Michael Wayne Graham

Expression patterns of vascular-specific promoters RolC and Sh in transgenic potatoes and their use in engineering PLRV-resistant plants.

The expression patterns of GUS fusion constructs driven by the Agrobacterium rhizogenes RolC and the maize Sh (Shrunken; sucrose synthase-1) promoters were examined in transgenic potatoes (cv. Atlantic). RolC drove high-level gene expression in phloem tissue, bundle sheath cells and vascular parenchyma, but not in xylem or non-vascular tissues. Sh expression was exclusively confined to phloem tissue. Potato leafroll luteovirus (PLRV) replicates only in phloem tissues, and we show that when RolC is used to drive expression of the PLRV coat protein gene, virus-resistant lines can be obtained. In contrast, no significant resistance was observed when the Sh promoter was used.

Adenine methylation atdam sites increases transient gene expression in plant cells

Escherichia coli encodes two major DNA methylation systems:dam, which produces 6-methyladenine; anddcm, which produces 5-methylcytosine. About 1–2% of adenine and cytosine residues in plasmid DNAs prepared inE. coli are methylated by these systems. Since DNA methylation profoundly influences gene expression in eukaryotes, we were interested in determining whether these bacterially encoded modifications might also effect plant gene expression in experimental systems. We therefore examined the influence ofdam anddcm methylation on gene expression from four GUS fusion constructs in transient assays in protoplasts and microprojectile-bombarded whole tissues. In these constructs, GUS expression was driven by promoter regions derived from theArabidopsis alcohol dehydrogenase (Adh1), maize ubiquitin (Ubil1), rice actin (Act1) and CaMV 35S genes. We show that methyladenine produced bydam methylation increased gene expression from constructs based on theAdh1, Ubi1 andAct1 genes. The increase in gene expression ranged from three-fold forUbi1 andAdh1 in protoplasts to 50-fold forAct1 in bombarded wheat tissues. Expression of a 35S. GUS construct was, however, insensitive todam methylation.dcm methylation had little if any effect on transient gene expression for any of these constructs. We provide indirect evidence that the critical sites of adenine methylation lie within sequences from the promoter regions, suggesting thatdam methylation increases transcription rate. These results have important experimental implications and also raise the intriguing possibility that methyladenine might play a role in the regulation of gene expressionin vivo.