ATMT transformation efficiencies with native promoters in Botryosphaeria kuwatsukai causing ring rot disease in pear

Botryosphaeria kuwatsukai is an important fungal pathogen affecting pear fruits. However, infection processes of this fungus are still unclear. This study seeks to develop the fungal transformation of B. kuwatsukai by Agrobacterium tumefaciens-mediated transformation (ATMT), assess the reliability of appropriate vectors and examine the infection processes in vitro using a GFP labeled strain of B. kuwatsukai. To establish a highly effective transformation system in B. kuwatsukai, binary vectors containing various lengths of H3 promoters and TEF promoters fused with GFP and hygromycin B resistance gene cassettes were constructed. These cassettes were integrated into the genomic DNA of B. kuwatsukai with high transformation frequency by the ATMT method. Transformants showed strong expression of GFP and hygromycin B resistance genes in cells. Furthermore, we investigated if native promoters are more suitable to govern marker genes than other general promoters used in other filamentous fungi. The results obtained herein demonstrate that the vectors constructed in this study can be utilized with high transformation rate. Microscopic examinations also reveal that fungal hyphae undergo morphological changes during the infection process resulting in biotrophic stage of infected host cells. Our results provide genetic insights to further explore the infection processes of B. kuwatsukai.