Search

The LATE EMBRYOGENESIS ABUNDANT PROTEINs (LEAPs) are a class of noncatalytic, intrinsically disordered proteins with a malleable structure. Some LEAPs exhibit a protein and/or membrane binding capacity and LEAP binding to various targets has been positively correlated with abiotic stress tolerance. Regarding the LEAPs’ presumptive role in protein protection, identifying client proteins (CtPs) to which LEAPs bind is one practicable means of revealing the mechanism by which they exert their function. To this end, we used phage display affinity selection to screen libraries derived from Arabidopsis thalianaseed mRNA with recombinant orthologous LEAPs from Arabidopsis and soybean (Glycine max). Subsequent high throughput sequencing of DNA from affinity-purified phage was performed to characterize the entire sub-population of phage retained by each LEAP orthologue. This entailed cataloging in-frame fusions, elimination of false positives, and aligning the hits on the CtP scaffold to reveal domains of respective CtPs that bound to orthologous LEAPs. This approach (Paired-end PhAge Sequencing, or PEPA-Seq) revealed a subpopulation of the proteome constituting the CtP repertoire in common between the two DHNs orthologues (LEA14 and GmPm12) compared to BSA (unrelated binding control). The veracity of LEAP:CtP binding for one of the CtPs (LEA14 and GmPM12 self-association) was independently assessed using temperature related intensity change (TRIC) analysis. Moreover, LEAP:CtP interactions for four other CtPs were confirmed in plantausing bimolecular fluorescence complementation (BiFC) assays. The results provide insights into the involvement of the DHN Y-segments and K-domains in protein binding.

Poly(A) polymerases are centrally involved in the process of mRNA 3′ end formation in eukaryotes. In animals and yeast, this enzyme works as part of a large multimeric complex to add polyadenylate tracts to the 3′ ends of precursor RNAs in the nucleus. Plant nuclear enzymes remain largely uncharacterized. In this report, we describe an initial analysis of plant nuclear poly(A) polymerases (nPAPs). An enzyme purified from pea nuclear extracts possesses many features that are seen with the enzymes from yeast and mammals. However, the pea enzyme possesses the ability to polyadenylate RNAs that are associated with polynucleotide phosphorylase (PNP), a chloroplast-localized enzyme involved in RNA turnover. Similar behavior is not seen with the yeast poly(A) polymerase (PAP). A fusion protein consisting of glutathione-S-transferase and the active domain of an Arabidopsis-encoded nuclear poly(A) polymerase was also able to utilize PNP, indicating that the activity of the pea enzyme was due to an interaction between the pea nPAP and PNP, and not to other factors that might copurify with the pea enzyme. These results suggest the existence, in plant nuclei, of factors related to PNP, and an interaction between such factors and poly(A) polymerases.

Eighty transgenic tobacco (Nicotiana tabacumL.) lines expressing either the tobacco vein mottling virus (TVMV) coat protein (CP) gene or the alfalfa mosaic virus (AMV) CP gene were evaluated for their agronomic performance with or without virus challenge. AH TVMV CP lines were characterized previously for their virus resistance following challenge with three different potyviruses. In the absence of virus challenge, the cured leaf yield of more than 80% of the TVMV CP lines was not significantly different from their nontransgenic counterparts; half of the poor performing lines were in one of the five genetic backgrounds. Similarly, most of the negative variation for yield among the AMV CP transgenic lines was within a single genetic background. Variation for other agronomic traits, including plant height, leaf size, and stalk diameter closely followed the results for yield. Following virus challenge, cured leaf yield of the most resistant transgenic lines was similar to the non‐challenged treatment. Among the AMV CP transgenic lines, yield loss following challenge with AMV was reduced from 22 to 30%. These results suggest that the primary selection criterion in a population of transgenic tobacco lines containing a viral CP gene should be for virus resistance, but it must be recognized that variation for agronomic performance may exist among the resistant selections, particularly in some genetic backgrounds.

