R. Oláh

Candidate plant gene homologues in grapevine involved in Agrobacterium transformation

The grapevine (Vitis vinifera) genome was analyzed in silico for homologues of plant genes involved in Agrobacterium transformation in Arabidopsis thaliana and Nicotiana spp. Grapevine homologues of the glucomannan 4-betamannosyltransferase 9 gene CslA-09 involved in bacterial attachment to the cell wall, homologues of reticulon-like proteins BTI1, 2, 3 and RAB8 GTPases, both involved in T-DNA transfer to the host cell, homologues of VirE2 interacting protein VIP1 that contributes to the targeting of T-DNA into the nucleus and to its integration, and homologues of the histone protein H2A, which promotes the expression of T-DNA encoded genes, were selected. Sequences homologous to the arabinogalactan-protein AtAGP17 were not found in the grape genome. Seventeen selected candidates were tested by semiquantitative RT-PCR analysis for changes in their expression levels upon inoculation with Agrobacterium tumefaciens C58. Of the tested homologues, the expression of VvRab8a, VvVip1a and two histone genes (VvHta2 and VvHta10) increased significantly, therefore we hypothesize that these might be involved in Agrobacterium transformation of V. vinifera.

Use of an intron containing grapevine gene as internal control for validation of cDNA synthesis in virus detection by RT-PCR

Detection of viruses in grapevine plants is important to prevent their spreading with propagating stocks. For this purpose PCR based protocols are commonly and routinely used. Since most grapevine viruses are RNA viruses, the detection procedure starts with RNA purification followed by cDNA synthesis prior to PCR. The cDNAs should be validated by an internal control for which usually constitutively expressed housekeeping genes are used. Here we publish a new set of primers designed in the Vitis vinifera phosphoenolpyruvate carboxylase gene to encompass two or three introns. PCR products amplified from remaining genomic DNA in the RNA samples and from cDNA can be clearly distinguished since cDNA-derived products are smaller in size compared to those amplified from genomic DNA. Thus these primers may be useful reference markers in RT-PCR-based virus detection assays both for the validation of cDNA synthesis and the detection of trace amounts of genomic DNA.