Biotechnology Institute, Zhejiang University, Hangzhou, 310058, China.
Agricultural College, Xinjiang Agricultural University, Urumuqi, 830052, China.
World J Microbiol Biotechnol. 2018 Nov 19;34(12):179. doi: 10.1007/s11274-018-2559-8.
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.
条斑球腔菌是一种重要的影响梨果实的真菌病原体。然而，该真菌的感染过程仍不清楚。本研究旨在通过农杆菌介导的转化（ATMT）开发条斑球腔菌的真菌转化，评估合适载体的可靠性，并使用 GFP 标记的条斑球腔菌菌株在体外检查感染过程。为了在条斑球腔菌中建立一个高效的转化系统，构建了含有不同长度 H3 启动子和 TEF 启动子与 GFP 和潮霉素 B 抗性基因盒融合的二元载体。这些盒通过 ATMT 方法整合到条斑球腔菌的基因组 DNA 中，具有较高的转化频率。转化子在细胞中表现出 GFP 和潮霉素 B 抗性基因的强烈表达。此外，我们还研究了天然启动子是否比其他丝状真菌中使用的其他通用启动子更适合调控标记基因。本研究结果表明，本研究构建的载体可以以较高的转化效率利用。显微镜检查还表明，在感染过程中，真菌菌丝经历形态变化，导致感染宿主细胞的生物营养阶段。我们的研究结果为进一步探索条斑球腔菌的感染过程提供了遗传见解。
