Development of a Magnetic Nanoparticle-mediated Genetic Transformation Technology for Alternaria solani and Functional Analysis of the Ubiquitin-proteasome Gene AsPR45
ZHAO Yu-Jiao*, FU Ai-Hua*, ZHANG Jia-Ying, LIU Wen-Wen, YANG Qiao-Mei, JIANG Dong-Mei, TANG Wei**, LI Jian-Mei**
School of Life Sciences/Yunan Key Laboratory of Potato Biology, Yunnan Normal University, Yunnan 650500, China
Abstract：The ubiquitin-proteasome system (UPS) is a critical protein degradation pathway in fungal cells, playing a vital role in regulating protein homeostasis and maintaining cellular functions. Previous studies have shown that during the early stages (0~72 h) of Alternaria solani infection in potato (Solanum tuberosum), the expression of the AsPR45 gene, which encoded the P45 subunit of the 26S proteasome in A. solani, was significantly upregulated. To further explore the function of the AsPR45 gene, this study first optimized a genetic transformation method for A. solani mediated by magnetic nanoparticles (MNPs). Using this transformation assay, a premature termination construct of AsPR45 was developed based on the pTOR-mRFP vector backbone and introduced into the wild-type strain TA-0410. Functional validation was carried out on 2 positive mutant strains. The transformation efficiency was the highest when the mass ratio of MNPs to the pTOR-mRFP vector was 1∶4, with binding at 37 ℃ for 30 min, followed by co-incubation with spores at room temperature for 1.5 h, achieving a transformation rate of (36.77±2.91)%. Biological function assays revealed that premature termination mutants of AsPR45 exhibited no significant difference in hyphal growth rate compared with the wild-type strain. However, the mutants produced less melanin in hyphae, had a significantly reduced conidiation rate, and showed markedly decreased pathogenicity on the leaves of potato cultivar 'Desiree'. Additionally, the EC50 values for difenoconazole resistance differed significantly between the wild-type strain and the mutants. These results indicated that AsPR45 was a key gene in the ubiquitination pathway in A. solani, playing an important role in fungal morphogenesis, potato infection, and stress resistance. This study not only provides a technical foundation for functional gene research in A. solani but also contributes to the identification of potential fungicide targets, accelerating the development of strategies for controlling potato early blight disease.
赵玉娇, 付爱华, 张佳滢, 刘文雯, 杨巧梅, 姜冬美, 唐唯, 李健美. 利用纳米磁珠介导的茄链格孢菌遗传转化技术的建立及其泛素化蛋白酶基因AsPR45功能研究[J]. 农业生物技术学报, 2025, 33(9): 2036-2048.
ZHAO Yu-Jiao, FU Ai-Hua, ZHANG Jia-Ying, LIU Wen-Wen, YANG Qiao-Mei, JIANG Dong-Mei, TANG Wei, LI Jian-Mei. Development of a Magnetic Nanoparticle-mediated Genetic Transformation Technology for Alternaria solani and Functional Analysis of the Ubiquitin-proteasome Gene AsPR45. 农业生物技术学报, 2025, 33(9): 2036-2048.
[1] 陈伟帅, 高利, 刘太国, 等. 2015. 小麦抗病遗传多样性对条锈病的调控效应[J]. 植物保护, 41(06): 185-190. (Chen W S, Gao L, Liu T G, et al.2015. Regulatory effects of genetic diversity for disease resistance in wheat on stripe rust[J]. Plant Protection, 41(06): 185-190.) [2] 胡懋, 曾杨璇, 苗华彪, 等. 2021. 根癌农杆菌介导真菌遗传转化的研究及应用[J]. 微生物学通报, 48(11): 4344-4363. (Hu M, Zeng Y X, Miao H B, et al.2021. Research and application of Agrobacterium tumefaciens-mediated fungal genetic transformation[J]. Acta Microbiologica Sinica, 48(11): 4344-4363.) [3] 贾艳晶, 郭鑫, 成仿云. 2020. 纳米磁珠介导牡丹花粉转基因技术的初步分析[J]. 分子植物育种, 18(08): 2577-2584. (Jia Y J, Guo X, Cheng F Y.2020. Preliminary analysis on transgenic technology of pollen-mediated by magnetic nanoparticles in tree peony[J]. Molecular Plant Breeding, 18(8): 2577-2584.) [4] 卢艳敏. 2013. 磁性纳米颗粒作为载体在基因转染中的研究进展[J]. 生物技术通讯, 24(05): 736-740. (Lu Y M.2013. Progress of magnetic nanoparticles as gene vector[J]. Letters in Biotechnology, 24(5): 736-740.) [5] 刘丽丽, 朱杰华, 崔亚婧, 等. 2014. 培养条件对茄链格孢产孢的影响[J]. 