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The ZmRCP-1 Promoter of Maize Provides Root Tip Specific Expression of Transgenes in Plantain

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The ZmRCP-1 Promoter of Maize Provides Root Tip Specific Expression of Transgenes in Plantain

Stephen O Onyango et al. J Biol Res (Thessalon).

Abstract

Background: Bananas and plantains (Musa spp.) provide 25 % of the food energy requirements for more than 100 million people in Africa. Plant parasitic nematodes cause severe losses to the crop due to lack of control options. The sterile nature of Musa spp. hampers conventional breeding but makes the crop suitable for genetic engineering. A constitutively expressed synthetic peptide in transgenic plantain has provided resistance against nematodes. Previous work with the peptide in potato plants indicates that targeting expression to the root tip improves the efficacy of the defence mechanism. However, a promoter that will provide root tip specific expression of transgenes in a monocot plant, such as plantain, is not currently available. Here, we report the cloning and evaluation of the maize root cap-specific protein-1 (ZmRCP-1) promoter for root tip targeted expression of transgenes that provide a defence against plant parasitic nematodes in transgenic plantain.

Results: Our findings indicate that the maize ZmRCP-1 promoter delivers expression of β-glucuronidase (gusA) gene in roots but not in leaves of transgenic plantains. In mature old roots, expression of gusA gene driven by ZmRCP-1 becomes limited to the root cap. Invasion by the nematode Radopholus similis does not modify Root Cap-specific Protein-1 promoter activity.

Conclusions: Root cap-specific protein-1 promoter from maize can provide targeted expression of transgene for nematode resistance in transgenic plantain.

Keywords: Nematode invasion; Plantain; Root specific promoter; Transgenic defence; β-glucuronidase.

Figures

Fig. 1
Fig. 1
Schematic presentation and validation of plasmid constructs. a pBI-RCP-1:GUS- the maize Root Cap-specific Protein-1 promoter (RCP1p) drives expression of the β-glucuronidase (gusA) gene. b pBI121- the cauliflower mosaic virus CaMV35S promoter (35Sp) drives expression of the gusA gene. For both constructs the nopaline synthase promoter (NOSp) was used to drive expression of the neomycin phosphotransferase II (nptII) gene and the nopaline synthase terminator (NOSt) was used as the 3΄ poly-A signal for both the selectable marker nptII and gusA genes. Left (LB) and right (RB) borders of the T-DNA and translation start sites with the direction of transcription are also indicated. c Polymerase chain reaction screen of Agrobacterium tumefaciens strain EHA105 colonies (1–9) transformed with pBI-RCP-1:GUS or pBI121. RCP1 primers amplify a 2 kbp fragment and the 35S primers amplify an 835 bp fragment. M—molecular size marker with sizes of bands indicated; P—positive control using the plasmid used for transformation
Fig. 2
Fig. 2
Regeneration of transgenic lines of plantain cv. ‘Gonja manjaya’. a Embryogenic cells infected with EHA105 Agrobacterium tumefaciens cells harbouring pBI-RCP-1:GUS or pBI121 construct. b Proliferation of Agrobacterium-infected cells on kanamycin selective medium. c Embryos developing on kanamycin selective medium. d Germinating transgenic seedlings from mature embryos on selective medium. e Transgenic plantlets on rooting medium. f Transgenic plants in pots in the glasshouse
Fig. 3
Fig. 3
PCR analysis of transgenic plantain lines. Positive transgenic lines screened by PCR for a 528 bp region of the gusA gene. Amplification from the pBI121 plasmid was used as a positive control (P) and amplification from the genomic DNA of a non-transformed plantain was used as a negative control (NT). Twenty RCP-1:GUS lines (R) and 15 35S:GUS lines (C) were positive. 100 bp plus DNA ladder (M) used to assess size of amplified bands
Fig. 4
Fig. 4
Histochemical GUS assay of leaves and roots of young transgenic lines of plantain cultivar ‘Gonja manjaya’. a Leaf of a 35S:GUS line. b Root tip of a 35S:GUS line. c Leaf of a RCP-1:GUS line. d Root tip of a RCP-1:GUS line. e Leaf of a non-transgenic control f Root tip of a non-transgenic control. Scale bar 5 mm
Fig. 5
Fig. 5
Histochemical GUS assay performed on nematode infected roots of transgenic plantains at 28 days post infection. a GUS stained nematode infected root of 35S:GUS line. b GUS stained nematode infected root of RCP-1:GUS line. c GUS stained uninfected root of RCP-1:GUS line Scale bar 5 mm
Fig. 6
Fig. 6
Root invasion assay for nematode infection. Establishment in root tips of ‘Gonja manjaya’ at 14 days post infection by Radopholus similis, Helicotylenchus multicinctus and Meloidogyne incognita

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