Transformation of tobacco with a lupin chitinase gene under control of a stress inducible promoter

Abstract

Chitinases are a diverse family of proteins occurring in plants. Their function varies considerably, with certain chitinases having been associated with development. The majority however, are pathogenesis related (PR) proteins that have been shown to play a role during plant pathogen interactions. This has lead to many investigations on the use of chitinases in providing transgenic disease resistance. These studies are usually done using a constitutive expression system. This however stands in contrast with the natural defense system were PR gene expression is usually only upregulated when the plant is exposed to abiotic and/or biotic stress factors. The constitutive expression is therefore not ideal as it increases ‘cost’ penalties due to the energy being spent expressing the gene. In this study however, an inducible expression system was applied using a stress inducible promoter AtGSTF6 derived from Arabidopsis thaliana, to drive Lupinus albus IF3 chitinase expression when the plants are under pathogen attack. The construct AtGSTF6-IF3 was inserted into the binary vector pCAMBIA 2300 and transformed into Nicotiana Tabacum cv JR6 by Agrobacterium-mediated transformation. To demonstrate the functionality of such a construct, an expression study was done on transgenic N. Tabacum to determine transcription and in vitro chitinase enzyme activity. The data revealed that IF3 chitinase gene transcription from lupin plants was achieved in N. Tabacum. Nine of the twelve lines that tested positive for chitinase gene transcription after hydrogen peroxide treatment, showed increased chitinase activity. With the success of showing increased chitinase activity, these lines were subjected to a detached leaf assay with Rhizoctonia solani AG2, which causes leaf target spot disease. The assay showed that six of the nine lines identified as having increased chitinase activity showed reductions in lesion areas. More specifically, three of the four lines showing more than a five-fold increase in chitinase activity compared to the untransformed N. Tabacum, showed significant lesion reduction. The AtGSTF6-IF3 construct can therefore be recommended to increase disease resistance in N. Tabacum towards Rhizoctonia solani AG2 after showing both expression and increased disease resistance in certain transgenic lines. Copyright