Plant defence genes expressed in tobacco and yeast

[摘要] Pathogen devastation of food products has been the topic of extensive research effortsworldwide. Fungal infections are foremost amongst these pests, contributing not only tolosses in product yield, but also significantly affecting the quality thereof. It is not surprisingthen that producers of these foodstuffs and their derived products continually strivetowards the highest possible product quality. Therefore, it remains imperative thatsatisfactory methods are implemented to control these fungal pathogens. The currentstrategies are all hampered by drawbacks, and severe crop losses are still experienced.New technologies are being explored; one such technology is the genetictransformation of plant species. This method has enabled scientists to introduce foreigngenes, with known functions and predictable outcomes, into plants. Genes identified to beinvolved in disease resistance have become the focus of numerous research effortsconcerned with the improvement of the plant's innate defence response. This study aimedto enhance disease resistance to fungal pathogens by means of the genetic transformationof two genes previously shown to be involved in disease resistance. These genes encodepolygalacturonase-inhibiting proteins (PGIPs) from Phaseolus vulgaris and resveratrolsynthase from Vitis vinifera. PGIPs specifically inhibit the action of fungalpolygalacturonases (PGs), which are enzymes responsible for the hydrolytic breakdown ofplant cell walls. These enzymes were also found to be the first enzymes that are secretedby fungal pathogens during infection of the host plant. Additionally, PGIP-PG interactionresults in the existence of molecules involved in the activation of plant defence responses.Resveratrol, the product of resveratrol synthase, exerts its antifungal action by destructionof the microbial cellular membranes. These mentioned genes were transformed alone, andin combination, into Nicotiana tabacum and the resultant transgenic lines were evaluatedfor enhanced disease resistance and for possible synergistic effects between thetransgenes.Several independent transgenic lines were regenerated with genes integrated into thetobacco genome. Almost all the plants harbouring only pgip or vst1 genes also expressedthese genes at a high frequency. Some non-expressing lines were identified from thetransgenic plants that had integrated both genes, but several lines were obtainedexpressing both transgenes. Good correlations were observed between transgene productactivity and enhanced resistance to the fungus Botrytis cinerea in an antifungal in plantaassay. Lines showing the highest PGIP activity and resveratrollevels were more resistantto the pathogen, leading to disease resistance of up to 80% seven days after inoculation incomparison to an untransformed control. These lines maintained their strong inhibition,even three weeks post-inoculation, showing a complete halt in disease development andfungal growth. These results provide good indications of the efficacy of these transgenesin the upregulation of plant defence. However, the study will have to be expanded to include even more transgenic lines to elucidate the possible synergistic effects of thesegenes.In an additional pilot study, genes encoding for precursors and for the formation ofresveratrol were introduced into the yeast Saccharomyces cerevisiae. The resultantrecombinant yeast strains were evaluated for their ability to produce the phenolicsubstance, resveratrol. This compound has been implicated in beneficial aspects relatingto human health, including positive effects on atherosclerosis and platelet aggregation as adirect result of its antioxidant and anti-inflammatory activities.Recombinant yeast strains were constructed that expressed genes coding forcoenzyme A ligase and resveratrol synthase. These strains were shown to be able toproduce the phenolic compound resveratrol from the precursors present in the yeast aswell as from the products introduced with the transformation. The resveratrol wascomplexed with an added glucose moiety. These results are extremely positive,considering the possibility of manipulating wine yeasts to produce resveratrol during thewine fermentation, thereby adding to the health aspects of both red and white wine. This isthe first report of the production of this compound by the introduction of genes necessaryfor its biosynthesis in a foreign host.This study has confirmed the importance of PGIPs and resveratrol in the effort toenhance disease resistance in plants through genetic transformation technology. It hasalso shown that the health benefits of resveratrol could be exploited more optimally in thewine industry, by producing wine yeasts with the ability to synthesise this importantantioxidant.

[发布机构] Stellenbosch University