Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance.

After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens. There is compelling evidence that SA plays a crucial role in triggering SAR. We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process. When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumulation of SA and SA glucoside compared to control plants. Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants. This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants.

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport.

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.

A virus-inducible tobacco gene encoding a glycine-rich protein shares putative regulatory elements with the ribulose bisphosphate carboxylase small subunit gene.

cDNA to an mRNA that is strongly induced in Samsun NN tobacco after tobacco mosaic virus (TMV) infection or salicylic acid treatment was used to probe a genomic blot and to screen a genomic library. The mRNA corresponds to a family of approximately eight genes, four of which were cloned. The sequence of the genes and flanking DNA in two clones was determined. One gene was found to contain an intron of 555 bp; S1-nuclease mapping studies indicated that this gene is expressed. The other gene is interrupted by an intron of 1,954 bp and is probably not expressed after TMV infection. The genes encode a protein of 109 amino acids with a putative N-terminal signal peptide of 26 amino acids. The protein contains a high proportion of glycine (25%) and charged amino acids (29%), suggesting that it may be a cell wall component. A comparison of the upstream sequences of the genes encoding the glycine-rich protein and the pathogenesis-related protein 1a showed only limited homology, although both genes are TMV- and salicylic acid-inducible. However, the upstream sequence of the glycine-rich protein gene contains a 64-bp inverted repeat that occurs in a similar position in the tobacco ribulose bisphosphate carboxylase small subunit gene.

Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants.

Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In addition, the mutations were introduced in the MP gene of AMV RNA 3 and the mutant RNAs were used to infect tobacco plants. Ten mutants were affected in one or more of the following functions of MP: the formation of tubular structures on the surface of protoplasts, association with the endoplasmic reticulum (ER) of suspension cells and epidermal cells, targeting to punctate structures in the cell wall of epidermis cells, movement from transfected cells to adjacent cells in epidermis tissue, cell-to-cell movement, or long-distance movement in plants. The mutations point to functional domains of the MP and support the proposed order of events in AMV transport. Studies with several inhibitors indicate that actin or microtubule components of the cytoskeleton are not involved in tubule formation by AMV MP. Evidence was obtained that tubular structures on the surface of transfected protoplasts contain ER- or plasmalemma-derived material.

Virus-induced gene expression for enzymes of ethylene biosynthesis in hypersensitively reacting tobacco.

A full-length cDNA clone (cEFE-26) encoding ethylene-forming enzyme (EFE) was isolated from a cDNA library, prepared from leaves of tobacco mosaic virus (TMV)-infected tobacco cultivar Samsun NN. The cDNA clone encodes a protein with 90% amino acid sequence similarity to established EFEs of tomato and other plants. By using cEFE-26 cDNA and the insert from cDNA clone pACC13 (B. A. Bailey, A. Avni, N. Li, A. K. Mattoo, and J. D. Anderson, Plant Physiol. 100:1615-1616, 1992) encoding tobacco 1-aminocyclopropane-1-carboxylic acid synthase as probes, it was established that tobacco contains small gene families for these proteins. Furthermore, RNA blot analyses indicated that transcript levels in leaves for the two ethylene pathway genes were elevated after infection with TMV. The results are discussed in relation to a possible signalling role of ethylene in induced resistance and gene expression for pathogenesis-related proteins.

Induction of a hypersensitive response by chimeric helicase sequences of tobamoviruses U1 and Ob in N-carrying tobacco.

Recently, the helicase domain of the Tobacco mosaic virus (TMV)-U1 replicase proteins (designated MOREHEL:U1) was identified as the elicitor of the N gene-mediated hypersensitive response (HR) in tobacco. In this study, we used agroinfiltration to express the equivalent MOREHEL domain of the non-HR-inducing tobamovirus strain TMV-Ob. It appeared that this MOREHEL:Ob sequence did not elicit a HR in N gene-carrying tobacco. Both MOREHEL sequences were divided into eight subdomains, and chimeras of MOREHEL sequences from U1 and Ob were constructed. Expression of these chimeric MOREHEL sequences revealed that, in the TMV-U1 MOREHEL sequence, at least four domains involved in full HR induction were present. The presence of at least three of these four domains seems a minimal requirement for HR induction. Two additional domains may play a minor role in HR induction. To study the elicitor function of the chimeras during the TMV life cycle, chimeric MOREHEL domains were introduced into full-length TMV cDNA clones. These constructs, however, were unable to establish an infection in Nicotiana benthamiana or Nicotiana tabacum plants.

Systemic resistance in Arabidopsis induced by rhizobacteria requires ethylene-dependent signaling at the site of application.

