ICAR-National Research Centre on Plant Biotechnology, New Delhi, India.
Hislop College, R.T.M Nagpur University, Nagpur, India.
Sci Rep. 2020 Mar 23;10(1):5243. doi: 10.1038/s41598-020-59027-x.
Rice blast resistance gene, Pi54 provides broad-spectrum resistance against different strains of Magnaporthe oryzae. Understanding the cellular localization of Pi54 protein is an essential step towards deciphering its place of interaction with the cognate Avr-gene. In this study, we investigated the sub-cellular localization of Pi54 with Green Fluorescent Protein (GFP) as a molecular tag through transient and stable expression in onion epidermal cells (Allium cepa) and susceptible japonica cultivar rice Taipei 309 (TP309), respectively. Confocal microscopy based observations of the onion epidermal cells revealed nucleus and cytoplasm specific GFP signals. In the stable transformed rice plants, GFP signal was recorded in the stomata, upper epidermal cells, mesophyll cells, vascular bundle, and walls of bundle sheath and bulliform cells of leaf tissues. These observations were further confirmed by Immunocytochemical studies. Using GFP specific antibodies, it was found that there was sufficient aggregation of GFP::Pi54protein in the cytoplasm of the leaf mesophyll cells and periphery of the epidermal cells. Interestingly, the transgenic lines developed in this study could show a moderate level of resistance to Xanthomonas oryzae and Rhizoctonia solani, the causal agents of the rice bacterial blight and sheath blight diseases, respectively. This study is a first detailed report, which emphasizes the cellular and subcellular distribution of the broad spectrum blast resistance gene Pi54 in rice and the impact of its constitutive expression towards resistance against other fungal and bacterial pathogens of rice.
水稻稻瘟病抗性基因 Pi54 对不同的稻瘟病菌株具有广谱抗性。了解 Pi54 蛋白的细胞定位是解析其与同源 Avr 基因相互作用位置的关键步骤。在这项研究中，我们通过洋葱表皮细胞（Allium cepa）中的瞬时和稳定表达，以及对感病粳稻品种台北 309（TP309）的稳定转化，用绿色荧光蛋白（GFP）作为分子标记研究了 Pi54 的亚细胞定位。基于共焦显微镜观察洋葱表皮细胞，发现 GFP 信号具有核和细胞质特异性。在稳定转化的水稻植株中，GFP 信号记录在气孔、上表皮细胞、叶肉细胞、维管束以及叶片组织的鞘细胞和泡状细胞的壁上。免疫细胞化学研究进一步证实了这些观察结果。使用 GFP 特异性抗体发现，GFP::Pi54 蛋白在叶肉细胞的细胞质和表皮细胞的周围有足够的聚集。有趣的是，本研究中开发的转基因系能够对水稻细菌性条斑病和纹枯病的病原菌黄单胞菌和立枯丝核菌表现出中等水平的抗性。本研究首次详细报告了广谱稻瘟病抗性基因 Pi54 在水稻中的细胞和亚细胞分布，以及其组成型表达对水稻其他真菌和细菌病原体抗性的影响。
