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.
[1] 陈孝仁. 2007. 大豆疫霉侵染早期机制的分子解析[D]. 硕士学位论文, 南京农业大学, 导师: 郑小波, pp. 47-53. (Chen X R, 2007. Molecular dissection of the events occurred during Phytophthora sojae early infection of soybean[D]. Thesis for M.S., Nanjing Agricultural University, Supervisor: Zheng X B, pp. 47-53.) [2] 陈孝仁, 王源超, 张正光, 等. 2005. 大豆疫霉菌原生质体制备及再生菌株的生物学性状[J]. 南京农业大学学报, 28(04): 45-49. (Chen X R, Wang Y C, Zhang Z G, et al.2005. Protoplast preparation of Phytophthora sojae and biological characterization of regenerated cell[J]. Journal of Nanjing Agricultural University, 28(04): 45-49.) [3] 李灿辉, 杨文丽, 王军. 2002. 论马铃薯的文化意义和社会影响[J]. 云南师范大学学报(哲学社会科学版), 34(2):122-128. (Li C H, Yang W L, Wang J.2002. Talk about the cultural significance and social influence of potato[J]. Journal of Yunnan Normal University (Philosophy and Social Sciences Edition), 34(2): 122-128.) [4] 龙昊, 汪天虹, 刘宣, 等. 2006. 瑞氏木霉pyrG基因缺陷型菌株筛选以及GnTI和VHb基因表达研究[C]//. 第二届中国青年学者微生物遗传学学术研讨会论文集, pp. 117-118. (Long H, Wang T H, Liu X, et al.2006. Screening of pyrG gene-deficient strains of Trichoderma reesei and study on the expression of GnTI and VHb genes[C]. Proceedings of the 2nd Chinese Young Scholars Symposium on Microbial Genetics, pp. 117-118.) [5] 王伟伟, 肖燕, 张艺夕, 等. 2018. 马铃薯晚疫病菌原生质体制备及再生体系的研究[J]. 生物技术通报, 34(4): 77-82. (Wang W W, Xiao Y, Zhang Y X, et al.2018. Preparation and regeneration method of Phytophthora infestans protoplast in potato[J]. Biotechnology Bulletin, 34(4): 77-82.) [6] 缪云琴, 孟然然, 唐唯, 等. 2016. 马铃薯晚疫病菌交配型检测方法比较[J]. 西南农业学报, 29(07): 1525-1529. (Miao Y Q, Meng R R, Tang W, et al .2016. Comparison of methods for detecting mating type in Phytophthora infestans[J]. Southwest China Journal of Agricultural Sciences, 29(07): 1525-1529.) [7] 许志刚, 胡白石. 2021. 普通植物病理学[M]. 北京: 高等教育出版社, pp. 18-32. (Xu Z G, Hu B S.2021. General Plant Pathology[M]. Higher Education Press, Beijing, China, pp. 18-32.) [8] 郑小波. 1997. 疫霉菌及其研究技术[M]. 北京: 中国农业出版社, pp. 85-91. (Zheng X B.1997. Phytophthora and its research technology[M]. China Agricultural Press, Beijing, China, pp. 85-91.) [9] 赵竟男, 江梅, 苏晓庆. 2008. 灭蚊真菌贵阳腐霉原生质体的制备和再生[J]. 贵阳医学院学报, 33(2): 111-114. (Zhao J N, Jiang M, Su X Q.2008. Study on the preparation and regeneration of protoplasts of Pythium guiyangensis, a mosquito-killing fungus[J]. Journal of Guiyang Medical College, 33(02): 111-114.) [10] 赵伟, 杨新宇, 董莎萌, 等. 2011. 外源合成dsRNA介导大豆疫霉PsCdc14基因沉默后对孢子囊发育的影响[J]. 中国科学: 生命科学, 41(12): 1177-1184. (Zhao W, Yang X Y, Dong S M, et al.2011. Transient silencing mediated by in vitro synthesized double-stranded RNA indicated that PsCdc14 is required for sporangia development in soybean root rot pathogen[J]. Science of China: Life Sciences, 41(12): 1177-1184.) [11] 祝菊澧, 梁静思, 王伟伟, 等. 2020. 马铃薯致病疫霉研究进展[J]. 微生物学通报, 47(03): 952-966. (Zhu J L, Liang J S, Wang W W, et al.2020. Research progress in Phytophthora infestans, pathogen of potato late blight[J]. Microbiology China, 47(3): 952-966.) [12] Bottin A, Larche L, Villalba F, et al.1999. Green fluorescent protein (GFP) as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phythphthora parasitica var. nicotianae[J]. FEMS Microbiology Letter, 176(1): 51-56. [13] Cao J, Qiu M, Ye W, et al.2022. Phytophthora sojae transformation based on the CRISPR/Cas9 system[J]. Bio -protocol, 12(2): e4352. [14] Clarke D D.1984. Phytophthora: Its biology, taxonomy, ecology and pathology[J]. Physiologial Plant Pathology, 24(2): 254-255. [15] Cvitanich C, Judelson H S.2003. Stable transformation of the oomycete, Phytophthora infestans, using microprojectile bombardment[J]. Current Genetics, 42(4): 228-235. [16] Danies G, Myers K, Mideros MF, et al.2017. An ephemeral sexual population of Phytophthora infestans in the Northeastern United States and Canada[J]. PLOS ONE,9(12), 395-400. [17] Dai T, Xu Y, Yang X, et al.2021. An improved transformation system for Phytophthora cinnamomi using green fluorescent protein[J]. Frontiers in Microbiology, 5(12): 1743-1746. [18] Fry W E, Goodwin S.1997. Re-emergence of potato and tomato late blight in the United States[J]. Plant Disease,81(12): 