Expression of a rice chitinase gene in transgenic banana ('Gros Michel', AAA genome group) confers resistance to black leaf streak disease

Abstract

Transgenic banana (Musa acuminata 'Gros Michel') integrating either of two rice chitinase genes was generated and its resistance to Black Leaf Streak disease caused by the fungus Mycosphaerella fijiensis was tested using a leaf disk bioassay. PCR screening indicated the presence of the hpt selectable marker gene in more than 90 % of the lines tested, whereas more than three quarters of the lines contained the linked rice chitinase gene resulting in a co-transformation frequency of at least 71.4 %. Further, a unique stable integration of the transgenes in each line revealed some false negative PCR results and the expected co-transformation frequency of 100 %. The transgene insert number per line ranged from 1 to 5 and single transgene insert lines (25 % of all) were identified. Considerable delay in disease development (up to 63 days post-incoculation) over a monitoring period of 108 days occurred in nine lines with extracellularly targeted chitinase out of 17 transgenic lines tested and their necrotic leaf area decreased by 73-94 % compared to the untransformed susceptible control line. Finally, correlation between symptom development and rice chitinase expression was confirmed in two lines by Western analysis. The potential of rice chitinase genes to enhance resistance against M. fijiensis in banana was demonstrated as well as the usefulness of the leaf disk bioassay for early disease screening in transgenic banana lines.

Fig. 1

Southern blot analysis for the integration of the rcg3 (a) or rcc2 (b) genes in independent transgenic ‘Gros Michel’ lines. Total DNA (10 μg) was digested with HindIII and hybridized with an rcg3 or rcc2-specific probe that spans part of the enhanced 35S promoter and the 5′ end of the rcg3 or rcc2 gene. A red arrowhead represents an rcg3 or rcc2 integration. MW: DIG-labeled molecular size marker (Roche); Co-: untransformed control plant; Co+: HindIII digested pBI333-EN4-RCG3 (a) or pBI333-EN4-RCC2 (b) plasmid controls (10 pg each); numbered lanes: independent transgenic lines a 01:GM.RCG3.01; 04:GM.RCG3.04; 06:GM.RCG3.06; 09:GM.RCG3.09; 10:GM.RCG3.10; 15:GM.RCG3.15; 20:GM.RCG3.20; 21:GM.RCG3.21; 24:GM.RCG3.24; 29:GM.RCG3.29; 31:GM.RCG3.31; 32:GM.RCG3.32; 35:GM.RCG3.35; 38:GM.RCG3.38; 39:GM.RCG3.39; b 02: GM.RCC2.02; 14: GM.RCC2.14

Fig. 2

Leaf disk bioassay with Mycosphaerella fijiensis on 9-month old greenhouse ‘Gros Michel’ banana plants transformed with one of two rice chitinase genes. Twelve leaf disks (5 × 5 cm) were placed on 0.4 % (w/v) water agar supplemented with GA₃ (8 mg L⁻¹). The abaxial surface of ten replicates per line was sprayed with suspension of M. fijiensis conidiospores (2 × 10⁴ mL⁻¹) and the other two served as non-inoculated internal controls. Untransformed ‘Gros Michel’ and ‘Calcutta 4’ plants were susceptible and resistant controls, respectively. Images were taken at seven time points (39, 53, 63, 73, 84, 95 and 108 days post inoculation = dpi). GM.RCG3.09 and GM.RCG3.20 were the two most susceptible transgenic lines, and GM.RCG3.10 and GM.RCG3.32 were the two most resistant ones in comparison with the untransformed ‘Gros Michel’ and ‘Calcutta4’ controls. RCG3, transgenic lines with rice chitinase rcg3

Fig. 3

Average infected area of leaf disks 39, 63, 84, and 108 days after Mycosphaerella fijiensis inoculation. Experimental details under Fig. 2. Each entry is the average (±SD) of ten replicate images analyzed by ASSESS software. At each time point the data were subjected to Kruskal–Wallis test which pointed out nine transgenic lines with significantly high level of resistance against M. fijiensis (except for line GM.RCG3.24 at 53 dpi and 63 dpi) and eight susceptible transgenic lines (framed in red rectangles). RCG3, transgenic lines with rice chitinase rcg3 and RCC2, transgenic lines with rice chitinase rcc2; dpi, days post inoculation

Fig. 4

RCG3 expression in non-infected leaf disks of the most BLSD resistant transgenic ‘Gros Michel’ lines at two time points as detected by Western blot analysis. TP1: start of leaf disk assay, day 1; TP4: end of leaf disk assay, day 78; Lane 1 and 7 GM untransformed control; Lane 2 GM.RCG3.01; Lane 3 GM.RCG3.10; Lane 4 GM.RGC3.15; Lane 5 GM.RCG3.21; Lane 6 and 13 MagicMark™ Western Protein Standard (Invitrogen); Lane 8 GM.RCG3.24; Lane 9 GM.RCG3.29; Lane 10 GM.RCG3.31; Lane 11 GM.RCG3.32; Lane 12 empty lane. Rectangular boxes show the positive lines