|Norelli, John (jay) - Jay|
Submitted to: Journal of the American Society for Horticultural Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2002
Publication Date: 7/20/2002
Citation: KO, K., NORELLI, J.L., REYNOIRD, J., BROWN, S. T4 LYSOZYME AND ATTACIN GENES ENHANCE RESISTANCE OF TRANSGENIC 'GALAXY' APPLE AGAINST ERWINIA AMYLOVORA (BURR.). JOURNAL OF AMERICAN SOCIETY OF HORTICULTURE SCIENCE. July 2002; Vol. 127, no. 4; pgs. 515-519. Interpretive Summary: This manuscript reports research aimed at increasing the resistance of 'Galaxy' apple (a commercially popular red-colored sport of 'Gala') to fire blight (a destructive plant disease). Genes for two different antibacterial proteins, called lysozyme and attacin, were transferred to 'Galaxy' using biotechnology methods. The lysozyme used is native to a bacterial virus and dthe attacin gene used is native to the giant silkworm moth. The genes were transferred both singly, to observe their individual effects on resistance, and in combination, to observe if they had enhanced activity when combined together. The genes were transferred such that the lysozyme gene was expressed constitutively (always expressed in all plant tissues), while the attacin gene was expressed by a regulatory element that was found to be both constitutive and with enhanced activity after wounding. Using methods of biochemical analysis it was confirmed that the genes were transferred to oall the transgenic plants studied and that both genes were transcribed (made into RNA messages for protein synthesis) in these plants. In addition, it was shown that attacin protein was produced in transgenic plants. Some of the transgenic plants containing the lysozyme gene or the attacin gene developed significantly less disease when challenged with the fire blight pathogen, indicating that these genes are useful in enhancing apple's resistance to fire blight disease. However, plants containing both genes showed no significant reduction in disease, indicating there was no advantage in combining these genes in the plant.
Technical Abstract: Genes encoding T4 lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of 'Galaxy' apple, enhanced resistance to Erwinia amylovora (Burr.). In these plasmids, T4L generally was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream domain and the untranslated leader sequence of alfalfa mosaic virus RNA4, and attE gene was controlled by the potato proteinase inhibitor II (Pin2) promoter. All transgenic lines were screened by polymerase chain reaction (PCR) for T4L and attE genes, and a double antibody sandwich enzyme linked immunosorbent assay for neomycin phosphotransferase II. Amplification of T4L and attE genes was observed in reverse transcriptase-PCR, indicating that these genes were transcribed in all tested transgenic lines containing each gene. The attacin protein was detected in all attE transgenic lines. The expression of attE under the Pin2 promoter was constitutive but higher level of expression were observed after mechanical wounding. Some T4L and attE transgenic lines had significant disease reduction compared to non-transgenic 'Galaxy'. However, transgenic lines containing both attE and T4L genes were not significantly more resistant than non-transgenic 'Galaxy', indicating that there was no synergy between attE and T4L in enhancing resistance to E. amylovora.