菌物学报, 33(3): 659‐667. (Liu L L, Zhu J H, Cui Y J, et al.2014. Effects of culture conditions on sporulation of Alternaria solani[J]. Journal of Mycology, 33(3): 659‐667.) [6] 孙国胜, 杨宇, 许可翠, 等. 2021. 纳米磁性颗粒介导的辣椒遗传转化体系建立[J]. 核农学报, 35(06): 1307-1312. (Sun G S, Yang Y, Xun K C, et al.2021. Establishment of genetic transformation system of pepper mediated by magnetic nanoparticles[J]. Journal of Nuclear Agricultural Sciences, 35(06): 1307-1312.) [7] 宋博, 张丽娟, 朱晓锋, 等. 2022. 香梨果萼黑斑病菌Alternaria alternata遗传转化体系的构建及GFP标记菌株的获得[J]. 植物病理学报, 52(01): 97-103. (Song B, Zhang L J, Zhu X F.2022. Development of efficient genetic transformation system in Alternaria alternata and its application for strain labeled with GFP[J]. Journal of Plant Pathology, 52(01): 97-103.) [8] Beliaev D V, Yuorieva N O, Tereshonok D V, et al.2021. High resistance of potato to early blight is achieved by expression of the pro-smamp1 gene for hevein-like antimicrobial peptides from common chickweed (Stellaria media)[J]. Plants (Basel, Switzerland), 10(7): 1395. [9] Birch P R, Bryan G, Fenton B, et al.2012. Crops that feed the world 8: potato: Are the trends of increased global production sustainable?[J]. Food Security, 4(4): 477-508. [10] Bundock P, den Dulk-Ras A, Beijersbergen A, et al.1995. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae[J]. The EMBO Journal, 14(13): 3206-3214. [11] Campos H, Ortiz O.2019. The potato crop: Its agricultural, nutritional and social contribution to humankind[M]. Switzerland: Springer International Publishing. [12] Cheng L, Watt R, Piper P W, et al.1994. Polyubiquitin gene expression contributes to oxidative stress resistance in respiratory yeast (Saccharomyces cerevisiae)[J]. Molecular & General Genetics, 243(3): 358-362. [13] de Groot M, Bundock P, Hooykaas P, et al.1988. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology, 16: 839-842. [14] del Pozo J C, Dharmasiri S, Hellmann H, et al.2002. AXR1-ECR1-dependent conjugation of RUB1 to the Arabidopsis Cullin AtCUL1 is required for auxin response[J]. The Plant Cell, 14(2): 421-433. [15] Ding S, Meinholz K, Cleveland K, et al.2019. Diversity and virulence of Alternaria spp. causing potato early blight and brown spot in Wisconsin[J]. Phytopathology, 109(3): 436-445. [16] Dong Y H, Wang S T.2022. Agrobacterium tumefaciens-mediated transformation method for Fusarium oxysporum[J]. Methods in Molecular Biology, 2391: 63-73. [17] Fonseka D L, Gudmestad N C.2016. Spatial and temporal sensitivity of Alternaria species associated with potato foliar diseases to demethylation inhibiting and anilino-pyrimidine fungicides[J]. Plant Disease, 100(9): 1848-1857. [18] Gangavaram L P, Mchunu N, Ramakrishnan P, et al.2009. Improved electroporation-mediated non-integrative transformation of Thermomyces lanuginosus[J]. Journal of Microbiological Methods, 77(2): 159-164. [19] 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(1): 32-40. [20] Jiang J, Guo X, Tan H, et al.2023. Transcriptome sequencing leads to an improved understanding of the infection mechanism of Alternaria solani in potato[J]. BMC Plant Biology, 23(1): 120. [21] 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. [22] Li Q, Feng Y, Li J, et al.2024. Multi-omics approaches to understand pathogenicity during potato early blight disease caused by Alternaria solani[J]. Front Microbiol, 15: 1357579. [23] Liu N, Chen G Q, Ning G A, et al.2016. Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae[J]. Microbiological Research, 182:40-48. [24] Noh W, Kim S W, Dong-Won B, et al.2010. Genetic introduction of foreign genes to Pleurotus eryngii by restriction enzyme-mediated integration[J]. Journal of Microbiology, 48(2): 253-256. [25] Odilbekov F, Selga C, Ortiz R, et al.2020. QTL mapping for resistance to early blight in a tetraploid potato population[J]. Agronomy. 