The expression of yeast antifungal genes in tobacco as possible pathogenesis-related proteins

[摘要] ENGLISH ABSTRACT:The resistance of plants to infection by phytopathogenic microorganisms is theresult of multiple defence reactions comprising both constitutive and induciblebarriers. While disease is the exception, such exceptions can be costly andeven devastating. In particular, fungal diseases remain one of the major factorslimiting crop productivity worldwide, with huge losses that need to be weighedup against massive cash inputs for pesticide treatments.Part of the defence reactions of plants is the synthesis ofpathogenesis-related proteins, such as the plant hydrolases, glucanases andchitinases. In recent years, attention has been paid to the implementation ofthese proteins in plant transformation schemes. The rationale for this approachwas that these antimicrobial agents not only degrade the main cell wallcomponents of fungi, but also produce glucosidic fragments that act as elicitorsof the biosynthesis of defence metabolites by the host. Furthermore, sincethese active antimicrobial agents are individually encoded by single genes,these defence systems should and have been shown to be highly amenable tomanipulation by gene transfer.In this study, yeast glucanases from Saccharomyces cerevisiae wereevaluated for their potential as antifungal proteins. The glucanases tested fortheir antifungal activity against Botrytis cinerea were the yeast EXG1 and BGL2genes, encoding an exoglucanase and an endoglucanase respectively. An invitro assay performed on these glucanases indicated that exoglucanase had amore detrimental effect on B. cinerea hyphal development and growth than theendoglucanase; the former caused typical disruption of the cells and leakage ofcell material. The yeast exoglucanase was subsequently subcloned into a plantexpression cassette containing the strong constitutive 358 promoter, yieldingplasm ids pEXG1 and pMJ-EXG1. The pMJ-EXG1 construct targeted theexoglucanase to the apoplastic region with a signal peptide from anantimicrobial peptide from Mirabilis jalapa, Mj-AMP2. The pEXG1 andpMJ-EXG1 constructs were mobilised into Agrobacterium tumefaciens tofacilitate the subsequent tobacco transformation, which yielded transgenic tobacco lines designated E and MJE respectively. Transgene integration wasconfirmed with southern blot and PCR analyses for both the E and MJE lines.The expression and heterologous production of the EXG1-encodedexoglucanase in the E-transgenic lines was shown with northern blots andactivity assays respectively. Moreover, the high level of expression of the yeastexoglucanase led to a decrease in susceptibility of the E lines to B. cinereainfection in comparison to the untransformed tobacco controls. An averagedecrease in disease susceptibility of 40% was observed in an in plantadetached leaf assay. Crude protein extracts from the E lines were alsoanalysed in an in vitro quantitive fungal growth assay, inhibiting in vitro fungalgrowth by average 20%, thus further confirming the antifungal nature of theyeast exoglucanase.Although integration of the MJ-EXG1 expression cassette was confirmed,no mRNA levels could be detected with northern blot or RT-PCR analysis of theMJE lines. These lines also did not show any in vitro antifungal activities or adecrease in susceptibility to B. cinerea infection in the detached leaf assay. It issuspected that this result is possibly linked to gene silencing, a phenomenonquite frequently associated with heterologous and/or overexpression ofglucanases in plant hosts. It appears as if the targeted overexpression to theapoplastic space triggered the gene silencing response, since the intracellularlyoverexpressed product was produced and shown to display activity. The yeastexoglucanase thus joins the list of silenced glucanases in overexpressionstudies in plants.Overall, this study confirmed the antifungal characteristics of theSaccharomyces exoglucanase and provides valuable information of thepossibility of utilising yeast glucanases in a transgenic environment. Adecrease in the susceptibility of tobacco to B. cinerea infection, as shown by theoverexpressed EXG1-encoded exoglucanases, merits further investigation intothe use of this gene in the engineering of disease-resistant crops.

[发布机构] Stellenbosch University