Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon, 34134, Republic of Korea.
Center for Eco-Friendly New Materials, Korea Research Institute of Chemical Technology, Daejeon, 34114, Republic of Korea.
Genes Genomics. 2024 Feb;46(2):253-261. doi: 10.1007/s13258-023-01486-3. Epub 2024 Jan 18.
Interactions of plants with biotic stress factors including bacteria, fungi, and viruses have been extensively investigated to date. Plasmodiophora brassicae, a protist pathogen, causes clubroot disease in Cruciferae plants. Infection of Chinese cabbage (Brassica rapa) plants with P. brassica results in the formation of root galls, which inhibits the roots from absorbing soil nutrients and water. Sugar, the major source of carbon for all living organisms including pathogens and host plants, plays an important role in plant growth and development.
To explore the roles of BrSWEET2, BrSWEET13, and BrSWEET14 in P. brassicae resistance, Arabidopsis thaliana T-DNA knockout mutants sweet2, sweet13, and sweet14 were employed.
To isolate total RNA from the collected root nodules, the root tissues washed several times with running water and frozen tissues with liquid nitrogen. Total RNA was extracted using the Spectrum™ Plant Total RNA Kit (SIGMA) and cDNA was synthesized in a 20 μl reaction volume using the ReverTra Ace-α- kit (TOYOBO). Real-time PCR was performed in a 10 μl reaction volume containing 1 μl of template DNA, 1 μl of forward primer, 1 μl of reverse primer, 5 μl of 2× iQTM SYBR Green Supermix (BioRad), and 2 μl of sterile distilled water. The SWEET genes were genotyped using BioFACT™ 2× TaqBasic PCR Master Mix 2.
Both sweet2 and sweet14 showed strong resistance to P. brassicae compared with wild-type Arabidopsis and Chinese cabbage plants and sweet13 mutant plants. Pathogenicity assays indicated that the SWEET2 gene plays an important role in clubroot disease resistance in higher plants.
迄今为止，植物与包括细菌、真菌和病毒在内的生物胁迫因素的相互作用已经得到了广泛的研究。原生动物病原体菜豆胞囊线虫会引起十字花科植物的根肿病。感染白菜（Brassica rapa）植物会导致根结的形成，从而抑制根部吸收土壤养分和水分。糖是包括病原体和宿主植物在内的所有生物的主要碳源，在植物生长和发育中起着重要作用。
探索 BrSWEET2、BrSWEET13 和 BrSWEET14 在抵抗菜豆胞囊线虫中的作用，使用拟南芥 T-DNA 敲除突变体 sweet2、sweet13 和 sweet14。
从收集的根瘤中分离总 RNA，用自来水冲洗根组织数次，并用液氮冷冻组织。使用 Spectrum™ 植物总 RNA 试剂盒（SIGMA）提取总 RNA，并使用 ReverTra Ace-α-试剂盒（TOYOBO）在 20 μl 反应体积中合成 cDNA。实时 PCR 在 10 μl 反应体积中进行，其中包含 1 μl 模板 DNA、1 μl 正向引物、1 μl 反向引物、5 μl 2× iQTM SYBR Green Supermix（BioRad）和 2 μl 无菌蒸馏水。使用 BioFACT™ 2× TaqBasic PCR Master Mix 2 对 SWEET 基因进行基因分型。
与野生型拟南芥和白菜植物以及 sweet13 突变体植物相比，sweet2 和 sweet14 均表现出对菜豆胞囊线虫的强抗性。致病性试验表明，SWEET2 基因在高等植物的根肿病抗性中起着重要作用。
