White pine weevil (Pissodes strobi) biological performance is unaffected by the jasmonic acid or wound-induced defense response in Norway spruce (Picea abies).

In eastern Canada, the white pine weevil (Pissodes strobi Peck) is a pest of several native pine and spruce species and of the introduced species, Norway spruce (Picea abies Karst). We evaluated the feeding activities, oviposition and rate of adult emergence of white pine weevil on field-grown Norway spruce subjected to jasmonic acid or wounding pretreatments. We also monitored the host-plant reaction to white pine weevil attack, jasmonic acid and wounding treatments by quantifying several mono- and sesquiterpenes in bark and characterizing some molecular aspects of the terpenoid response. Two cDNA sequences were identified that had a high percentage of identity with genes encoding monoterpene or sesquiterpene synthases. Both putative terpene synthase genes showed distinctive profiles in Norway spruce bark and needles following all treatments. Although the Norway spruce trees showed different physiological responses to mechanical wounding and white pine weevil attack, transcript activity of the gene encoding terpenoid synthase and consequent accumulation of terpenoid resin did not significantly affect the weevils' feeding activities, oviposition or rate of adult emergence.

Bacterial disease resistance of transgenic hybrid poplar expressing the synthetic antimicrobial peptide D4E1.

Important losses in poplar productivity occur because of susceptibility to microbial pathogens. To enhance disease resistance in susceptible genotypes, the gene coding for D4E1, a synthetic antimicrobial peptide consisting of 17 amino acid residues, was introduced into poplar (Populus tremula L. x Populus alba L.) via Agrobacterium-mediated transformation. Four kanamycin-resistant transformants were selected based on significant accumulation of the D4E1 transcript and confirmed by reverse transcription-polymerase chain reaction and RNA dot-blot analysis. These transgenic poplar lines were tested for resistance to Agrobacterium tumefaciens, Xanthomonas populi pv. populi and Hypoxylon mammatum (Wahl.) Miller. One transgenic poplar line, Tr23, bearing the highest transcript accumulation for the D4E1 gene, showed a significant reduction in symptoms caused by A. tumefaciens and X. populi. However, none of the transgenic poplar lines showed a significant difference in disease response to the fungal pathogen H. mammatum.

Isolation and characterization of a dehydrin gene from white spruce induced upon wounding, drought and cold stresses.

A cDNA clone encoding a dehydrin gene was isolated from a cDNA library prepared from white spruce (Picea glauca) needle mRNAs. The cDNA, designated PgDhn1, is 1159 nucleotides long and has an open reading frame of 735 bp with a deduced amino acid sequence of 245 residues. The PgDhn1 amino acid sequence is highly hydrophilic and possesses four conserved repeats of the characterized lysine-rich K-segment (EKKGIMD-KIKEKLPG), and an 8-serine residue stretch prior to the first lysine-rich repeat that is common to many dehydrins. The DEYGNP conserved motif is, however, absent in the PgDhn1 sequence. In unstressed plants, the highest level of transcripts was detected in stem tissue and not fully expanded vegetative buds. PgDhn1 expression was also clearly detected in reproductive buds, at various stages of development. The mRNAs corresponding to PgDhn1 cDNA were induced upon wounding and by jasmonic acid (JA) and methyl jasmonate (MeJa) treatments. Upon drought stress, increased transcript accumulation was observed in needle tissue reaching a maximum level 48 h after treatment. Treatments of seedlings with abscisic acid or ethephon also resulted in high levels of transcript accumulation in needle tissue. Finally, cold induction of PgDhn1 transcripts was also detected as early as 8 h after treatment.

Toxoplasma gondii: structure and characterization of the 26S ribosomal RNA and peptidyl transferase domain.

The 26S, 5.8S, and the intergenic spacer ribosomal DNAs (rDNA) of Toxoplasma gondii have been cloned and completely sequenced from both DNA strands. The length of the large subunit was found to be 3487 nucleotides and the 5.8S was 153 nucleotides long. These formed the large rRNA subunit of Toxoplasma and were mapped in the rDNA unit known to be repeated 110 times in a head-to-tail fashion in the genome. Primer extension analysis and multiple alignments localized the 5' end point of the two rRNAs. Comparisons with Toxoplasma rDNA by nucleic acid homology studies gave 76% similarity with the dinoflagellate Prorocentrum micans, 66% with yeast Saccharomyces cerevisiae, and 64% with the ciliate Tetrahymena thermophila. Similarity was apparent in the conserved core structure of the large subunit rRNA and divergent sequences were identified in the so-called divergent domains. Construction of a secondary structure model of the peptidyl transferase center of the large rRNA revealed similarities with the same domain from other life forms.

Detection of the pathogenic parasite Toxoplasma gondii by specific amplification of ribosomal sequences using comultiplex polymerase chain reaction.

Amplification of DNA sequences from ribosomal DNA (rDNA) was tested as a specific and sensitive method for the detection of small numbers of Toxoplasma gondii tachyzoite cells. We applied the polymerase chain reaction (PCR) on the basis of detection of the 110-fold repetitive rDNA as a target by using (i) DNA sequences within the small ribosomal subunit known to be universal and conserved in all eukaryotes and (ii) small ribosomal subunit and intergenic spacer rDNA sequences known to be T. gondii species specific. The level of sensitivity obtained from a crude cell lysate allowed the detection of as few as one parasite visualized directly as a specific PCR product in agarose gels. By using a combination of universal and T. gondii species-specific primers, we propose a comultiplex-based PCR approach as a new diagnostic tool. The combination of sensitivity, specificity, and built-in positive and negative PCR controls should make detection of the rDNA sequences by comultiplex PCR a useful clinical test for the diagnosis of toxoplasmosis and for epidemiological studies. Finally, the idea of a built-in positive control to support or counter the T. gondii-specific PCR result is novel and is a notable advance.