Frans Th. Brederode

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.

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.

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.

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-Is 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.

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-1s mode of action in PR-1a gene expression is discussed.

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.

Studies on sequence homology between the RNA's of alfalfa mosaic virus.

Abstract Four major and at least ten minor RNA species are present in preparations of alfalfa mosaic virus (AMV). To study the base sequence homology of these RNAs by competition hybridization, virus-specific double-stranded RNA (ds-RNA) was isolated from infected leaves. Determination of the fractions of 3 H-labeled AMV-RNA displaced from the hybrid by purified preparations of the unlabeled major RNAs (B-, M-, Tb- and Ta-RNA) revealed the presence of unique base sequences in the genome fragments B-, M- and Tb-RNA. The size of the base sequences common to B- and M-RNA, if any, is less than 200 nucleotides. A possible redundancy between Tb-RNA and B- or M-RNA could not be established because of a contamination of the Tb-RNA preparation with minor RNA species. A preparation of unlabeled minor RNA species was able to displace all 3 H-labeled AMV-RNA from the hybrid, showing the presence of all virus-specific base sequences in this material. Unlabeled preparations of Ta- and Tb-RNA completely interfered with the annealing of 3 H-labeled Ta-RNA to ds-RNA. This confirms the conclusion of earlier work that the Ta-RNA sequences are present in Tb-RNA. However, evidence was obtained indicating that Ta-RNA is heterogeneous and contains all Ta-RNA sequences. The nature of the small interference of B- and M-RNA with the annealing of the 3 H-labeled Ta-RNA preparation to ds-RNA is discussed. A comparison between the base sequences of four AMV strains revealed an almost complete homology. This may be correlated with the fact that components of different strains can be exchanged without loss of infectivity. No base sequence homology was observed between AMV and other viruses with a tripartite genome: brome mosaic virus, cowpea chlorotic mottle virus, cucumber mosaic virus, and tobacco streak virus.