Next generation RNA sequencing to identify overexpressed transporter genes in Zymoseptoria tritici field strains. Zymoseptoria tritici

Septoria leaf blotch is a worldwide threat for wheat and mainly controlled by the application of synthetic fungicides. The fungal pathogen responsible for this disease, Zymoseptoria tritici, was shown as highly adaptable to its host plant, but also to fungicide challenge. Over the past decades it developed resistance to most fungicides due to target site modifications. Recently isolated strains showed cross-resistance to diverse fungicides and to unrelated drugs, suggesting a resistance mechanism that seems rarer in phytopathogenic fungi, known as multidrug resistance (MDR) in other organisms. In this study we show for two Z. tritici MDR strains, MDR6 and MDR7, enhanced prochloraz efflux sensitive to the modulators amitryptiline and chlorpromazine. Efflux was also inhibited by verapamil in the MDR7strain. Transcriptomics revealed several overexpressed transporter genes in both MDR strains, out of which the expression of the MgMFS1 transporter gene was the strongest and constitutively high in tested MDR field strains. Its inactivation in the MDR6 strain abolished resistance to fungicides with different modes of action revealing its involvement in the MDR phenomenon in Z. tritici. Overall design: A total of four strains were compared, two sensitive (IPO323, S6) and two MDR strains (09-ASA-3apz; 09-CB01) with three replicates each. All strains were grown in liquid YPD medium to exponential growth.

壳针孢叶斑病（Septoria leaf blotch）对小麦构成全球性威胁，当前主要通过施用合成杀菌剂进行防治。引发该病害的致病菌小麦壳针孢（Zymoseptoria tritici）不仅对寄主植物具有极强适应性，同时也能应对杀菌剂的选择压力。在过去数十年间，该病菌因靶标位点突变而对绝大多数杀菌剂产生了抗药性。近期分离得到的菌株对多种杀菌剂及非同类药剂均表现出交互抗药性，这暗示其存在一种在植物病原真菌中较为罕见的抗药性机制——该机制在其他生物中被称为多药抗药性（multidrug resistance, MDR）。本研究针对两株小麦壳针孢MDR菌株MDR6与MDR7展开分析，发现其咪鲜胺（prochloraz）外排作用增强，且该外排过程可被调节剂阿米替林（amitryptiline）与氯丙嗪（chlorpromazine）抑制。在MDR7菌株中，维拉帕米（verapamil）同样可抑制其外排作用。转录组学（transcriptomics）分析显示，两株MDR菌株中均存在多个过表达的转运蛋白编码基因，其中MgMFS1转运蛋白基因的表达水平最高，且在所有受试田间MDR菌株中均呈组成型高表达。敲除MDR6菌株中的该基因后，其对不同作用机制杀菌剂的抗药性完全消失，证实该基因参与了小麦壳针孢的MDR抗药性过程。实验设计概况：本研究共对比4株菌株，包括2株敏感菌株（IPO323、S6）与2株MDR菌株（09-ASA-3apz、09-CB01），每组设置3次生物学重复。所有菌株均在液体YPD培养基（YPD medium）中培养至指数生长期。