1349-1357. [19] Fry W.2008. Phytophthora infestans: The plant (and R gene) destroyer[J]. Molecular Plant Pathology, 9(3): 385-402. [20] Fry W E, McGrath M T, Seaman A.2013. The 2009 late blight pandemic in the eastern United States causes and results[J]. Plant Disease, 97(3): 296-306. [21] Jeger M J, Viljanen-Rollinson S L H.2001. The use of the area under the disease-progress curve (AUDPC) to assess quantitative disease resistance in crop cultivars[J]. Theoretical and Applied Genetics, 102: 32-40. [22] Judelson H S, Michelmore R W.1991. Transient expression of genes in the oomycete Phytophthora infestans using Bremia lactucae regulatory sequences[J]. Current Genetics, 19(6): 453-459. [23] Judelson H S, Coffey M D, Arredondo F R, et al.1993. Transformation of the oomycete pathogen Phytophthora megasperma f. sp. glycinea occurs by DNA integration into single or multiple chromosomes[J]. Current Genetics, 23(3): 211-218. [24] Kassaw A, Belete E, Abera M.2021. Potato late blight (Phytophthora infestans) incidence and severity influenced by cropping practices adopted in North Wollo, Eastern Amhara, Ethiopia[J]. Indian Phytopathology, 74(4): 981-991. [25] Latijnhouwers M, Govers F.2003. A Phytophthora infestans G protein β subunit is involved in sporangia formation[J]. Eukaryotic Cell, 2(5): 971-977. [26] Li Y, Van D L T A J, Evenhuis A, et al.2012. Population dynamics of Phytophthora infestans in the Netherlands reveals expansion and spread of dominant clonal lineages and virulence in sexual offspring[J]. G3: Genes| Genomes| Genetics, 2(12): 1529-1540. [27] Li Y, Shen H, Zhou Q, et al.2017. Changing ploidy as a strategy: The Irish potato famine pathogen shifts ploidy in relation to its sexuality[J]. Molecular Plant-microbe, 30(1):45-52. [28] Liu J, Wang Z K, Sun H H, et al.2017. Characterization of the Hog1 MAPK pathway in the enotmopathogenic fungus Beauveria bassiana[J]. Environmental Microbiology, 19(5): 1808-1821. [29] Mort-Bontemps M, Fevre M.1997. Transformation of the oomycete Saprolegnia monoica to hygromycin-B resistance[J]. Current Genetics, 31(3): 272-275. [30] Mcleod A, Fry B A, Zuluaga A P, et al.2008. Toward improvements of oomycete transformation protocols[J]. Journal of Eukaryotic Microbiology, 55(2): 103-109. [31] Sheperd S J, van West P, Gow N A R.2003. Protcomic analysis of asexual development of Phtopthora palmivora[J]. Mycological Research, 107(4): 395-400. [32] Siammour A, Mauchmani B, Mauch F.2013.Quantification of induced resistance against Phytophthora species expressing GFP as a vital marker: β-aminobutyric acid but not BTH protects potato and Arabidopsis from infection[J]. Molecular Plant Pathology, 4(4):237-248. [33] VanWest P, deJong A J, Judelson H S, et al.1998. The ipiO gene of Phytophthora infestans is highly expressed in invading hyphae during infection[J]. Fungal Genetics and Biology, 23(2): 126-138. [34] VanWest P, Shepherd S J, Walker C A, et al.2008. Internuclear gene silencing in Phytophthora infestans is established through chromatin re-modelling[J]. Microbiology, 154(5): 1482-1490. [35] VanWest P, Reid B, Campbell T A, et al.1999. Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora palmivora[J]. FEMS Microbiology Letters, 178(1): 71-80. [36] Vijn I, Govers F.2003. Agrobacterium tumefaciens mediated transformation of the oomycete plant pathogen Phytophthora infestans[J]. Molecular Plant Pathology, 4(6): 459-467. [37] Wang Z, Tyler B M, Liu X.2018. Protocol of Phytophthora capsici transformation using the CRISPR-Cas9 system[J]. Plant Pathogenic Fungi and Oomycetes: Methods and Protocols, 1848: 265-274. [38] Weiland J J.2003. Transformation of Pythium aphanidermatum to geneticin resistance[J]. Current Genetics, 2(2): 191-199. [39] Yi S Y, Kim Y J, Hwang B K.1993. Protoplast formation and regeneration from mycelia of Phytophthora capsici[J].The Korean Journal of Mycology, 21(1): 1-8. [40] Yang L, Mclellan H, Naqvi S, et al.2016. Potato NPH3/RPT2-like protein StNRL1, targeted by a Phytophthora infestans RXLR effector, is a susceptibility factor[J]. Plant Physiology, 171(1): 645-657.
