菰黑粉菌细胞核和线粒体的荧光标记菌株构建

doi:10.3969/j.issn.1674-7968.2024.05.019

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Abstract Stem swollen of Zizania latifolia was associated with the successful infection of Ustilago esculenta, which depended on the dimorphic transition of U. esculenta. The dynamic changes of the nucleus and mitochondria during the process of dimorphic transition remain unknown. This study will focus on the coding gene for histone H1 and the coding sequence for the mitochondrial targeting signal (MTS) in the nucleus, respectively. After connecting them with the GFP and mCherry fluorescent protein genes, respectively, fluorescent labeling vectors for the nucleus and mitochondria was constructed. Wild-type strains UeT14 and UeT55 of U. esculenta as experimental materials to genetic transformation by Agrobacterium tumefaciens-mediated transformation (ATMT), and the recombinant strains that stably expressed red and green fluorescent proteins in nucleus or mitochondria were obtained: UeT55-H1-GFP, UeT14-H1-mCherry, UeT55-MTS-GFP and UeT14-MTS-mCherry. In UeT55-H1-GFP and UeT14-H1-mCherry strains, bright and round fluorescent spots could be seen in the center of the spores, which were co-located with the blue fluorescence formed by DAPI. In UeT55-MTS-GFP and UeT14-MTS-mCherry strains, small dot green or red fluorescence could be seen in the spores, and the green fluorescence was co-located with the red fluorescence formed by MitoTracker. In this study, the stable expression of fluorescent protein in nucleus or mitochondria was obtained, which provides an important reference and material for studying the molecular mechanism of growth, development, fusion and infection of U. esculenta.

Key words： Ustilago esculenta Zizania latifolia Nucleus Mitochondria Fluorescent protein labeling

Received: 27 March 2023

ZTFLH:

S645.2

Corresponding Authors: **zhye@cjlu.edu.cn

About author:: * These authors contributed equally to this work

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	TANG Jin-Tian
	YANG Fu-Rong
	ZHANG Lei-Lei
	LI Yu-Kang
	ZHANG Ya-Fen
	FU Hui-Lan
	XIA Wen-Qiang
	CUI Hai-Feng
	YE Zi-Hong

Cite this article:

TANG Jin-Tian,YANG Fu-Rong,ZHANG Lei-Lei, et al. Construction of Fluorescently Labeled Strains of Nucleus and Mitochondria for Ustilago esculenta[J]. 农业生物技术学报, 2024, 32(5): 1188-1197.

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https://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2024.05.019 OR https://journal05.magtech.org.cn/Jwk_ny/EN/Y2024/V32/I5/1188