Root colonization of Arabidopsis thaliana by the nonpathogenic, rhizosphere-colonizing, biocontrol bacterium Pseudomonas fluorescens WCS417r has been shown to elicit induced systemic resistance (ISR) against Pseudomonas syringae pv. tomato (Pst). The ISR response differs from the pathogen-inducible systemic acquired resistance (SAR) response in that ISR is independent of salicylic acid and not associated with pathogenesis-related proteins. Several ethylene-response mutants were tested and showed essentially normal symptoms of Pst infection. ISR was abolished in the ethylene-insensitive mutant etr1-1, whereas SAR was unaffected. Similar results were obtained with the ethylene-insensitive mutants ein2 through ein7, indicating that the expression of ISR requires the complete signal-transduction pathway of ethylene known so far. The induction of ISR by WCS417r was not accompanied by increased ethylene production in roots or leaves, nor by increases in the expression of the genes encoding the ethylene biosynthetic enzymes 1-aminocyclopropane-1-carboxylic (ACC) synthase and ACC oxidase. The eir1 mutant, displaying ethylene insensitivity in the roots only, did not express ISR upon application of WCS417r to the roots, but did exhibit ISR when the inducing bacteria were infiltrated into the leaves. These results demonstrate that, for the induction of ISR, ethylene responsiveness is required at the site of application of inducing rhizobacteria.

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.

Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco.

cDNA clones of messenger RNAs for acidic and basic chitinases were isolated from libraries of tobacco mosaic virus-infected Samsun NN tobacco and petunia. The tobacco cDNA clones for acidic chitinase fell into two different groups, whereas all petunia cDNA clones had the same sequence. Also, tobacco genomic clones were isolated and one was characterized. This genomic clone, corresponding to one of the cDNA clones, showed that this acidic chitinase gene contains two introns. The amino acid sequences of the acidic chitinases from tobacco, as deduced from the cDNA clones, fully agreed with partial sequences derived from peptides obtained from purified tobacco-derived pathogenesis-related proteins PR-P and PR-Q. The deduced amino acid sequences showed that PR-P and PR-Q are 93 and 78%, respectively, identical to the petunia enzyme. All deduced chitinase sequences indicated the presence of an NH2-terminal, highly hydrophobic signal peptide. In addition, the polysaccharide-binding domain present at the NH2-terminus of basic chitinases from mature tobacco is not present in these acidic chitinases. Furthermore, the complete coding sequence for the petunia chitinase, constructed downstream of the cauliflower mosaic virus 35S promoter, was used to transform tobacco. The resulting chimeric gene was constitutively expressed, and the petunia enzyme was targeted to the extracellular fluid. In contrast, a basic chitinase of tobacco, expressed from a chimeric gene, was found in total leaf extracts but not in preparations of extracellular fluid.

Isolation and characterization of the immunoglobin of pike (Esox lucius L.).

The purification of the immunoglobul in from pike serum and its physicochemical characterization is presented. The immunoglobul in was prepared by means of gel filtration and ion exchange chromatography. Measurements in the analytical ultracentrifuge showed a sedimentation constant of 15.0 S. A molecular weight of 650.000 was calculated. The immunoglobulin was composed of heavy and light chains of molecular weights 60.000 and 22.500, respectively. It is likely that the immunoglobulin of pike is composed of 8 heavy and 8 light chains and possesses a tetrameric structure. The heavy chains contain 9.2% sugars and amino sugars. The amino acid composition of the chains is similar to that of other fish immunoglobulins.

Genetic dissection of the multiple functions of alfalfa mosaic virus coat protein in viral RNA replication, encapsidation, and movement.

Coat protein (CP) of alfalfa mosaic virus (AMV) binds as a dimer to the 3' termini of the three genomic RNAs and is required for initiation of infection, asymmetric plus-strand RNA accumulation, virion formation, and spread of the virus in plants. A mutational analysis of the multiple functions of AMV CP was made. Mutations that interfered with CP dimer formation in the two-hybrid system had little effect on the initiation of infection or plus-strand RNA accumulation but interfered with virion formation and reduced or abolished cell-to-cell movement of the virus in plants. Six of the 7 basic amino acids in the N-terminal arm of CP (positions 5, 6, 10, 13, 16, and 25) could be deleted or mutated into alanine without affecting any step of the replication cycle except systemic movement in plants. Mutation of Arg-17 interfered with initiation of infection (as previously shown by others) and cell-to-cell movement of the virus but not with plus-strand RNA accumulation or virion formation. The results indicate that in addition to the RNA-binding domain, different domains of AMV CP are involved in initiation of infection, plus-strand RNA accumulation, virion formation, cell-to-cell movement, and systemic spread of the virus.

