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.
Figure 2: Reverse transcriptase PCR analysis of transgenic plants. (a) Using GFP:Pi54 specific primers in transgenic Pi54OX and non-transgenic (NT)-control TP 309; Lane 1: 500 bp ladder, Lane 2–6: Pi54OX 1,3,4, 5 and 6, Lane 7: NT- control TP309; Lane 8: pRTV construct, Lane 9: No template control. (b) Internal control; Actin gene amplification in transgenicPi54OX and NT-control TP 309; Lane 1: 500 bp ladder, Lanes 2–6: Pi54OX 1,3,4 5 and 6, Lane 7: NT-control TP309, Lane 8: No template control. (c) CT values during qRT- PCR using GFP::Pi54 specific primers in transgenic Pi54OX and non-transgenic (NT)-control TP 309; Lane 1: NT-control TP309; Lane 2–6: Pi54OX 1,3,4, 5 and 6.