Blast resistance gene Pi54 over-expressed in rice to understand its cellular and sub-cellular localization and response to different pathogens

Abstract

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.

Figure 1

Stages of rice genetic transformation using biolistic approach. (a) Induction of embryogenic calli from mature rice seeds, (b) calli subjected to bombardment, (c) proliferating calli on hygromycin selection medium; arrowhead show the actively proliferating calli, (d) shoot regeneration from hygromycin resistant calli, (e) rooting of plantlets (f) transgenic plants grown in pots under controlled conditions.

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.

Figure 3

Functional complementation analysis Pi54-overexpression lines with highly virulent strains of M. oryzae, (a) MG-79 and (b) Mo-ni-25.

Figure 4

Transient expression and localization analysis of GFP::Pi54 in onion epidermal cells. Specimens were observed under Leica SP5 Confocal microscope at 488 nm excitation and 560 nm emission spectra. (a,b) GFP::Pi54 fusion protein expressing in nucleus, cytoplasmic and membrane; Scale bar: 50 µm; (c) Positive control: CaMV 35S-GFP; Scale bar: 75 µm.

Figure 5

Horizontal sections of rice leaves. (a) Transgenic rice leaf showing GFP signal in Guard cells (GC), Long cells (LC) and Short cell (ShC) in epidermis; scale bar 10 µm. (b) Peeled epidermis from transgenic rice leaf showing GFP expression in dumbbell shaped Guard cells (GC) & Silica cells (SC); scale bar 10 µm. (c) Non-transgenic rice leaf showing no GFP signal in Guard cells (GC), Long cells (LC) and Short cell (ShC) in epidermis; scale bar 10 μm.

Figure 6

Transverse section of transgenic rice leaf observed under Leica SP5 confocal microscope at 488 nm excitation and 560 nm emission spectra. (a) transgenic plant leaf showing GFP signals in mesophyll cell (MC), upper (UE) and lower epidermis (LE), vascular bundle (VB), cell wall of bundle sheath cells (BSC) and bulliform cells (BC); Scale bar: 25 µm. (b) Non-transgenic leaf showing no GFP signal; Scale bar: 25 µm.

Figure 7

Immunocytochemistry analysis of transgenic rice leaf. (a) Immunofluorescence (green fluorescence) patters obtained in mesophyll cell (MC), upper (UE) and lower epidermis (LE), vascular bundle cell wall (VB), cell wall of bundle sheath cells (BSC) and bulliform cells (BC); Scale bar: 25 µm.(b) Non-transgenic leaf; no binding immunofluorescence of primary secondary antibody; Scale bar: 25 µm.