We have introduced chimeric genes containing polyadenylation signals from a human gene and two animal virus genes into tobacco cells. We see, in all three cases, inefficient and ‘aberrant’ utilization of the foreign polyadenylation signals. We find that a chimeric gene carrying the polyadenylation site of the human growth hormone gene is polyadenylated at three sites in the vicinity of the site that is polyadenylated in human cells. A chimeric gene containing the polyadenylation site from the adenovirus 5 E1A gene is polyadenylated at a site 11 bases downstream from that reported in animal cells. A gene carrying the polyadenylation site from the SV40 early region is polyadenylated some 80 bases upstream from the site that is polyadenylated in animal cells. In all three cases, related mRNAs ending at flanking ‘authentic’ plant polyadenylation sites can be detected, indicating that the foreign polyadenylation signals are inefficiently utilized in tobacco cells.

Mutants of tobacco vein mottling virus (TVMV) were constructed in which the tyrosine residue (Tyr1860) that links the VPg to the viral RNA was changed to phenylalanine or serine or was inverted in position with the adjacent glycine residue. In another mutant, the tyrosine residue nearest to Tyr1860 (Tyr1867) was changed to a phenylalanine residue. The resulting mutants were tested for their ability to infectNicotiana tabacumplants or protoplasts. The Tyr1860 mutants did not accumulate to detectable levels in infected plants when tested by ELISA and Northern blot analysis. Moreover, the Tyr1860-associated mutants were not infectious in protoplasts, indicating that mutations involving the linking amino acid of the TVMV VPg abolished viral replication. In contrast to the Tyr1860 mutants, transcripts from the mutation of Tyr1867 to a phenylalanine residue infected both protoplasts and plants. Analysis of progeny RNA from plants inoculated with the Tyr1867 mutant indicated that a reversion to wild type had occurred in systemically infected leaves.

We have expressed the putative RNA-dependent RNA polymerase encoded by the potyvirus tobacco vein mottling virus (TVMV) inEscherichia colias a glutathioneS-transferase fusion protein. As prepared, the fusion protein possessed the poly(U) polymerase activity that is a hallmark of other picornavirus-encoded polymerases. In addition, this protein was able to utilize full-length TVMV RNA as a template for RNA synthesis. A fusion protein containing a mutation in the highly conserved GDD motif of the polymerase (GDD → ADD) possessed 7% of the activity of the wild type. Our results confirm that the presumed polymerase encoded by TVMV is in fact an RNA-dependent RNA polymerase and that the GDD motif so widely seen in viral polymerases has an important function in the TVMV protein.

The interaction of potato virus X (PVX) and potato virus Y (PVY) in tobacco causes a synergistic disease characterizedby a dramatic increase in symptom severity, a change in the regulation of PVX RNA replication, and an increase in accumulation of PVX. In this study we demonstrate that PVX also interacts synergistically with three other members of the potyvirus group of plant viruses, tobacco vein mottling virus (TVMV), tobacco etch virus (TEV), and pepper mottle virus. These synergisms resemble the classic PVX/PVY synergism with respect to both the increase in host response and the change in PVX replication. To determine if the induction of PVX/potyviral synergism requires potyviral genome replication per se or if the response is mediated by expression of one or more potyviral genes, we used tobacco plants stably transformed with various subsets of the TVMV genome. PVX infections of transgenic plants expressing the 5′-proximal region of the TVMV genome, including the protease-1, helper component protease, and protein-3 genes, result in symptoms resembling those of PVX/potyviral synergism. A similar synergistic-like response occurs when transgenic tobacco plants expressing the analogous but smaller region from the 5′-proximal region of the TEV genome were infected with PVX. Replication of PVX RNA is altered in transgenic plants expressing 5′-proximal sequences of either TVMV or TEV, and in a manner similar to that observed in double infections. These results indicate that replication of the potyviral genome is not required for PVX/potyviral synergism and that the response is mediated by expression of potyviral sequences which have been localized to the 5′-proximal third of the genomic RNAs of both TVMV and TEV.

The two-hybrid system was used to test for pairwise interactions between the tobacco vein mottling virus (TVMV)-encoded RNA-dependent RNA polymerase (or NIb protein) and two other TVMV-encoded proteins: the NIa protein, which consists of genome-linked protein (VPg) and proteinase domains, and the viral coat protein (CP). Using this approach, we find that the NIb protein interacts with both the NIa protein and the CP in yeast cells. Moreover, we find that a mutation in the conserved GDD domain of the NIb protein diminishes the NIb–CP interaction but not the NIb–NIa interaction. Likewise, mutations in the vicinity of the NIa protein to which the genomic RNA is covalently attached eliminate the NIb–NIa interaction. We conclude that the NIb protein interacts with the VPg domain of the NIa protein and that this interaction requires a functional RNA attachment site. This interaction may be important for the initiation of viral RNA synthesis in infected cells. We also conclude that the CP interacts with the NIb in a manner that is sensitive in changes in the highly conserved GDD motif. The role of this interaction in the functioning of the NIb protein or the CP is unclear, but may involve regulation of viral RNA synthesis in infected cells.

