Chikara Masuta

Morphological changes and hypomethylation of DNA in transgenic tobacco expressing antisense RNA of the S-adenosyl-l-homocysteine hydrolase gene

S-adenosyl-l-homocysteine hydrolase (SAHH) is a key enzyme in the regulation of intracellular methylation reactions. To investigate the role of SAHH in methylation reactions and morphogenesis in planta, we have made transgenic plants expressing antisense RNA of tobacco SAHH. The transgenic plants displayed distinct morphological changes including a floral homeotic change. We hypothesized that the changes were caused by increased levels of cytokinin. In those transgenic plants, we observed that a repetitive DNA sequence appeared less methylated than controls. We speculated that altered gene expressions by the hypomethylation of DNA might be involved in the changes.

Isolation and analysis of a genomic clone encoding a pokeweed antiviral protein

Partial cDNAs encoding a pokeweed antiviral protein were obtained by polymerase chain reaction from the poly(A)+ RNA of seeds, leaves, and roots using two specific primers based on the amino acid sequence of a pokeweed antiviral protein from the seeds (PAP-S). Using the cDNAs as a radioactive probe, 17 and 39 positive plaques were isolated from libraries containing the genomic DNA of Phytolacca americana digested with Bam HI partially and completely, respectively. The plaques were grouped into nine types by Southern hybridization. The type α genomic clone encodes a protein of 294 amino acids. Its amino acid sequence is similar but not identical to that of PAP-S. A comparison of the two amino acid sequences suggested that the deduced protein contains extrapeptides of 24 and 9 amino acids at the NH2 and the COOH terminals, respectively. The putative protein was expressed in Escherichia coli and shown to depurinate the specific adenine of wheat 25S rRNA, indicating that the protein encoded by a type α genomic clone is a functional protein exhibiting RNA N-glycosidase activity.

Molecular cloning of a cDNA clone for tobacco lipid transfer protein and expression of the functional protein in Escherichia coli

A cDNA clone encoding a lipid transfer protein (LTP) was isolated from tobacco by screening a library with a PCR‐amplified spinach LTP gene, DNA sequence analysis showed a large open reading frame (344 bp) encoding a polypeptide of 114 amino acids. The first 23 amino acids of the deduced protein have the characteristics of a signal peptide for protein secretion or targeting into dense microbody‐like vesicles. The cDNA clone was then inserted into an expression vector, pMAL, and expressed in E. coli as a fusion with the maltose binding protein (MBP). The MBP‐LTP fusion protein was purified to homogeneity and subjected to factor Xa cleavage to yield the LTP domain. A lipid transfer assay demonstrated that the resulting LTP was functional. The availability of the expression system in E. coli will facilitate the elucidation of in vivo function(s) of plant LTPs.

Reciprocal phenotype alterations between two satellite RNAs of cucumber mosaic virus

Cucumber mosaic virus Y satellite RNA (Y-satRNA) induces distinctive yellow mosaic symptoms on tobacco, whereas S19 satellite RNA (S19-satRNA) causes an attenuated green mosaic on tobacco, although they show considerable sequence identity. Biological assays of infectious chimeric satellite RNA molecules synthesized from cDNA clones of Y-satRNA and S19-satRNA using common restriction sites showed that the determinant for the induction of yellow mosaic symptoms lies in the BstXI-NheI fragment, in which 14 nucleotide differences are found between the two satellite RNAs. To define more precisely the yellow mosaic determinant(s) in this fragment, several site-directed mutants of Y-satRNA were created. The replacement of AUU, at nucleotides 191 to 193 in Y-satRNA, with GC, which mimics the S19-satRNA sequence at the corresponding site, abolished the ability of Y-satRNA to elicit a yellow mosaic. Conversely, a mutant RNA molecule derived from S19-satRNA in which GC at nucleotides 192 and 193 was changed to AUU induced the yellow mosaic symptoms. Thus, the phenotypes of two satellite RNAs on tobacco can be altered reciprocally by changing the sequences in this limited region.