10(5): 728. [26] Oh Y, Franck W L, Han S O, et al.2012. Polyubiquitin is required for growth, development and pathogenicity in the rice blast fungus Magnaporthe oryzae[J]. PLOS One, 7(8): e42868. [27] O'Mahony P J, Oliver M J.1999. The involvement of ubiquitin in vegetative desiccation tolerance[J]. Plant Molecular Biology, 41(5): 657-667. [28] Pervaiz A, Sajid Z A, Yousaf S, et al.2023. Micro tuberization potential of Jasmonic acid, kinetin and putrescine in potato (Solanum tuberosum L.)[J]. American Journal of Potato Research, 100: 184-191 [29] Roig P, Gozalbo D.2003. Depletion of polyubiquitin encoded by the UBI4 gene confers pleiotropic phenotype to Candida albicans cells[J]. Fungal Genetics and Biology, 39(1): 70-81. [30] Rolland S, Jobic C, Fevre M, et al.2003. Agrobacterium-mediated transformation of Botrytis cinerea, simple purification of monokaryotic transformants and rapid conidia-based identification of the transfer-DNA host genomic DNA flanking sequences[J]. Current Genetics, 44(3): 164-171. [31] Schmey T, Tominello-Ramirez C S, Brune C, et al.2024. Alternaria diseases on potato and tomato[J]. Molecular Plant Pathology, 25(3): e13435. [32] Shi X, Xie X, Guo Y, et al.2024. A fungal core effector exploits the OsPUX8B.2-OsCDC48-6 module to suppress plant immunity[J]. Nature Communications, 15(1): 2559. [33] Sosa-Acosta J R, Iriarte-Mesa C, Ortega G A, et al.2020. DNA-iron oxide nanoparticles conjugates: Functional magnetic nanoplatforms in biomedical applications[J]. Topics in Current Chemistry (Cham), 378(1): 13. [34] Stokstad E.2019. The new potato[J]. Science (New York, N.Y.), 363(6427): 574-577. [35] Wang C, Wang J, Zhang D, et al.2023. Identification and functional analysis of protein secreted by Alternaria solani[J]. PLOS ONE, 18(3): e0281530. [36] Wang C, Zhang D, Cheng J, et al.2022. Identification of effector CEP112 that promotes the infection of necrotrophic Alternaria solani[J]. BMC Plant Biology, 22(1): 466. [37] Wang Y, Zhao X, Du W, et al.2017. Production of transgenic mice through sperm-mediated gene transfer using magnetic nano-carriers[J]. Journal of Biomedical Nanotechnology, 13(12): 1673-1681. [38] Wang Z P, Zhang Z B, Zheng D Y, et al.2022. Efficient and genotype independent maize transformation using pollen transfected by DNA-coated magnetic nanoparticles[J]. Journal Integrative Plant Biology, 64(6): 1145-1156. [39] Wolters P J, Faino L, van den Bosch T B M, et al.2018. Gapless genome assembly of the potato and tomato early blight pathogen Alternaria solani[J]. Molecular Plant-microbe Interactions: MPMI, 31(7): 692-694. [40] Xue W, Haynes K G, Qu X.2019. Characterization of early blight resistance in potato cultivars[J]. Plant Disease, 103(4): 629-637. [41] Zeng L R, Vega-Sánchez M E, Zhu T, et al.2006. Ubiquitination-mediated protein degradation and modification: An emerging theme in plant-microbe interactions[J]. Cell Research, 16(5): 413-426. [42] Zhang D, He J Y, Haddadi P, et al.2018. Genome sequence of the potato pathogenic fungus Alternaria solani HWC-168 reveals clues for its conidiation and virulence[J]. BMC Microbiology, 18(1): 176. [43] Zhang J, Liu C, Xie Y, et al.2017. Enhancing fructooligosaccharides production by genetic improvement of the industrial fungus Aspergillus niger ATCC 20611[J]. Journal of Biotechnology, 249: 25-33. [44] Zhang Y, Zhou Q, Tian P, et al.2020. Induced expression of CYP51 associated with difenoconazole resistance in the pathogenic Alternaria sect. on potato in China[J]. Pest management science, 76(5): 1751-1760. [45] Zhao W, Zhou T, Zheng H Z, et al.2018. Yeast polyubiquitin gene UBI4 deficiency leads to early induction of apoptosis and shortened replicative lifespan[J]. Cell Stress Chaperones, 23: 527-537. [46] Zhao X, Meng Z, Wang Y, et al.2017. Pollen magnetofection for genetic modification with magnetic nanoparticles as gene carriers[J]. Nature Plants, 3(12): 956-964.
