An improved detection method for the Rhopalosiphum padi virus (RhPV) allows monitoring of its presence in aphids and movement within plants.

Rhopalosiphum padi virus (RhPV) is an insect RNA virus that infects aphids, reducing their lifespan and fecundity. It can be transmitted vertically between aphids and horizontally via the plant. An improved detection method for the virus in aphids and plants using RT-PCR was developed; this allowed individual aphids to be tested for RhPV. Testing of R. padi aphids collected from different sites in Sweden revealed the presence of RhPV in wild aphid populations for the first time in Europe. Virus could be detected in several life stages of R. padi, including sexual individuals and eggs, establishing an over-wintering route for the virus. Using RT-PCR, systemic transport of the virus in plants was tracked. Virus spread from the aphid feeding site to all parts of the plant, including roots, within 7 days, and could be acquired by virus-free aphids feeding on the same plant.

Animal and plant members of a gene family with similarity to alkaloid-synthesizing enzymes.

Here we describe novel members of a gene family which have similarity to strictosidine synthase (SS), one of the key enzymes in the production of monoterpene indole alkaloids. In addition to the first animal member of the family described previously (Drosophila hemomucin), a second Drosophila member has been identified, which appears to differ in subcellular distribution from hemomucin. In Arabidopsis, SS-like genes form a multigene family, compatible with a possible function as antifeedants and antibacterial compounds. In Caenorhabditis, two members have been identified and one member each in mouse and human. Interestingly, the human SS-like gene is strongly expressed in the brain, the very organ many of the indole alkaloids act upon.

A novel flower-specific Arabidopsis gene related to both pathogen-induced and developmentally regulated plant beta-1,3-glucanase genes.

Beta-1,3-glucanases are usually associated with plant defense responses, although some are also developmentally or hormonally regulated. We characterized two Arabidopsis genes linked in a tandem array, BG4 and BG5, encoding putative novel isoforms of beta-1,3-glucanase. The deduced polypeptides, BG4 and BG5, were highly similar to each other (89% amino acid identity) but only moderately related (32 to 41% amino acid identity) to the different categories of previously characterized beta-1,3-glucanases, suggesting that BG4 and BG5 may represent a novel class of beta-1,3-glucanases in plants. Neither of the genes was responsive to pathogen or SA induction in contrast to the previously identified Arabidopsis beta-1,3-glucanases, nor could we detect any developmental or hormonally induced expression in the vegetative parts of the plants. Both RNA blot and in situ hybridization data demonstrated that the BG4 gene was specifically expressed in the style and septum of the ovary, suggesting that the corresponding protein is involved in the reproductive process of the plant.

Identification of in vivo processing intermediates and of splice junctions of tRNAs from maize chloroplasts by amplification with the polymerase chain reaction.

Total RNA from chloroplasts of maize seedlings was used for polymerase chain reaction (PCR) mediated amplification of tRNA precursors and of mature tRNAs encoded by the two split tRNA genes of the ribosomal spacer (tRNA(lle)GAU and tRNA(Ala)UGC) and the single intron-containing tRNA(Gly)UCC gene. Sequence analysis of DNAs amplified from the mature tRNAs by combinations of exon specific primers allows unambiguous identification of the respective splice junctions. Primer combinations in which 5'- or 3'-flanking precursor tRNA sequences are included, leads to the amplification of processing intermediates in which 5'-terminal extensions are still present, whereas no PCR products corresponding to 3'-terminal extensions could be detected. From this it is concluded that in chloroplasts the 5'-terminal endonucleolytic cleavage by RNase P occurs as one of the final steps in the tRNA processing pathway of which the endonucleolytic cleavage at the 3' side probably occurs prior to the splicing of the intron sequences.

Functional in vivo verification in E. coli of promoter activities from the rDNA/tDNA(Val)(GAC) leader region of Zea mays chloroplasts.

Restriction fragments containing upstream sequences of the rRNA operon from Zea mays chloroplasts were tested for promoter activity in vivo by insertion into an E. coli promoter-probe vector. The expression of this vector's reporter gene, which codes for alkaline phosphatase, was stimulated more than 1,500-fold upon linkage with the chloroplast rRNA promoter. Site specific mutagenesis of the invariant T of the -10 sequence of this promoter reduced the expression of the reporter gene to 2% of the wild type. This indicates that the chloroplast rRNA promoter, which directs transcriptional initiation 117 bp upstream of the 16S rRNA gene, is also active in the bacterial system. A restriction fragment further upstream containing the gene for tRNA(Val) (GAC) also showed strong promoter activity (29% as compared with the rRNA promoter). This promoter activity probably reflects the chloroplast promoter directing the synthesis of the tRNA(Val) (GAC) primary transcript. Surprisingly, this restriction fragment also displayed promoter activity (13% compared with the rRNA promoter) in reverse orientation.