A conserved hairpin structure in Alfamovirus and Bromovirus subgenomic promoters is required for efficient RNA synthesis in vitro.

The coat protein gene in RNA 3 of alfalfa mosaic virus (AMV; genus Alfamovirus, family Bromoviridae) is translated from the subgenomic RNA 4. Analysis of the subgenomic promoter (sgp) in minus-strand RNA 3 showed that a sequence of 37 nt upstream of the RNA 4 start site (nt +1) was sufficient for full sgp activity in an in vitro assay with the purified viral RNA-dependent RNA-polymerase (RdRp). The sequence of nt -6 to -29 could be folded into a potential hairpin structure with a loop represented by nt -16, -17, and -18, and a bulge involving nt -23. By introducing mutations that disrupted base pairing and compensatory mutations that restored base pairing, it was shown that base pairing in the top half of the putative stem (between the loop and bulge) was essential for sgp activity, whereas base pairing in the bottom half of the stem was less stringently required. Deletion of the bulged residue A-23 or mutation of this residue into a C strongly reduced sgp activity, but mutation of A-23 into U or G had little effect on sgp activity. Mutation of loop residues A-16 and A-17 affected sgp activity, whereas mutation of U-18 did not. Using RNA templates corresponding to the sgp of brome mosaic virus (BMV; genus Bromovirus, family Bromoviridae) and purified BMV RdRp, evidence was obtained indicating that also in BMV RNA a triloop hairpin structure is required for sgp activity.

Nematode transmission of tobacco rattle virus serves as a bottleneck to clear the virus population from defective interfering RNAs.

DI7 is a defective interfering RNA derived from RNA 2 of tobacco rattle tobravirus (TRV) isolate PpK20. Tobacco was transformed with DI7 cDNA fused to the CaMV 35S promoter. Upon infection of the transgenic plants with TRV isolate PpK20 or the serologically unrelated isolate PaY4, the transgenic DI7 RNA started to accumulate at high levels and strongly interfered with accumulation of wild-type (wt) RNA 2. When DI7 transgenic plants infected with isolate PpK20 were used as source plants in nematode-transmission experiments, the vector Paratrichodorus pachydermus efficiently transmitted virus to healthy bait plants. However, the nematodes transmitted only the wt virus present in the transgenic source plants, whereas virus particles containing the abundant, accumulated DI7 RNA were excluded from transmission. Evidence is presented that wt RNA 2 and DI7 RNA are encapsidated in cis by their encoded CPs, which are known to be functional and nonfunctional in transmission, respectively. This mechanism would result in defective interfering RNAs, which rapidly arise after mechanical transmission of the virus in the laboratory, being eliminated from tobraviruses under natural field conditions. Also this mechanism which acts with nematode transmitted virus isolates contrasts with that of vector-transmission of defective potyviruses and luteoviruses by wt helper viruses.

A conformational switch at the 3' end of a plant virus RNA regulates viral replication.

3' untranslated regions of alfamo- and ilar-virus RNAs fold into a series of stem-loop structures to which the coat protein binds with high affinity. This binding plays a role in initiation of infection ('genome activation') and has been thought to substitute for a tRNA-like structure that is found at the 3' termini of related plant viruses. We propose the existence of an alternative conformation of the 3' ends of alfamo- and ilar-virus RNAs, including a pseudoknot. Based on (i) phylogenetic comparisons, (ii) in vivo and in vitro functional analyses of mutants in which the pseudoknot has been disrupted or restored by compensatory mutations, (iii) competition experiments between coat protein and viral replicase, and (iv) investigation of the effect of magnesium, we demonstrate that this pseudoknot is required for replication of alfalfa mosaic virus. This conformation resembles the tRNA-like structure of the related bromo- and cucumo-viruses. A low but specific interaction with yeast CCA-adding enzyme was found. The existence of two mutually exclusive conformations for the 3' termini of alfamo- and ilar-virus RNAs could enable the virus to switch from translation to replication and vice versa. The role of coat protein in this modulation and in genome activation is discussed.

Mutation of GT-1 binding sites in the Pr-1A promoter influences the level of inducible gene expression in vivo.

