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Effect of Autophagy-related Gene PlATG12 on the Growth, Development and Pathogenicity of Peronophythora litchii |
CHEN Tai-Xu1,2, YANG Cheng-Dong1,2, YU Ge1,2, LUO Man-Fei1,2, ZHANG Xue1,2, LYU Lin1,2, CHEN Qing-He1,2,* |
1 Sanya Nanfan Research Institute/School of Tropical Agriculture and Forestry, Hainan University, Sanya 572025, China; 2 Key Laboratory of Green Prevention and Control of Tropical Agricultural and Forestry Biological Disasters, Ministry of Education, Haikou 570228, China. |
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Abstract Autophagy plays an important role in plant resistance to pathogen infection. The autophagy-related protein 12 (ATG12), is mainly involved in the formation and extension of the double-layer membrane of autophagy. However, its role in plant pathogens, especially Oomycetes, has been rarely reported. In order to clarify the role of Peronophythora litchii PlATG12 (GeneBank No. OR909654) in the growth and development, and pathogenesis, the PlATG12 knockout mutants were obtained by CRISPR/Cas9 gene editing and PEG mediated protoplast transformation technology, and the in situ complementation was performed. Phenotypic assay results showed that compared with the wild-type strain SHS3 (WT), the average mycelial growth rate of the ΔPlatg12-77 and ΔPlatg12-315 mutants was decreased by 6.50% and 7.49%, respectively, and the average sporangium number was reduced by 33.86% and 34.02%, respectively. Meanwhile, the zoospore release rate of the mutants was significantly lower than those of WT after incubating at 12 ℃ for 0.5 and 2 h. In addition, the oospore production was only 20.25% and 22.00% of the WT, respectively, and the oospore of type Ⅲ and type Ⅳ increased significantly. The pathogenicity of PlATG12 knockout mutants on litchi (Litchi chinensis) leaves was lower than that of WT. However, the phenotypic defects of PlATG12 knockout mutants were rescued after in situ complementation of PlATG12. These results indicated that autophagy-related gene PlATG12 plays a key role in the growth and development, and pathogenesis of P. litchii. This study provides theoretical basis for the functions of autophagy pathway in the pathogenic process of oomycetes.
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Received: 28 August 2023
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Corresponding Authors:
* qhchen@hainanu.edu.cn
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[1] 杜红岩. 2023. 尖孢镰刀菌自噬基因的克隆与功能分析[D]. 硕士学位论文, 河南农业大学, 导师: 赵莹, 文才艺. pp. 24-46. (Du H Y.2023. Cloning and functional analysis of autophagy related genes in Fusarium oxysporum[D]. Thesis for M. S., Henan Agricultural University, Supervisor: Zhao Y, Wen C Y. pp. 24-46.) [2] 姜子德, 习平根, 冼继东, 等. 2011. 对未来五年我国荔枝植保研究的思考[J]. 中国热带农业, 5: 61-63. (Jiang Z D, Xi P G, Xian J D, et al.2021. Thoughts on litchi plant protection research in the next five years[J]. China Tropical Agriculture, 5: 61-63.) [3] 孔广辉, 冯迪南, 李雯, 等. 2021. 荔枝霜疫病的研究进展[J]. 果树学报, 38(04): 603-612. (Kong G H, Feng D N, Li W, et al.2021. Research progress in studies on the downy blight disease in litchi[J]. Journal of Fruit Science, 38(04): 603-612.) [4] 雷志火. 2020. 不同荔枝品种对荔枝霜疫病抗病性研究[D]. 硕士学位论文, 华南农业大学, 导师: 潘汝谦, pp. 32-38. (Lei Z H.2020. Resistance of litchi cultivars to litchi downy blight caused by Peronophythora litchii[D]. Thesis for M.S., South China Agricultural University, Supervisor: Pan R Q, pp. 32-38.) [5] Che R M, Liu C H, Wang Q, et al.2023. The Valsa mali effector Vm1G-1794 protects the aggregated MdEF-Tu from autophagic degradation to promote infection in apple[J]. Autophagy. 19(6): 1745-1763. [6] Chen L L, Zhang X, Wang W, et al.2017. Network and role analysis of autophagy in Phytophthora sojae[J]. Scientific Reports. 7(1): 1879. [7] Dikic I, Elazar Z.2018. Mechanism and medical implications of mammalian autophagy[J]. Nature Reviews Molecular Cell Biology. 19(6): 349-364. [8] Fukuda T, Ebi Y, Saigusa T, et al.2020. Atg43 tethers isolation membranes to mitochondria to promote starvation-induced mitophagy in fission yeast[J]. Elife. 9: e61245. [9] Fukuda T, Furukawa K, Maruyama T, et al.2023. The mitochondrial intermembrane space protein mitofissin drives mitochondrial fission required for mitophagy[J]. Molecular Cell. 83(12): 2045-2058. e9. [10] Fukuda T, Kanki T.2021. Atg43, a novel autophagy-related protein, serves as a mitophagy receptor to bridge mitochondria with phagophores in fission yeast[J]. Autophagy. 