We have purified a novel factor (PAP-III) that is a component of a multisubunit poly(A) polymerase from pea seedlings. This factor consists of one or more polypeptides with molecular masses of about 105 kDa and of a population of associated RNAs that can serve as substrates for polyadenylation. When these RNAs are separated from the 105-kDa polypeptides, polyadenylation becomes dependent upon exogenously added RNA. This RNA-dependent activity does not require the presence of a polyadenylation signal in the substrate, indicating that the activity under study is a nonspecific polyadenylation activity. One or more of the 105-kDa polypeptides could be cross-linked to the products of polyadenylation labeled with [α-32P]ATP and to exogenously added labeled RNAs. Cross-linking of the 105-kDa polypeptides to the products of polyadenylation was not affected by the presence of exogenously added competitors, whereas cross-linking to exogenous RNAs was diminished by excesses of RNA homopolymers. Exogenous RNAs could be polyadenylated by the combination of PAP-I + PAP-III, and this activity was diminished if the binding of the exogenous RNAs to PAP-III was prevented. We conclude from these studies that PAP-III is an RNA binding protein, that polyadenylation by the poly(A) polymerase occurs while the substrate RNAs are associated with this protein, and that the pea poly(A) polymerase can only polyadenylate those RNAs that are associated with PAP-III.

The genome‐linked protein‐proteinase (NIa) genes of three potyviruses (tobacco vein mottling virus[TVMV], tobacco etch virus[TEV], and potato virus Y [PVY]) were introduced into tobacco (Nicotiana tabacumL.) cultivar Burley 21 and transgenic plant lines were evaluated for viral symptoms and virus accumulation. Plant lines that expressed the TVMV NIa gene were resistant to infection by TVMV; inoculated plants did not develop disease symptoms after inoculation, and virus did not accumulate. Most lines that carried the TEV NIa and PVY NIa genes did develop symptoms after inoculation with TEV and PVY, respectively, but these symptoms abated with time (a response termed as recovery). Three genes, each consisting of two NIa coding regions (TEV NIa‐PVY NIa, TEV NIa‐TVMV NIa, and TVMV NIa‐PVY NIa) were also introduced into Burley 21. Transgenic lines that carried either TEV NIa‐PVY NIa or TEV NIa‐TVMV NIa constructs either did not develop disease symptoms or exhibited the process of recovery after inoculation with TEV. TEV NIa‐TVMV NIa and TVMV NIa‐PVY NIa lines recovered from an initial infection after inoculation with TVMVL. Likewise, TEV NIa‐PVY NIa and TVMV NIa‐PVY NIa transformed lines recovered from an initial infection after inoculation with PVY. These results demonstrate that different potyvirus NIa‐proteinase genes can be used for protection against virus infection and that multiple NIa genes may be used to produce plants that are protected against more than one potyvirus.

There are no known genetic sources of resistance to alfalfa mosaic virus (AMV) in the genus Nicotiana. In this communication, we describe how we genetically engineered an AMV (strain 425) coat protein (CP) gene into three commercial burley tobacco (N. tabacumL.) genotypes, BY 21, TN 86, and KY 8959, and the evaluation of the transgenic lines in greenhouse and field experiments. In a replicated greenhouse trial, 11 AMV‐CP transgenic lines in a BY 21 background were resistant to two AMV isolates, AMV‐425 and AMV‐KY, but the lines were less resistant to another isolate, AMV‐NC. In a 1994 field experiment, we found that nine BY 21 AMV‐CP transgenic lines were resistant to AMV‐KY when inoculated both before and after transplanting. The level of resistance varied among the nine lines. In 1995, 10 randomly selected AMV‐CP lines in each of the three genetic backgrounds were evaluated in a replicated field trial. Mean infection scores of each set of lines were significantly less than those of their non‐transgenic counterparts, but there were no significant differences among these three mean infection scores. However, there were significant differences among lines within a genetic background for their response to AMV infection. High sensitivity to AMV infection, found in genotypes TN 86 and KY 8959 that possess an endogenous gene for resistance to potyviruses, was not evident in their AMV‐CtrPa nsgenic lines. The CP mediated resistance to AMV was effective in reducing viral symptoms in the field and it may provide a valuable source for AMV resistance in tobacco.

