvirus (TEV) RNA-dependent RNA polymerase (NIb) has been shown to interact with the proteinase domain of the VPg-proteinase (NIa). To investigate the significance of this interaction, a Saccharomyces cerevisiae two-hybrid assay was used to isolate conditional NIa mutant proteins with temperature-sensitive (ts) defects in interacting with NIb. Thirty-six unique tsNIa mutants with substitutions affecting the proteinase domain were recovered. Most of the mutants coded for proteins with little or no proteolytic activity at permissive and nonpermissive temperatures. However, three mutant proteins retained proteolytic activity at both temperatures and, in two cases (tsNIa-Q384P and tsNIa-N393D), the mutations responsible for the ts interaction phenotype could be mapped to single positions. One of the mutations (N393D) conferred a ts-genome-amplification phenotype when it was placed in a recombinant TEV strain. Suppressor NIb mutants that restored interaction with the tsNIa-N393D protein at the restrictive temperature were recovered by a two-hybrid selection system. Although most of the suppressor mutants failed to stimulate amplification of genomes encoding the tsNIa-N393D protein, two suppressors (NIb-I94T and NIb-C380R) stimulated amplification of virus containing the N393D substitution by approximately sevenfold. These results support the hypothesis that interaction between NIa and NIb is important during TEV genome replication.

Selectable viruses and altered susceptibility mutants in Arabidopsis thaliana.

The genetic basis for susceptibility or nonsusceptibility of plants to viruses is understood poorly. Two selectable tobacco etch virus (TEV) strains were developed for identification of Arabidopsis thaliana mutants with either gain-of-susceptibility or loss-of-susceptibility phenotypes. These strains conferred a conditional-survival phenotype to Arabidopsis based on systemic expression of herbicide resistance or proherbicide sensitivity genes, thereby facilitating mass selections and screens for Arabidopsis mutants that enhance or suppress TEV replication, cell-to-cell movement, or long-distance movement. A multicomponent mechanism that restricts systemic invasion of TEV was identified through isolation of gain-of-susceptibility mutants with alterations at two loci.

A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing.

Posttranscriptional gene silencing (PTGS) in plants inactivates some aberrant or highly expressed RNAs in a sequence-specific manner in the cytoplasm. A silencing mechanism similar to PTGS appears to function as an adaptive antiviral response. We demonstrate that the P1/HC-Pro polyprotein encoded by tobacco etch virus functions as a suppressor of PTGS. A locus comprised of a highly expressed beta-glucuronidase (GUS) transgene was shown to exhibit PTGS. Genetic crosses and segregation analyses revealed that a P1/ HC-Pro transgene suppressed PTGS of the GUS sequence. Nuclear transcription assays indicated that the silencing suppression activity of P1/HC-Pro was at the posttranscriptional level. These data reveal that plant viruses can condition enhanced susceptibility within a host through interdiction of a potent defense response.

Genetic evidence for an essential role for potyvirus CI protein in cell-to-cell movement.

The potyvirus cylindrical inclusion (CI) protein, an RNA helicase required for genome replication, was analyzed genetically using alanine-scanning mutagenesis. Thirty-one mutations were introduced into the CI protein coding region of modified tobacco etch virus (TEV) genomes expressing either beta-glucuronidase or green fluorescent protein reporters. Twelve of the mutants were replication-defective in protoplast inoculation assays. Among the 19 replication-competent mutants, several possessed cell-to-cell or long-distance movement defects in tobacco plants. Two mutants, AS1 and AS8, were restricted to single cells in inoculated leaves despite genome amplification levels that were equivalent to that of parental virus. Other mutants, such as AS9 and AS14, were able to move cell to cell slowly but were debilitated in long-distance movement. These data provide genetic evidence for a direct role of CI protein in potyvirus intercellular movement, and for distinct roles of the CI protein in genome replication and movement. In combination with high-resolution ultrastructural analyzes and previous genetic data, these results support a model in which CI protein interacts directly with plasmodesmata and capsid protein-containing ribonucleoprotein complexes to facilitate potyvirus cell-to-cell movement.

Identification and characterization of a locus (RTM1) that restricts long-distance movement of tobacco etch virus in Arabidopsis thaliana.

Screens of Arabidopsis thaliana for susceptibility to tobacco etch virus (TEV) revealed that each of 10 ecotypes were able to support genome replication and cell-to-cell movement in inoculated leaves. However, only four ecotypes, including C24 and La-er, supported complete infections in which TEV was able to replicate and move from cell to cell and long distances through the vasculature. The rates of cell-to-cell movement of a reporter-tagged TEV strain (TEV-GUS) in inoculated leaves of C24 and Columbia (Col-3) were similar, and infection foci continued to expand in both ecotypes through 10 days post-inoculation. No visible or microscopic hypersensitive or cell death responses were evident in inoculated leaves of Col-3 plants. Infection of neither C24 nor Col-3 plants with TEV-GUS resulted in induction of PR-1a gene expression, which is normally associated with active defence responses and systemic acquired resistance. The genetic basis for the restriction of long-distance movement of TEV-GUS in Columbia was investigated using C24 x Col-3 crosses and backcrosses and using La-er x Col-0 recombinant inbred lines. A dominant locus conditioning the restricted TEV infection phenotype was identified on chromosome 1 between markers ATEAT1 and NCC1 at approximately 14 cM in both genetic analyses. This locus was designated RTM1 (restricted TEV movement 1). It is proposed that RTM1 mediates a restriction of long-distance movement through a mechanism that differs substantially from those conditioned by the dominant resistance genes normally associated with gene-for-gene interactions.

Secondary structures in the capsid protein coding sequence and 3' nontranslated region involved in amplification of the tobacco etch virus genome.

The 3'-terminal 350 nucleotides of the tobacco etch potyvirus (TEV) genome span the end of the capsid protein (CP)-coding sequence and the 3' nontranslated region (NTR). The CP-coding sequence within this region contains a 105-nucleotide cis-active element required for genome replication (S. Mahajan, V. V. Dolja, and J. C. Carrington, J. Virol. 70:4370-4379, 1996). To investigate the sequence and secondary structure requirements within the CP cis-active region and the 3' NTR, a systematic linker-scanning mutagenesis analysis was done. Forty-six mutations, each with two to six nucleotide substitutions, were introduced at consecutive hexanucleotide positions in the genome of a recombinant TEV strain expressing a reporter protein (beta-glucuronidase). Genome amplification activity of each mutant in the protoplast cell culture system was measured. Mutations that severely debilitated genome amplification were identified throughout the CP-coding cis-active sequence and at several distinct locations within the 3' NTR. However, based on a computer model of RNA folding, mutations that had the most severe effects mapped to regions that were predicted to form base-paired secondary structures. Linker-scanning mutations predicted to affect either strand of a base-paired structure within the CP-coding cis-active sequence, a base-paired structure between two segments of the CP-coding cis-active sequence and a contiguous 14-nucleotide segment of the 3' NTR, and a base-paired structure near the 3' terminus of the 3' NTR inactivated genome amplification. Compensatory mutations that restored base pair interactions in each of these regions restored amplification activity, although to differing levels depending on the structure restored. These data reveal that the 3' terminus of the TEV genome consists of a series of functionally discrete sequences and secondary structures and that the CP-coding sequence and 3' NTR are coadapted for genome amplification function through a requirement for base pair interactions.

Viral invasion and host defense: strategies and counter-strategies.

The outcome of infection of plants by viruses is determined by the net effects of compatibility functions and defense responses. Recent advances reveal that viruses have the capacity to modulate host compatibility and defense functions by a variety of mechanisms.