PITG_02860 Gene Silencing and Functional Identification in Phytophthora infestans
WANG Na1,2,*, ZHANG Yang-Qian1,*, TAN Chen1, XU Pei1,2, LUO Zhan-Hong4, GAO Jian-Li3, TANG Wei1,2,**, LIU Jing1,2,**
1 School of Life Science, Yunnan Normal University, Kunming 650500, China; 2 Joint Academy of Potato Science, Yunnan Normal University, Kunming 650500, China; 3 Wenshan Academy of Agriculture Sciences, Wenshan 663099, China; 4 Yunnan Yinmore Modern Agriculture Co., Ltd., Kunming 650500, China
Abstract Mating system is an important trait in oomycetes, effector PITG_02860 was segregated and linked with the mating type of A1 of Phytophthora infestans. In order to investigate the function of the P. infestans PITG_02860 gene, protoplasts transformation protocol was optimized under the conditions of the mycelium cultured with soybean juice for 72 h, and then lysed by the complex enzyme (10 mg/mL lysing enzyme, 5 mg/mL cellulase) at 26 ℃ for 45 min, resulted in the highest rate of preparation of protoplasts, which was up to (31.5±2.36) protoplasts/μL; and the regeneration of the protoplasts in the medium of rye could reach to (3.34±0.20)%. Based on this transformation system, the PITG_02860 gene silencing vector was constructed with the vector pTOR-mRFP as the backbone and transformed into strain 88069. Functional verification of the 2 silenced strains revealed that, compared to the wild-type, the number of oospores produced during sexual reproduction significantly decreased and the rate of oospore deformity significantly increased (P<0.05), the growth of hyphae was hindered, the number of zoospores significantly decreased (P<0.05), and the pathogenicity to potato (Solanum tuberosum) leaves was significantly reduced (P<0.05). The above results indicated that PITG_02860 was not only a key gene for the normal development of sexual reproduction of P. infestans, but also an important virulence function gene when infecting potatoes. The study provides new ideas for sexual reproduction occurrence and virulence mechanism of P. infestans.
WANG Na,ZHANG Yang-Qian,TAN Chen, et al. PITG_02860 Gene Silencing and Functional Identification in Phytophthora infestans[J]. 农业生物技术学报, 2024, 32(6): 1404-1414.
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