A plant polyadenylation signal consists of three distinct components: a far-upstream element (FUE) that can control utilization of several polyadenylation sites, one or more near-upstream elements (NUEs) that control utilization of each site in a transcription unit, and polyadenylation site (CSs) themselves. NUEs have previously been suggested to be related to the mammalian polyadenylation signal AAUAAA. However, many plant genes do not contain AAUAAA-like motifs near their polyadenylation sites. To better understand the nature of NUEs, we conducted a systematic analysis of the NUE for one polyadenylation site (site 1) in the pea rbcS-E9 gene; this NUE lacks an AAUAAA motif. Linker substitution studies showed that the NUE for site 1 in this gene resides in the sequence AAAUGGAAA. Single-nucleotide substitutions in this domain had modest effects on the functioning of this NUE. Replacement of part of this sequence with the sequence AAUAAA increased the efficiency of this NUE. However, alteration of nucleotides immediately 3' of the AAUAAA reversed this effect. Our results indicate that the NUE for site 1 consists of as many as 9 nucleotides, that these 9 bases do not include an element that is intolerant of single base changes, that the sequence AAUAAA can function as a NUE for site 1, and that sequences flanking AAUAAA can affect the efficiency of functioning as a NUE.

We have introduced the SV40 small t intron into tobacco cells as part of a cauliflower mosaic virus 35S promoter-chloramphenicol acetyltransferase-SV40 transcription unit. We find that the small t intron is efficiently and accurately spliced in transgenic tobacco cells that carry this transcription unit. Our results indicate that there is substantial conservation of RNA processing signals between plants and animals, more than has been previously assumed. They also suggest that pre-mRNA processing in plants requires multiple branch sites for efficient processing.

We have used a cDNA clone of the polyvirus, tobacco vein mottling virus, to construct 19 mutants bearing 12-nt insertions in the viral genome. These mutants display a variety of phenotypes in inoculated tobacco plants or protoplasts. All mutants with insertions in P3, Cl, 6K, Nla, or Nlb failed to produce detectable amounts of progeny viral RNA in protoplasts or plants which suggests that they all may be directly involved in replication. Mutants (one in P1 and one in HCpro) presumably affected in polyprotein processing also did not replicate in plants or protoplasts. Seven mutants, with insertions in the 5′ noncoding region, P1, HCpro, or CP regions of the genome, were able to infect protoplasts. Three of the 7 mutants (1 in the 5′ noncoding region and 2 in HCpro) were able to infect protoplasts but not plants. The remaining 4 mutants replicated in protoplasts and were able to cause systemic infection in plants. The mutation in the CP had no effect on virus accumulation or symptom development in inoculated plants, whereas the other 3 (1 in P1 and 2 in HCpro) induced cyclical patterns of symptom expression. These symptoms ranged from very mild to wild-type-like as new leaves emerged and, as the plants continued to grow, this pattern was repeated. These results support the assignment of roles in replication to five coding regions in the genome and demonstrate that sequence alterations in many parts of other regions of the viral genome may have pronounced effects on replication and the expression of disease symptoms.

We have characterized the polyadenylation signal from the octopine synthase (ocs) gene. This signal directs mRNA 3′ end formation at a number of distinct sites. A combination of deletion and linker-substitution analyses revealed that each of these sites is controlled by multiple upstream sequence elements. Upstream sequences relatively far (>80 nt) from the ocs poly[A] sites were found to be needed for functioning of these sites. Upstream sequences nearer to poly[A] sites were also found to be involved in mRNA 3′ end formation in the ocs gene. In addition, a set of novel elements that mediates 3′ end choice was uncovered by deletion analysis of sequences downstream from the ocs polyadenylation sites. Our experiments indicate mRNA 3′ end formation in the ocs is controlled by a complex series of cis-acting signals, and suggest that the process of mRNA 3′ end formation might be linked to transcription termination.