Homology between the proteins encoded by tobacco mosaic virus and two tricornaviruses.

A comparison was made of the amino acid sequences of the proteins encoded by RNAs 1 and 2 of alfalfa mosaic virus (A1MV) and brome mosaic virus (BMV), and the 126K and 183K proteins encoded by tobacco mosaic virus (TMV). Three blocks of extensive homology of about 200 to 350 amino acids each were observed. Two of these blocks are located in the A1MV and BMV RNA 1 encoded proteins and the TMV encoded 126K protein; they are situated at the N-terminus and C-terminus, respectively. The third block is located in the A1MV and BMV RNA 2 encoded proteins and the C-terminal part of the TMV encoded 183K protein. These homologies are discussed with respect to the functional equivalence of these putative replicase proteins and a possible evolutionary connection between A1MV, BMV and TMV.

Altered balance of the synthesis of plus- and minus-strand RNAs induced by RNAs 1 and 2 of alfalfa mosaic virus in the absence of RNA 3.

The synthesis of viral plus-strand and minus-strand RNAs in cowpea protoplasts inoculated with mixtures of alfalfa mosaic virus nucleoproteins (B, M, Tb, and Ta) was analyzed by the Northern blotting technique. A mixture of B, M, and Tb induced the synthesis of plus-strand RNAs 1, 2, 3, and 4 and three minus-strand RNAs corresponding to RNAs 1, 2, and 3, respectively. Compared to this complete infection, a mixture of B and M induced the synthesis of a reduced amount of plus-strand RNAs 1 and 2 and a greatly enhanced amount of minus-strand RNAs 1 and 2. No detectable viral RNA synthesis was induced by mixtures of B and Tb or M and Tb. It is concluded that expression of genomic RNAs 1 and 2 results in the formation of a replicase activity that produces roughly equal amounts of viral plus- and minus-strand RNAs and that an RNA 3-encoded product, possibly the coat protein, is responsible for a switch to an asymmetric production of viral plus-strand RNA. The observation that no minus-strand corresponding to the subgenomic RNA 4 is produced suggests that recognition of the genome segments by the viral replicase involves sequences outside the 3'-terminal regions that are homologous to RNA 4.

Sequence comparison and secondary structure analysis of the 3' noncoding region of flavivirus genomes reveals multiple pseudoknots.

Sequences of 191 flavivirus RNAs belonging to four sero-groups were used to predict the secondary structure of the 3' noncoding region (3' NCR) directly upstream of the conserved terminal hairpin. In mosquito-borne flavivirus RNAs (n = 164) a characteristic structure element was identified that includes a phylogenetically well-supported pseudoknot. This element is repeated in the dengue and Japanese encephalitis RNAs and centers around the conserved sequences CS2 and RCS2. In yellow fever virus RNAs that contain one CS2 motif, only one copy of this pseudoknotted structure was found. The conserved pseudoknotted element is absent from the 3' NCR of tick-borne virus RNAs, which altogether adopt a secondary structure that is very different from that of mosquito-borne virus RNAs. The strong conservation of the pseudoknot in mosquito-borne flavivirus RNAs implies a stronger relationship between these viruses than concluded from previous secondary structure analyses. The role of the (tandem) pseudoknots in flavivirus replication is discussed.

Structural elements of the 3'-terminal coat protein binding site in alfalfa mosaic virus RNAs.

The 3'-terminal of the three genomic RNAs of alfalfa mosaic virus (AIMV) and ilarviruses contain a number of AUGC-motifs separated by hairpin structures. Binding of coat protein (CP) to such elements in the RNAs is required to initiate infection of these viruses. Determinants for CP binding in the 3'-terminal 39 nucleotides (nt) of AIMV RNA 3 were analyzed by band-shift assays. From the 5'- to 3'-end this 39 nt sequence contains AUGC-motif 3, stem-loop structure 2 (STLP2), AUGC-motif 2, stem-loop structure 1 (STLP1) and AUGC-motif 1. A mutational analysis showed that all three AUGC-motifs were involved in CP binding. Mutation of the A- and U-residues of motifs 1 or 3 had no effect on CP binding but similar mutations in motif 2 abolished CP binding. A mutational analysis of the stem of STLP1 and STLP2 confirmed the importance of these hairpins for CP binding. Randomization of the sequence of the stems and loops of STLP1 and STLP2 had no effect on CP binding as long as the secondary structure was maintained. This indicates that the two hairpins are not involved in sequence-specific interactions with CP. They may function in a secondary structure-specific interaction with CP and/or in the assembly of the AUGC-motifs in a configuration required for CP binding.

