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.

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.

Isolation of a wheat cDNA clone for an abscisic acid-inducible transcript with homology to protein kinases.

Increases in the plant hormone abscisic acid (ABA) initiate water-stress responses in plants. We present evidence that a transcript with homology to protein kinases is induced by ABA and dehydration in wheat. A 1.2-kilobase cDNA clone (PKABA1) was isolated from an ABA-treated wheat embryo cDNA library by screening the library with a probe developed by polymerase chain reaction amplification of serine/threonine protein kinase subdomains VIb to VIII. The deduced amino acid sequence of the PKABA1 clone contains the features of serine/threonine protein kinases, including homology with all 12 conserved regions of the catalytic domain. PKABA1 transcript levels are barely detectable in growing seedlings but are induced dramatically when plants are subjected to dehydration stress. The PKABA1 transcript can also be induced by supplying low concentrations of ABA, and coordinate increases in ABA levels and PKABA1 mRNA occur when seedlings are water-stressed. Identification of this ABA-inducible transcript with homology to protein kinases provides a basis for examining the role of protein phosphorylation in plant responses to dehydration.

Optically pure abscisic Acid analogs-tools for relating germination inhibition and gene expression in wheat embryos.

We report an examination of the structural requirements of the abscisic acid (ABA) recognition response in wheat dormant seed embryos using optically pure isomers of ABA analogs. These compounds include permutations to the ABA structure with either an acetylene or a trans bond at C-4 C-5, and either a single or double bond at the C-2' C-3' double bond. (R)-ABA and the three isomers with the same configuration at C-1' as natural ABA were found to be effective germination inhibitors. The biologically active ABA analogs exhibited differential effects on ABA-responsive gene expression. All the ABA analogs that inhibited germination induced two ABA-responsive genes, wheat group 3 lea and dhn (rab). However, (R)-ABA and (S)-dihydroABA were less effective in inducing the ABA-responsive gene Em within the time that embryonic germination was inhibited.

Molecular cloning and expression of abscisic Acid-responsive genes in embryos of dormant wheat seeds.

Hydrated dormant cereal seeds do not germinate even when environmental conditions are favorable for germination. By using cDNA cloning and differential screening, we have identified mRNAs from five gene families that are abundant in the embryos of imbibed, but developmentally arrested wheat (Triticum aestivum L.) seeds. Gene transcript levels of these mRNAs are maintained and even increase in embryos of imbibed dormant seeds for as long as the seeds remain dormant. In contrast, transcript levels decline in nondormant seeds after imbibition and disappear as germination occurs. All the identified genes are ABA responsive. Based on these data we conclude that wheat seeds in the hydrated dormant state exhibit prolonged expression of ABA-responsive genes.