Infection of Nicotiana tabacum Samsun NN with tobacco mosaic virus (TMV) results in a hypersensitive plant response and leads to systemic acquired resistance (SAR). The induction of SAR is mediated by the plant hormone salicylic acid (SA) and is accompanied by the induced expression of a number of genes including the pathogenesis-related (PR) gene 1a. Previously, it has been found that TMV infection and SA treatment resulted in a reduction of binding of nuclear protein GT-1 to far-upstream regions (-902 to -656) of the PR-1a gene. To test if GT-1 is a negative regulator of PR-1a gene expression, the effects of mutations in the seven putative GT-1 binding sites in this region were studied in vitro using dimethyl sulfate interference footprinting and band shift assays. This showed that at least one of the seven sites is indeed a GT-1 binding site. However, when tested in transgenic plants, the mutations did not result in constitutive expression of the chimeric PR-1a/GUS transgene, while inducible expression after SA treatment was decreased. The results suggest that binding of GT-1-like proteins to far-upstream PR-1a promoter regions indeed influences gene expression. A possible model for GT-1's mode of action in PR-1a gene expression is discussed.

The 3' untranslated region of alfalfa mosaic virus RNA3 contains a core promoter for minus-strand RNA synthesis and an enhancer element.

The 3' untranslated regions (UTRs) of the three genomic RNAs of alfalfa mosaic virus consist of a 3' homologous sequence of 145 nt and upstream unique sequences 18-34 nt in length. Mutations were made in the 3' UTR of a cDNA clone of RNA3. Point mutations in five AUGC motifs which interfere with specific binding of coat protein to the 3' UTR had no effect on template activity of RNA3 for minus-strand RNA synthesis in vitro by purified viral RNA-dependent RNA polymerase (RdRp). Deletion analysis showed that the 3' homologous sequence of 145 nt was sufficient for a low level of template activity in the in vitro RdRp assay and a similarly low level of RNA3 accumulation in plants. The presence of an additional sequence of nucleotides 145-165 from the 3' end of RNA3 enhanced template recognition by RdRp in vitro and accumulation of RNA3 in vivo to wild-type levels.

Mutations in coat protein binding sites of alfalfa mosaic virus RNA 3 affect subgenomic RNA 4 accumulation and encapsidation of viral RNAs.

The 3'-untranslated regions (3'-UTRs) of the three RNAs of alfalfa mosaic virus (AMV) contain a specific binding site for coat protein (CP) and act as a promoter for minus-strand RNA synthesis by the purified AMV RNA-dependent RNA polymerase (RdRp) in an in vitro assay. Binding of CP to the viral RNAs is required to initiate infection. The sequence of the 3'-terminal 39 nucleotides of AMV RNA 3 can be folded into two stem-loop structures flanked by three single-stranded AUGC sequences and represents a CP binding site. Mutations in this sequence that are known to interfere with CP binding in vitro were introduced into an infectious clone of RNA 3, and mutant RNA transcripts were used as templates in the in vitro RdRp assay and to infect protoplasts and plants. Mutation of AUGC motif 2 or disruption of the stem of the 3'-proximal hairpin 1 interfered with CP binding in vitro but not with minus-strand promoter activity in vitro or replication of RNA 3 in vivo. However, hairpin 1 appeared to be essential for encapsidation of RNA 3. Reversion of three G-C base pairs in hairpin 1 had no effect on CP binding but interfered with minus-strand promoter activity in vitro and with RNA 3 replication in vivo. It is concluded that the viral RdRp and CP recognize different elements in the 3'-UTRs of AMV RNAs. Moreover, several mutations that interfered with CP binding in vitro interfered with the accumulation in vivo of RNA 4, the subgenomic messenger for CP, but not with the accumulation of RNA 3.

Functional equivalence of common and unique sequences in the 3' untranslated regions of alfalfa mosaic virus RNAs 1, 2, and 3.

The 3' untranslated regions (UTRs) of alfalfa mosaic virus (AMV) RNAs 1, 2, and 3 consist of a common 3'-terminal sequence of 145 nucleotides (nt) and upstream sequences of 18 to 34 nt that are unique for each RNA. The common sequence can be folded into five stem-loop structures, A to E, despite the occurrence of 22 nt differences between the three RNAs in this region. Exchange of the common sequences or full-length UTRs between the three genomic RNAs did not affect the replication of these RNAs in vivo, indicating that the UTRs are functionally equivalent. Mutations that disturbed base pairing in the stem of hairpin E reduced or abolished RNA replication, whereas compensating mutations restored RNA replication. In vitro, the 3' UTRs of the three RNAs were recognized with similar efficiencies by the AMV RNA-dependent RNA polymerase (RdRp). A deletion analysis of template RNAs indicated that a 3'-terminal sequence of 127 nt in each of the three AMV RNAs was not sufficient for recognition by the RdRp. Previously, it has been shown that this 127-nt sequence is sufficient for coat protein binding. Apparently, sequences required for recognition of AMV RNAs by the RdRp are longer than sequences required for CP binding.

