Transformation with pBC-bR Phleo resulted in 60 Phleo-resistant colonies, 34% of which were PCR-positive strains (Table 3), while transformation with the HP1 construct yielded an average of 3 transformants from 10 colonies (30%) (Table 4). Discussion Since protoplast-based and Agrobacterium-mediated transformation methods are complex and time-consuming, we set out to develop new and simple transformation methods for B. cinerea. We tested different transformation methods, two of which were based on published transformation protocols (electroporation and blasting) and one which is a newly developed

method and is based on wounding-mediated transformation LY294002 manufacturer of sclerotia. Electroporation did not yield any results despite repeated attempts under

various conditions. In addition, there is no published protocol for B. cinerea and other labs that have tried this method have not reported positive results. Of the other two methods described, transformation of sclerotia was efficient (15-50%), easy to perform, required CB-5083 no dedicated instruments or reagents, and colonies appeared after a relatively short time. A significant advantage of this method is the possibility of storing sclerotia for long periods but obviously, it can only be used on strains and species which form sclerotia. The second method, bombardment of DNA with high-pressure air blasted directly onto the growing hyphal tips, also demonstrated good efficiency (30-40%) and took only a short time, as has also been shown for S. sclerotiorum [12]. Unlike conventional bombardment, this method employs a DNA solution that contains a surfactant, which may assist in DNA penetration into the cells [17, 18], Crenigacestat molecular weight rather than solid particles such as tungsten or gold [22]. However, it should be noted that this method requires a specialized instrument. Both methods required small amounts of DNA construct, which can be a significant advantage in terms of cost and throughput, and both methods demonstrated Terminal deoxynucleotidyl transferase facilitated, high efficiency gene targeting (50-60%). One possible explanation for the positive results with the sclerotium and blast methods is the fact that they impose minimal stress

on the cells. In contrast, the electroporation method requires diversion of metabolism to cell-wall regeneration and membrane recovery, and both of these processes may result in a significant stress response. We believe that these reproducible and reliable transformation procedures will increase the efficiency of transformation, will simplify and improve our ability to resolve gene function in this important phytopathogen, and can be easily calibrated for additional fungi. Conclusions In this study we describe two alternative protocols–direct hyphal transformation by blasting and wounding-mediated transformation of sclerotia, which are fast, simple and reproducible and might improve functional analysis in B. cinerea and other sclerotium-forming fungi.