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.

Tobacco mosaic virus 126-kDa protein increases the susceptibility of Nicotiana tabacum to other viruses and its dosage affects virus-induced gene silencing.

The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein-green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.

The Fed-1 (CAUU)4 element is a 5' UTR dark-responsive mRNA instability element that functions independently of dark-induced polyribosome dissociation.

Darkness rapidly induces a decline in the stability and translation of the pea Ferredoxin-1 (Fed-1) mRNA in transgenic tobacco. Direct half-life measurement showed that mutation of the (CAUU)4 stabilizes Fed-1 mRNA in the dark. (CAUU)1, a feature more common in plant 5' UTRs than (CAUU)4, confers slight light-responsive mRNA accumulation. At least three but less than 11 CAUU repeats near the 5' end of the 5' UTR are required for full light-responsive accumulation. Furthermore, 26 nt of the 5' UTR, including the (CAUU)4 repeat, is sufficient to confer a significant approximately 2.5-fold increase in light-regulated mRNA accumulation when fused to the 5' end of a heterologous plant mRNA. A mutation of the (CAUU)4 repeat that compromises light-regulated mRNA stability changes in vitro the accessibility of the region to ribonuclease V1 and ribonuclease A suggesting the geometry formed by the repeat may be important for instability. Finally, dark-induced Fed-1 mRNA instability occurs even when most of the mRNA is retained on polyribosomes, and thus is likely an independent event regulated by darkness.

Light control of nuclear gene mRNA abundance and translation in tobacco.

Photosynthetic signals modulate expression of nuclear genes at the levels of mRNA transcription, mRNA stability, and translation. In transgenic tobacco (Nicotiana tabacum), the pea (Pisum sativum) Ferredoxin 1 (Fed-1) mRNA dissociates from polyribosomes and becomes destabilized when photosynthesis is inhibited by photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We used polymerase chain reaction suppressive-subtractive hybridization to identify similarly regulated endogenous tobacco genes. This screen identified 14 nuclear-encoded tobacco mRNAs whose light-induced increase in abundance is suppressed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Sequence analysis of the cognate cDNAs revealed that nine of the mRNAs encode putative chloroplast-targeted proteins. We asked whether the abundance of these mRNAs was regulated transcriptionally or posttranscriptionally. Of the five mRNAs with sufficient abundance to detect using nuclear run-on assays, we observed transcriptional regulation of alpha-tubulin, thiazole biosynthetic enzyme, and pSKA10 (an unknown gene). Photosystem A subunit L and, to a lesser extent, alpha-tubulin and pSKA10 mRNAs, may also be stabilized in the light. In contrast, Rubisco small subunit mRNA abundance appears to be transcriptionally up-regulated but posttranscriptionally down-regulated in the light. To determine whether, like Fed-1 mRNA, the mRNAs identified in this screen were translationally responsive to light, we characterized the polyribosome association of these mRNAs in the light and after a 15-min dark treatment. A subset of the mRNAs showed dramatic dark-induced polyribosome dissociation, similar to Fed-1 mRNA, and all of the mRNAs showed at least slight polyribosome dissociation. Thus, both posttranscriptional and translational regulation appear to be important mechanisms regulating the expression of many nuclear-encoded mRNAs encoding proteins involved in photosynthesis.