Replicase-mediated resistance to alfalfa mosaic virus.

Tobacco plants transformed with the P1 and P2 replicase genes of alfalfa mosaic virus (AIMV) have been shown to produce functional replicase proteins, permitting their infection with AIMV inocula lacking the genome segments encoding P1 and P2, respectively. To see whether expression of a mutant P2 protein would interfere with the assembly of a functional replicase complex, tobacco plants were transformed with modified P2 genes. When plants were transformed with a P2 gene encoding an N-terminally truncated protein which mimicked the tobacco mosaic virus 54K protein, no resistance was observed with 10 independent lines of transformants. Similarly, when the GDD motif in the full-length P2 protein was changed into VDD, no resistance was observed in 14 transgenic lines. However, when the GDD motif was changed into GGD (5 lines), GVD (15 lines), or DDD (13 lines), 20 to 30% of the transgenic lines showed a high level of resistance to AIMV infection. This resistance was effective to inoculum concentrations of 10 to 25 micrograms/ml of virus and 100 micrograms/ml of viral RNA, causing severe necrosis of control plants. For all transgenic lines, the expression of the transgenes was analyzed at the RNA level. With the GGD, GVD, and DDD mutants, resistance was generally observed in plants with a relatively high expression level. This indicates that the resistance is due to the mutant replicase rather than to an RNA-mediated cosuppression phenomenon.

Plants transformed with a mutant alfalfa mosaic virus coat protein gene are resistant to the mutant but not to wild-type virus.

Transgenic tobacco plants expressing the wild-type (wt) coat protein (CP) gene of alfalfa mosaic virus (AIMV) have been shown to be resistant to infection with viral particles and RNAs or to infection with viral particles only. The difference in resistance of these plants to RNA inocula was found to correlate with a difference in the expression level of the transgene. Plants expressing a mutant AIMV CP with the N-terminal serine residue changed to glycine have been shown to be susceptible to infection with wt viral particles or RNAs. By site-directed mutagenesis of AIMV cDNA a viable mutant virus encoding CP with the same N-terminal mutation was obtained. Plants expressing wt or mutant CP were resistant to the mutant virus, demonstrating that a single amino acid substitution in CP did not permit the virus to overcome CP-mediated resistance. Although the mutant CP did not confer resistance to wt virus when expressed in transgenic plants, it was still effective in classical cross-protection: plants infected with the mutant virus were resistant to severe strain of AIMV. A model to explain the data is discussed.

Tobacco proteinase inhibitor I genes are locally, but not systemically induced by stress.

A cDNA library of tobacco mosaic virus (TMV)-infected tobacco was screened with polymerase chain reaction products obtained using a degenerate primer corresponding to proteinase inhibitor I (PI-I) of tomato and potato. The resulting clones encoded two highly similar, putative tobacco PI-I proteins, indicating that both genes identified in tobacco are probably expressed. The tobacco PI-I's were approximately 50% identical to wound-inducible potato and tomato PI-I and 80% identical to an ethylene-regulated tomato PI-I. Northern blot analyses indicated that healthy tobacco leaf contains only minor amounts of PI-I mRNA, and that the inhibitor genes are induced by TMV infection, salicylate treatment, ethephon spraying, UV light irradiation and wounding. The results indicate that the tobacco PI-I genes are coordinately expressed with the genes for the basic pathogenesis-related proteins. Contrary to PI-I genes of tomato and potato, wound induction of the tobacco genes occurs only locally; the upper, unwounded leaves do not show any wound-induced PI-I gene expression.

Circadian expression and induction by wounding of tobacco genes for cysteine proteinase.