[1] 李妹芳, 冯海霞, 郭尚敬. 2008. 植物热激蛋白启动子的特点及应用[J]. 安徽农业科学, 36(29): 12605-12606.
(Li M F, Feng H X, Guo S J.2008. Characteristics and application of plant heat shock protein promoter[J]. Journal of Anhui Agricultural Sciences, 36(29): 12605-12606.)
[2] 刘丽萍. 2013. 希金斯刺盘孢致病缺陷型突变体的筛选及其T-DNA插入基因的功能分析[D]. 博士学位论文, 华中农业大学, 导师: 黄俊斌, pp. 26-45.
(Liu L P, 2013. Screening of pathogenicity-deficient mutants from a T-DNA insertional library of Colletotrichum higginslanum and characterized function analysis of pathogenesis-associate genes[D]. Thesis for Ph.D., Huazhong Agricultural University, Supervisor: Huang J B; pp. 1-121)
[3] 张雅芬, 卞加慧, 叶子弘, 等. 2022. 一种菰黑粉菌内源强启动子pHSP及其表达载体和应用[P]. 浙江省: CN112725341B, 2022-03-18.
(Zhang Y F, Bian J H, Ye Z H, et al.2022 An endogenous strong promoter pHSP of Zizania nigra and its expression vector and application[P]. Zhejiang Province: CN112725341B, 2022-03-18.)
[4] Basse C W.2010. Mitochondrial inheritance in fungi[J]. Current Opinion in Microbiology, 13(6): 712-719.
[5] Bortfeld M, Auffarth K, Kahmann R, et al.2004. The Ustilago maydis a2 mating-type locus genes lga2 and rga2 compromise pathogenicity in the absence of the mitochondrial p32 family protein Mrb1[J]. The Plant Cell, 16(8): 2233-2248.
[6] Dong Z, Long J, Huang L, et al.2019. Construction and application of an HSP70 promoter-inducible genome editing system in transgenic silkworm to induce resistance to Nosema bombycis[J]. Applied Microbiology and Biotechnology, 103: 9583-9592.
[7] Echauri-Espinosa R O, Callejas-Negrete O A, Roberson R W, et al.2012. Coronin is a component of the endocytic collar of hyphae of Neurospora crassa and is necessary for normal growth and morphogenesis[J]. PLOS ONE, 7(5): e38237.
[8] Guo H B, Li S M, Peng J, et al.2007. Zizania latifolia Turcz, cultivated in China[J]. Genetic Resources and Crop Evolution, 54: 1211-1217.
[9] Hasan R, Lv B, Uddin M J, et al.2022. Monitoring mycoparasitism of Clonostachys rosea against Botrytis cinerea using GFP[J]. Journal of Fungi, 8(6): 567.
[10] Hu Y, Plutz M, Belmont A S.2010. Hsp70 gene association with nuclear speckles is Hsp70 promoter specific[J]. Journal of Cell Biology, 191(4): 711-719.
[11] Jeong S Y, Dai W S.2008. The role of mitochondria in apoptosis[J]. BMB Reports, 41(1): 11-22.
[12] Kapuscinski J.1995. DAPI: a DNA-specific fluorescent probe[J]. Biotechnic & Histochemistry, 70(5): 220-233.
[13] Khan I A, Ning G, Liu X, et al.2015. Mitochondrial fission protein MoFis1 mediates conidiation and is required for full virulence of the rice blast fungus Magnaporthe oryzae[J]. Microbiological Research, 178: 51-58.
[14] Kubala M H, Kovtun O, Alexandrov K, et al.2010. Structural and thermodynamic analysis of the GFP: GFP‐nanobody complex[J]. Protein Science, 19(12): 2389-2401.
[15] Kummer E, Ban N.2021. Mechanisms and regulation of protein synthesis in mitochondria[J]. Nature Reviews Molecular Cell Biology, 22: 307-325.
[16] Li L, Schmelz M, Kellner E M, et al.2007. Nuclear labeling of Coccidioides posadasii with green fluorescent protein[J]. Annals of the New York Academy of Sciences, 1111(1): 198-207.
[17] Logan D C, Leaver C J.2000. Mitochondria‐targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells[J]. Journal of Experimental Botany, 51(346): 865-871.
[18] Lorang J M, Tuori R P, Martinez J P, et al.2001. Green fluorescent protein is lighting up fungal biology[J]. Applied and Environmental Microbiology, 67(5): 1987-1994.
[19] Lorenz H, Hailey D W, Lippincott S J.2006. Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization[J]. Nature methods, 3(3): 205-210.
[20] Maor R, Puyesky M, Horwitz B A, et al.1998. Use of green fluorescent protein (GFP) for studying development and fungal-plant interaction in Cochliobolus heterostrophus[J]. Mycological Research, 102(4): 491-496.
[21] Mahlert M, Vogler C, Stelter K, et al.2009. The a2 mating-type-locus gene lga2 of Ustilago maydis interferes with mitochondrial dynamics and fusion, partially in dependence on a Dnm1-like fission component[J]. Journal of cell science, 122(14): 2402-2412.
[22] Neubauer M, Zhu Z, Penka M, et al.2015. Mitochondrial dynamics in the pathogenic mold Aspergillus fumigatus: Therapeutic and evolutionary implications[J]. Molecular Microbiology, 98(5): 930-945.
[23] Okamoto K, Shaw J M.2005. Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes[J]. Annual Review of Genetics, 39: 503-536.
[24] Pérez-Martín J, Castillo-Lluva S, Sgarlata C, et al.2006. Pathocycles: Ustilago maydis as a model to study the relationships between cell cycle and virulence in pathogenic fungi[J]. Molecular Genetics and Genomics, 276: 211-229.
[25] Prasher D C.1995. Using GFP to see the light[J]. Trends in Genetics, 11(8): 320-323.
[26] Ruiz-Roldán M C, Köhli M, Roncero M I G, et al.2010. Nuclear dynamics during germination, conidiation, and hyphal fusion of Fusarium oxysporum[J]. Eukaryotic Cell, 9(8): 1216-1224.
[27] Sahoo D, Ummalyma S B, Okram A K, et al.2018. Effect of dilute acid pretreatment of wild rice grass (Zizania latifolia) from Loktak Lake for enzymatic hydrolysis[J]. Bioresource Technology, 253: 252-255.
[28] Viotti M, Nowotschin S, Hadjantonakis A K.2011. Afp::mCherry, a red fluorescent transgenic reporter of the mouse visceral endoderm[J]. Genesis, 49(3): 124-133.
[29] Wada H, Shintani D, Ohlrogge J.1997. Why do mitochondria synthesize fatty acids? Evidence for involvement in lipoic acid production[J]. Proceedings of the National Academy of Sciences of the USA, 94(4): 1591-1596.
[30] Wang J L, Zhang L, Gao L X, et al.2021. A bright, red-emitting water-soluble BODIPY fluorophore as an alternative to the commercial Mito Tracker Red for high-resolution mitochondrial imaging[J]. Journal of Materials Chemistry B, 9(41): 8639-8645.
[31] Wang Z H, Yan N, Luo X,et al.2020. Gene expression in the smut fungus Ustilago esculenta governs swollen gall metamorphosis in Zizania latifolia[J]. Microbial Pathogenesis, 143: 104-107.
[32] Yan N, Du Y M, Liu X M, et al.2018. Morphological characteristics, nutrients, and bioactive compounds of Zizania latifolia, and health benefits of its seeds[J]. Molecules, 23(7): 1561.
[33] Zhang Y F, Wu M, Ge Q W, et al.2019. Cloning and disruption of the UeArginase in Ustilago esculenta: Evidence for a role of arginine in its dimorphic transition[J]. BMC Microbiology, 19(1): 1-12.