Ben J.C. Cornelissen

Molecular characterization of messenger RNAs for 'pathogenesis-related' proteins 1a, 1b and 1c, induced by TMV infection of tobacco.

A cDNA library was made to poly(A)‐containing RNA from tobacco mosaic virus (TMV)‐infected Samsun NN tobacco plants and clones corresponding to mRNAs for the ‘pathogenesis‐related’ (PR) proteins 1a, 1b and 1c were identified. One clone was found to contain a complete copy of PR‐1b mRNA. The structural organization of this RNA is: a leader sequence of 29 nucleotides, an open reading frame of 504 nucleotides encoding a 30 amino acid long signal peptide and a 138 amino acid long mature protein, and a 3′‐non‐coding region of 235 nucleotides. Two other clones were found to contain partial copies of PR‐1a and PR‐1c mRNAs. The data indicate an ∼90% homology between the amino acid sequences of PR‐1a, ‐1b and ‐1c. Using one of the clones as probe it was shown that in the TMV‐inoculated lower leaves and the non‐inoculated upper leaves of a tobacco plant, the PR‐1 mRNAs become detectable from 2 and 8 days after inoculation, respectively.

Virus-induced synthesis of messenger RNAs for precursors of pathogenesis-related proteins in tobacco

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) induces a number of host‐encoded, so‐called pathogenesis‐related (PR‐) proteins, which are found in the intercellular space of the leaf and are associated with induced resistance. By immunoprecipitation of their in vitro translation products we were able to detect the mRNAs corresponding to a number of PR‐proteins in TMV‐infected tobacco, but not in healthy plants. Analysis by the Northern blot technique using cloned cDNA of PR1‐mRNAs as probe showed that the mRNAs for the closely related proteins PR1a, 1b and 1c occur at a low level in healthy tobacco; upon TMV infection this level is increased >100‐fold. The PR1‐specific probe did not hybridize to mRNAs corresponding to other PR‐proteins. Sequencing of the 5′‐terminal region of PR1‐mRNAs showed that PR1‐proteins are derived from precursors by removal of an N‐terminal signal peptide of 30 amino acids.

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.

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

SummaryThe 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.

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

SummaryA 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.

Structure of tobacco genes encoding thaumatin-like proteins

Two tobacco genes encoding thaumatin-like proteins were cloned and sequenced. Both genes are expressed after infection of tobacco with tobacco mosaic virus (TMV). Comparison of the upstream sequences of these genes with those of other TMV-inducible tobacco genes revealed limited regions of homology.

Induction of Plant Genes by Compatible and Incompatible Virus-Plant Interactions

cDNAs were cloned to one host mRNA induced by a compatible virus-plant interaction and several host mRNAs induced by an incompatible virus-plant interaction. Regulatory sequences in two of the corresponding genes were analysed and three of the inducible genes were constitutively expressed in transgenic tobacco to test a putative role of the encoded proteins in defence mechanisms.