Two sets of clones were isolated from a tobacco cDNA library, utilizing as a probe a PCR fragment obtained from tomato cDNA using a degenerate primer based on the sequence of tomato systemin. Contrary to expectation, the clones did not correspond to tobacco homologues of tomato pro-systemin. However, the cDNAs encoded two highly similar proteins with extensive structural homology to cysteine proteinases from a wide range of plant and animal species. Northern blot analyses showed that in unstressed tobacco leaf the genes for the putative proteinases are expressed according to a circadian rhythm. Furthermore, incision wounding enhances the expression approximately six-fold. Other forms of stress, such as infection with tobacco mosaic virus, treatment with ethephon or UV light do not result in induced expression of the tobacco cysteine proteinase genes.

Differential induction of acquired resistance and PR gene expression in tobacco by virus infection, ethephon treatment, UV light and wounding.

Genes for acidic, extracellular and basic, intracellular pathogenesis-related (PR) proteins of tobacco were studied for their response to tobacco mosaic virus (TMV) infection, ethephon treatment, wounding and UV light. The genes encoding the acidic PR proteins (PR-1, PR-2, PR-3, PR-4 and PR-5) responded similarly to the different forms of stress. They appeared to be highly inducible by TMV, moderately inducible by ethephon treatment and UV light and not inducible by wounding. The genes for the basic counterparts of PR-1, PR-2, PR-3 and PR-5 also displayed a common stress response. However, this response was different from that of the acidic PR proteins. Here, the highest induction was obtained upon ethephon treatment, while the other stress conditions resulted in somewhat lower levels of expression. Most genes for acidic PR proteins are systemically induced in the uninfected upper leaves of TMV-infected plants, whereas the genes encoding the basic PR proteins are not. Increased levels of resistance to TMV, comparable to resistance obtained by pre-infection with the virus, were found in UV-irradiated leaves but not in wounded or ethephon-treated leaves. This indicates that the basic PR proteins are not involved in resistance to TMV infection. Tobacco phenylalanine ammonia-lyase genes were not inducible by the various stress conditions. The implications of these findings in relation to the phenomenon of acquired resistance are discussed.

Tobacco and tomato PR proteins homologous to win and pro-hevein lack the "hevein" domain.

Clones corresponding to tobacco pathogenesis-related (PR) proteins PR-4 and tomato PR protein P2 were isolated from phage cDNA libraries of tobacco infected with tobacco mosaic virus and tomato infected with Cladosporium fulvum, respectively. The probe used in these screenings was a polymerase chain reaction product, synthesized on phage DNA from the tobacco cDNA library, using a synthetic oligonucleotide primer whose sequence corresponded to the partial amino acid sequence available for P2. The different cDNA sequences from the tobacco and tomato clones contained open reading frames for small proteins with 80-90% amino acid sequence identity. Both tobacco PR-4 and tomato P2 are synthesized as precursor proteins, with an N-terminal signal peptide involved in extracellular targeting. The proteins are highly similar to putative wound-induced proteins of potato (win) and to the precursor protein of hevein. However, in contrast to the hevein pro-protein and win proteins, PR-4 and P2 do not contain N-terminal, chitin-binding "hevein" domains. The tobacco and tomato genomes contain a limited number of genes corresponding to PR-4 or P2, whose expression is induced upon infection with the above-mentioned pathogens.

Genome structure of tobacco rattle virus strain PLB: further evidence on the occurrence of RNA recombination among tobraviruses.

RNA-2 (2196 nucleotides) of tobacco rattle virus (TRV) strain PLB was found to consist of a 5'-terminal sequence of 1376 nucleotides identical to the 5'-sequence of RNA-2 of TRV strain PSG and a 3'-terminal sequence of 820 nucleotides that is identical to the 3'-sequence of RNA-1 of strain PLB. Thus, in strains PLB and PSG the same coat protein gene is fused to different RNA-1 derived 3'-termini. By combining RNA-1 of TRV strain TCM with RNA-2 of strain PLB, a viable pseudo-recombinant was formed with genome segments that have nonidentical 3'-sequences. After 25 passages in tobacco each RNA retained its strain-specific 3'-sequences. This indicates that the perfect 3'-homology that occurs between the two genome segments of all natural TRV isolates analyzed so far is not a prerequisite for a stable genotype.

Homology between chitinases that are induced by TMV infection of tobacco.