Limited sequence variation in the leader sequence of RNA 4 from several strains of alfalfa mosaic virus.

The sequences of the 5'-noncoding regions of RNA 4 of seven strains of alfalfa mosaic virus were compared. In each case, the coat protein cistron was preceded by a leader sequence of 39 nucleotides (including the initiator codon). At position 26, an A-, G-, or U-residue was found. Otherwise the leader sequences are identical.

Cis-acting functions of alfalfa mosaic virus proteins involved in replication and encapsidation of viral RNA.

cDNA clones of RNAs 1 and 2 of alfalfa mosaic virus (AMV) were slightly modified to permit transcription of infectious RNAs with T7 RNA polymerase. Together with transcripts of an available clone of AMV RNA 3, these transcripts were used to study cis- and trans-acting functions of AMV proteins in protoplasts from nontransgenic tobacco plants and from plants transformed with the P1 and P2 genes, encoded by RNAs 1 and 2, respectively. Transgenic P1 was unable to complement mutations in the P1 gene in RNA 1, pointing to a cis-acting function of P1 in RNA 1 replication. A study of the replication of RNA 3 mutants in nontransgenic protoplasts revealed that coat protein (CP) expressed from RNA 3 in the inoculum is required in trans for replication and encapsidation of RNAs 1 and 2 but is required in cis for replication and encapsidation of RNA 3. CP is required in the inoculum to initiate infection of nontransgenic plants and protoplasts. When protoplasts expressing both P1 and P2 (P12 protoplasts) were infected with RNAs 1, 2, and 3, initiation of replication of RNAs 1 and 2 required the presence of CP in the inoculum, whereas the initiation of replication of RNA 3 did not. This demonstrated that CP expressed from RNA 3 cannot substitute for the early function of CP in the inoculum. The results showed that CP in the inoculum is required to permit viral minus-strand RNA synthesis, whereas CP expressed from RNA 3 after the initiation of infection is required for plus-strand RNA synthesis.

Biological activity of transcripts synthesized in vitro from full-length and mutated DNA copies of tobacco rattle virus RNA 2.

Full-length cDNA clones of RNA 2 of tobacco rattle virus (TRV) strain PLB have been cloned into the transcription vector pPM1. Products of in vitro transcription by Escherichia coli RNA polymerase, either capped or uncapped, were as infectious as native RNA 2 when coinoculated with RNA 1 of TRV strain TCM. At least 70% of the internal sequence of the cDNA could be deleted without reduction of the replication efficiency of the transcripts. Sequences of 340 nucleotides at the 5' end and 405 nucleotides at the 3' end of PLB RNA 2 were found to be sufficient for replication. The encapsidation of deletion mutants of PLB RNA 2 was investigated after addition of native PLB RNA 1 and RNA 2. Accumulation of these mutants was distinguished from that of wild-type RNA 2 by insertion of nonviral sequences in the deleted parts. Three mutant forms of RNA 2 with extensive deletions in the coat protein (CP) gene were replicated but failed to encapsidate, while mutants with nonviral sequences inserted downstream from the CP gene showed a large reduction in replication efficiency.

Time course of alfalfa mosaic virus RNA and coat protein synthesis in cowpea protoplasts.

A study was made of the time course of the synthesis of viral plus-strand RNA, minus-strand RNA, and coat protein in alfalfa mosaic virus-infected Cowpea protoplasts. The three genomic RNAs were synthesized at different rates, as were their corresponding minus-strands. We conclude that viral RNA synthesis is regulated both at the level of minus-strand production and the level of plus-strand production. The synthesis of subgenomic RNA 4 was slower than that of its corresponding genomic RNA (RNA 3), indicating that an additional function, expressed later in infection, is required for production of subgenomic coat protein messenger. The data support a model for RNA 4 synthesis involving internal initiation by the RNA polymerase at the intercistronic junction in minus-strand RNA 3. The temporal relationship of the synthesis of RNA 3, RNA 4, and coat protein is discussed.

Plant-virus-based vectors for gene transfer will be of limited use because of the high error frequency during viral RNA synthesis.

The error frequency during the RNA replication of alfalfa mosaic virus (AMV) was calculated to be significantly higher than 10(-5). It may be expected that RNA synthesis in general will have low fidelity compared to DNA synthesis. The low fidelity of RNA replication will severely restrict the usefulness of vectors for genetic engineering which are based on RNA viruses, viroids or DNA viruses which are replicated via an RNA intermediate (e.g. caulimoviruses). Spontaneous mutants selected by host shift were found to be much less stable than UV-induced mutants. This difference points to variations in fidelity during RNA synthesis, probably due to the local sequence of the template.