Cucumber mosaic virus-tolerant transgenic tomato plants expressing a satellite RNA

SummaryA satellite RNA (T73-satRNA) gave reduced symptom severity on tomato plants when coinoculated with an ordinary strain of cucumber mosaic virus (CMVO). cDNA for T73-satRNA was introduced into a binary vector (pTOK162) through a homologous recombination in an Agrobacterium tumefaciens cell, and then transferred to leaf disks of tomato. Stable integration and transcription of the cDNA in the regenerants were verified by Southern and northern blot hybridizations, respectively. Upon inoculation with CMV-O, the transformants exhibited very slight symptoms of CMV, grew normally, and finally set fruits, whereas untransformed wildtype tomato plants showed very severe symptoms, and their growth was retarded and formed few fruits. Agarose gel electrophoresis of total RNA from CMV-O-inoculated transformants detected RNA molecules corresponding to T73-satRNA.

RESISTANCE AGAINST CUCUMBER MOSAIC VIRUS IN PLANTS EXPRESSING THE VIRAL REPLICON

CMV RNAs 1 and 2 are considered to constitute the viral replicon. Tobacco plants were transformed with either RNA1 or RNA2 to produce plant lines V1 and V2, respectively. Plants homozygous for each of the RNAs were generated and crossed to produce V1V2 (V2V1) lines that expressed both RNA1 and RNA2. An RNase protection assay indicated that RNA1 and RNA2 multiplied in V1V2 (V2V1) plants. Surprisingly, V1V2 (V2V1) plants, unlike their parent lines, showed a remarkably high level of resistance to CMV; this resistance was more effective against RNA inoculation than against virion inoculation. Experiments using protoplasts showed that the resistance was expressed at the single cell level. All the data together suggested that the observed resistance does not fit the criteria for either ‘RNA‐mediated’ or ‘replicase‐mediated’ resistance.

Inducible expression by plant hormones of S-adenosyl-l-homocysteine hydrolase gene from Nicotiana tabacum during early flower bud formation in vitro

Abstract cDNA clones corresponding to genes induced during the early stage of flower bud formation in thin cell layer (TCL) cultures of tobacco were isolated by differential screening. Sequence analysis showed that one of the clones encoded S -adenosyl- l -homocysteine hydrolase (SAHH), which is known to be an enzyme regulating intracellular transmethylation reactions. Recently, it was also found to be a cytokinin-binding protein. An SAHH gene was isolated by screening a tobacco genomic library with the cDNA as a probe. Northern blot analysis showed that expression of the SAHH gene was greatest in pistils and roots, and could be induced by kinetin and IAA. Kinetin and IAA are required for in vitro flower differentiation in TCL culture. The promoter sequence of the gene was fused to the β-glucuronidase (GUS) reporter gene and introduced in suspension-cultured cells, which rendered expression of GUS inducible by kinetin. Possible involvement of SAHH in regulation of gene expression, cytokinin signal transduction and flower differentiation is discussed.

Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP)

Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes. A specific modification in the 25S rRNA of M. jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis. Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A4324 in rat 28S rRNA. The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes. The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin). However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes.Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells. Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.

Expression of a pokeweed antiviral protein in Escherichia coli and its characterization

Two expression vectors were constructed to produce a putative mature α‐pokeweed antiviral protein (α‐PAP) in Escherichia coli with its NH2‐ and COOH‐terminal extrapeptides excised. One was for its intracellular expression with a methionine at its NH2‐terminal. The other was for its secretion using an ompA signal peptide. The former product was purified from the total soluble proteins of the transformant with a yield of 1.74 mg/liter and the latter had a yield of 5.55 mg/liter. Both products exhibited RNA N‐glycosidase activity on wheat ribosomes and inhibitory activity to protein synthesis in a rabbit reticulocyte system.

Comparative study of screening with subtracted probe and differential screening on isolation of flower-specific cDNA clones from Nicotiana sylvestris

Abstract A simple and efficient screening procedure for isolation of tissue-specific cDNA clones is described. A plasmid cDNA library was constructed from mRNAs extracted from Nicotiana sylvestris flower buds. Flower-specific cDNA clones were identified after screening of the cDNA library with subtracted probe enriched for flower-specific sequences. Comparison of this procedure with the conventional differential screening indicated that it has the advantages of direct isolation of highly specific cDNA clones, as well as identification of less abundant specifically expressed clones. The isolated cDNA clones were classified into 14 independent cross-hybridization groups. Northern blot analysis with the cDNA clones as probes, distinguished three types of mRNA expression patterns: (1) specific expression in stamen during a restricted period of flower development; (2) expression in the reproductive organs; (3) expression in both the structural and reproductive flower organs. Sequencing revealed that 6 of the groups correspond to known genes, but the remaining 8 were not homologous to the sequences in the data bank.