Recently, four chitinases have been detected in tobacco mosaic virus (TMV) infected tobacco: two acidic chitinases that were identified as pathogenesis-related (PR) proteins P and Q and two basic chitinases (Legrand et al., Proc.Natl. Acad. Sci. USA, in press). Here, it was shown that P and Q are closely serologically related but not related to other known acidic tobacco PR proteins. Antisera to P and Q were used to characterize translation products of TMV-induced mRNAs that were hybrid-selected with cDNA clones described previously (Hooft van Huijsduijnen et al., EMBO J 5: 2057-2061, 1986). In this way cDNA clones corresponding to the acidic and basic chitinases were identified. The partial amino acid sequences of the acidic and basic tobacco chitinases that were represented in the clones, showed an approximately 70% homology to each other and to the sequence of a bean chitinase. Although the acidic and basic chitinases differ in apparent molecular weight, they were found to have homologous C-termini.Hybridization of cDNA probes to genomic blots indicated that the acidic and basic chitinases are each encoded by two to four genes in the amphidiploid genome of Samsun NN tobacco. A similar complexity was found for the genes encoding the tobacco PR protein that is homologous to the sweet-tasting protein thaumatin and to the bifunctional trypsin/α-amylase inhibitor from maize.

Analysis of the genome structure of tobacco rattle virus strain PSG.

The sequence of the 3'-terminal 2077 nucleotides of genomic RNA 1 and the complete sequence of genomic RNA 2 of tobacco rattle virus (TRV, strain PSG) has been deduced. RNA 2 (1905 nucleotides) contains a single open reading frame for the viral coat protein (209 amino acids), flanked by 5'- and 3'-noncoding regions of 570 and 708 nucleotides, respectively. A subgenomic RNA (RNA 4) was found to lack the 5'-terminal 474 nucleotides of RNA 2 and is the putative messenger for coat protein. The deduced RNA 1 sequence contains the 3'-terminal part of a reading frame that probably corresponds to the TRV 170K protein and reading frames for a 29K protein and a 16K protein. Proteins encoded by the first two reading frames show significant amino acid sequence homology with corresponding proteins encoded by tobacco mosaic virus. Subgenomic RNAs 3 (1.6 kb) and 5 (0.7 kb) were identified as the putative messengers for the 29K and 16K proteins, respectively. At their 3'-termini all PSG-RNAs have an identical sequence of 497 nucleotides; at the 5'-termini homology is limited to 5 to 10 bases.

Complete nucleotide sequence of alfalfa mosaic virus RNA 2.

Double-stranded cDNA of in vitro polyadenylated alfalfa mosaic virus (AlMV) RNA 2 has been cloned and sequenced. The use of an oligodeoxyribonucleotide corresponding to the known sequence of the 5'-end of RNA 2 to prime second-strand DNA synthesis, enabled us to construct the complete primary structure of AlMV RNA 2. The sequence of 2,593 nucleotides contains a long open reading frame for a protein of Mr 89,753 starting at the first AUG codon from the 5'-end. This coding region is flanked by a 5'-terminal sequence of 54 nucleotides and a 3'-noncoding region of 166 nucleotides which includes the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common.

Complete nucleotide sequence of alfalfa mosaic virus RNA 1.

Double-stranded cDNA of alfalfa mosaic virus (AlMV) RNA 1 has been cloned and sequenced. From clones with overlapping inserts, and other sequence data, the complete primary sequence of the 3644 nucleotides of RNA 1 was deduced: a long open reading frame for a protein of Mr 125,685 is flanked by a 5'-terminal sequence of 100 nucleotides and a 3' noncoding region of 163 nucleotides, including the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common. The two UGA-termination codons halfway RNA 1, that were postulated by Van Tol et al. (FEBS Lett. 118, 67-71, 1980) to account for partial translation of RNA 1 in vitro into Mr 58,000 and Mr 62,000 proteins, were not found in the reading frame of the Mr 125,685 protein.

Nucleotide sequences at the 5'-termini of the alfalfa mosaic virus RNAs and the intercistronic junction in RNA 3.

Nucleotide sequences at the 5'-termini of the alfalfa mosaic virus genomic RNAs and the intercistronic junction in RNA 3 were deduced and compared to identify possible common recognition signals for replicating enzymes in the corresponding minus-stranded viral RNAs. Homology between the 5'-terminal sequences is less than 11 nucleotides and no complementarity with the homologous sequence occurring at the 3'-end of the viral RNAs was observed. Homology between the 5'-terminus and intercistronic region in RNA 3 is compatible with the synthesis of subgenomic RNA 4 by internal initiation of transcription on the RNA 3 minus strands. The sequence around the intercistronic junction can be folded into a very stable secondary structure.

Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).