17(3): 826-827. [11] Fujioka Y, Noda N N, Fujii K, et al.2008. In vitro reconstitution of plant Atg8 and Atg12 conjugation systems essential for autophagy[J]. The Journal of Biological Chemistry. 283(4): 1921-1928. [12] Haller M, Hock A K, Giampazolias E, et al.2014. Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity[J]. Autophagy. 10(12): 2269-2278. [13] Hanada T, Noda N N, Satomi Y, et al.2007. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy[J]. The Journal of Biological Chemistry. 282(52): 37298-37302. [14] Hou J, Wang J J, Lin H Y, et al.2020. Roles of autophagy-related genes in conidiogenesis and blastospore formation, virulence, and stress response of Beauveria bassiana[J]. Fungal Biology. 124(12): 1052-1057. [15] Jiang L Q, Ye W W, Situ J J, et al.2017. A Puf RNA-binding protein encoding gene PlM90 regulates the sexual and asexual life stages of the litchi downy blight pathogen Peronophythora litchii[J]. Fungal Genetics and Biology. 98: 39-45. [16] Jiang S T, Chang A N, Han L T, et al.2020. Autophagy regulates fungal virulence and sexual reproduction in Cryptococcus neoformans[J]. Frontiers in Cell and Developmental Biology. 8: 374. [17] Kaufmann A, Beier V, Franquelim H G, et al.2014. Molecular mechanism of autophagic membrane-scaffold assembly and disassembly[J]. Cell. 156(3): 469-481. [18] Kershaw M J, Talbot N J.2009. Genome-wide functional analysis reveals that infection-associated fungal autophagy is necessary for rice blast disease[J]. Proceedings of the National Academy of Sciences of the USA. 106(37): 15967-15972. [19] Keulers T G, Koch A, van Gisbergen M W, et al.2022. ATG12 deficiency results in intracellular glutamine depletion, abrogation of tumor hypoxia and a favorable prognosis in cancer[J]. Autophagy. 18(8): 1898-1914. [20] Khalid A R, Zhang S M, Luo X M, et al.2021. Functional analysis of autophagy-related gene ATG12 in potato dry rot fungus Fusarium oxysporum[J]. International Journal of Molecular Sciences. 22(9): 4932. [21] Li G, Gong Z W, Dulal N, et al.2023. A protein kinase coordinates cycles of autophagy and glutaminolysis in invasive hyphae of the fungus Magnaporthe oryzae within rice cells[J]. Nature Communications. 14(1): 4146. [22] Lv W Y, Wang C Y, Yang N, et al.2017. Genome-wide functional analysis reveals that autophagy is necessary for growth, sporulation, deoxynivalenol production and virulence in Fusarium graminearum[J]. Scientific Reports. 7(1): 11062. [23] Mizushima N.2020. The ATG conjugation systems in autophagy[J]. Current Opinion in Cell Biology. 63: 1-10. [24] Mizushima N, Komatsu M.2011. Autophagy: Renovation of cells and tissues[J]. Cell. 147(4): 728-741. [25] Qiu M, Li Y N, Ye W W, et al.2021. A CRISPR/Cas9-mediated in situ complementation method for Phytophthora sojae mutants[J]. Molecular Plant Pathology. 22(3): 373-381. [26] Radoshevich L, Murrow L, Chen N, et al.2010. ATG12 conjugation to ATG3 regulates mitochondrial homeostasis and cell death[J]. Cell. 142(4): 590-600. [27] Situ J J, Xi P G, Lin L, et al.2022. Signal and regulatory mechanisms involved in spore development of Phytophthora and Peronophythora[J]. Frontiers in Microbiology.13: 984672. [28] Wang J R, Zhou G Q, Huang W X, et al.2022. Autophagy-related gene PlATG6a is involved in mycelial growth, asexual reproduction and tolerance to salt and oxidative stresses in Peronophythora litchii[J]. International Journal of Molecular Sciences. 23(3): 1839. [29] Wang Z W, Tyler B M, Liu X L.2018. Protocol of Phytophthora capsici transformation using the CRISPR-Cas9 system[J]. Methods in Molecular Biology (Clifton, N.J.). 1848: 265-274. [30] Werner A, Herzog B, Frey S, et al.2016. Autophagy-associated protein SmATG12 is required for fruiting-body formation in the filamentous Ascomycete Sordariama crospora[J]. PLOS ONE. 11(6): e0157960. [31] Ye W W, Wang Y, Shen D Y, et al.2018. Sequencing of the litchi downy blight pathogen reveals it is a Phytophthora species with downy mildew-like characteristics[J]. Molecular Plant-Microbe Interactions. 29(7): 573-583. [32] Zhou Y X, Chen L, Hu J, et al.2015. Resistance mechanisms and molecular docking studies of four novel QoI fungicides in Peronophythora litchii[J]. Scientific Reports.5: 17466. [33] Zhu H H, Situ J J, Guan T F, et al.2022. A C2H2 zinc finger protein PlCZF1 is necessary for oospore development and virulence in Peronophythora litchii[J]. International Journal of Molecular Sciences. 23(5): 2733. |
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