Transformation of the obligate biotrophic rust fungus Uromyces fabae

专性生物营养真菌与锈菌一样，是一类主要的病原菌，尤其是世界范围内重要的经济作物。然而，由于缺乏稳定转化的系统，对锈菌和其他专性生物营养物质的研究一直受到阻碍。我们已经建立了生物信息学作为一种向豆类锈菌Uromyces Fabae转基因的传递工具。来源于质膜ATPase基因的内源调控元件(UF-PMA1)被用来驱动颜色和选择标记的表达。GUS、GFP和DsRed已被确定为颜色标记，而内源基因琥珀酸脱氢酶1(SucDH1(H254Y))的突变形式被用作选择标记。该系统使用杀菌剂卡波辛作为选择剂，并允许在寄主植物蚕豆上直接选择转化子。然而，到目前为止，转基因在几轮繁殖后就消失了。我们正在建立基于农杆菌介导的基因传递的第二种转化方法。该系统使用与生物系统相同的控制元件、标记和选择程序，但具有提高转基因稳定性的优势。因此，未来的工作将集中在根癌农杆菌介导法的改进和将该方法推广到其他锈菌上。

Obligate biotrophic fungi, like the rust fungi, represent a major class of pathogens especially of economically important crops world wide. Yet, research on rust fungi and other obligate biotrophs has been hampered by the absence of a system for stable transformation. We have established Biolistics as a delivery tool for transgenes into the legume rust Uromyces fabae. Endogenous regulatory elements derived from the plasma membrane ATPase gene (Uf- PMA1) are used to drive expression of color and selection markers. GUS, GFP and DsRed have been established as color markers, while a mutated form of the endogenous gene succinate dehydrogenase 1 (SucDH1(H254Y)) is used as selection marker. The system uses the fungicide Carboxin as selective agent and allows a selection of transformants directly on the host plant Vicia faba. However, so far the transgene is lost after a couple of rounds of propagation. We are in the process of establishing a second transformation method based on Agrobacterium tumefaciens mediated gene delivery. This system uses the same control elements, markers and selection procedure as the biolistic system, but has the advantage of an increased stability of the transgenes. Future work will therefore center on the improvement of the A. tumefaciens mediated technique and on the extension of the method to other rust fungi.