Objective 1: Characterize VvMEl and VvME2 expression in grape leaves with regard to wounding or fungal attack.
The work thus far has shown that there are two malic enzyme genes in grape, VvMEl and VvME2. The VvMEl enzyme is a cytoplasmic form and VvME2 appears to be targeted to the plastid. Expression of grape malic enzyme is increased in leaves that are wounded and there is an additional increase when leaves are treated with elicitors after wounding. Using gene-specific probes we have shown that the plastid-targeted (VvME2) form is the one increased in leaves by wounding and elicitors, whereas the cytoplasmic form (VvMEl)remains relatively constant under these conditions.
Objective 2: Construct cDNA libraries from normal and elicitor-treated grape leaf tissue.
We isolated RNA from control grape leaves and ones treated with various elicitors and selected several samples from which to make the corresponding cDNA libraries. We made cDNA from the RNA by reverse transcription and are now prepared to attach linkers and make the libraries if this is indicated by the progress of the project (see above).
Objective 3: Carry out differential display experiments to identify mRNAs that are induced by wounding or fungal attack.
We isolated RNA from grape leaves that were either unwounded (control) or induced with various treatments. A cDNA library of the mRNAs was constructed using reverse transcriptase and we amplified a grape glutathione reductase from the cDNA. This result is important because it demonstrates that the RNA isolation and the reverse transcriptase step were successful. We designed a degenerate oligonucleotide specific to the region of phenylalanine ammonia lyase (PAL) encoding the active site of the protein. This oligonucleotide was used in conjunction with an “nv-poly-dT” primer to selectively amplify sequences from the cDNA. Seven bands were obtained and these are being sequenced to confirm that they represent PAL cDNAs. The experiments with glutathione reductase and PAL have shown that we have a working system and we are now proceeding to the more difficult step of amplifying non-specific sequences by random priming in the PCR amplification step.