Transgenic expression of the rice Xa21 pattern-recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum

Abstract

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern-recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21-mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar 'Gonja manjaya' (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa.

Keywords: Xa21; Xanthomonas campestris pv. musacearum; banana Xanthomonas wilt; rice pattern-recognition receptor; transgenic banana.

Figure 1

Regeneration of transgenic banana plants. a) Co-cultivation of embryogenic cells and Agrobacterium in liquid callus induction medium, b) Embryo maturation, c) Germination on selective medium supplemented with hygromycin, d) Putative transgenic shoots regenerated on selective medium, e) Transgenic shoots in proliferation media and f) Transgenic plants transferred to soil in small plastic cups in glass house.

Figure 2

a). Schematic representation of the T-DNA region of construct pCAMBIA1300-Ubi:Xa21 used for plant transformation. b) Representative picture showing PCR analysis of genomic DNA from transgenic banana lines and the non-transgenic plant using Xa21 specific primers and hygromycin phosphotransferase (hpt) specific primers. Amplified PCR product designations were shown on the right and product sizes were shown on the left. M - molecular weight marker, 1–12- transgenic plants, P - plasmid DNA, C - non-transgenic control plant.

Figure 3

Representative picture showing enhanced resistance of transgenic banana lines to Xanthomonas campestris pv. musacearum. Evaluation of transgenic lines using in vitro banana plantlets, a) Inoculated non-transgenic control plant, b–d) Inoculated transgenic plants (GX5, GX8 and GX26). Evaluation of transgenic banana plants in glasshouse, e) Non-transgenic control plant showing complete wilting after artificial inoculation, f-h) Transgenic plants (GX5, GX8 and GX26) showing no BXW symptoms after 60 days following inoculation of potted plants, i) Cut pseudostem of non-transgenic control plant showing yellow bacterial ooze of Xanthomonas campestris pv. musacearum after 60 days of artificial inoculation, j–k) Transgenic plants (GX8 and GX26) showing no bacterial ooze. All photographs were taken 60 days after artificial inoculation.

Figure 4

Bacterial population analysis of transgenic plants (GX8 and GX26) and control non-transgenic plants. a) Bacterial population growth analysis for 16 dpi. b) Bacterial population at 16 dpi in different section (from inoculation point, 5 cm above and 5 cm below point of inoculation) of midrib of inoculated leaves. The data presented is the average of 6 samples at each time point. The error bar represents the standard deviation.

Figure 5

RT-PCR and qRT-PCR of 12 transgenic lines (GX3, GX5, GX8, GX15, GX26, GX36, GX37, GX46, GX57, GX62, GX74 and GX76) and non-transgenic control plant. a) RT-PCR of transgenic banana lines using primers specific to Xa21 and 25S ribosomal gene. Amplified RT-PCR product designations were shown on the right and products sizes were shown on the left. M - molecular weight marker, NTC - non-transgenic control, b) Relative transcript levels of the Xa21 gene in transgenic lines in comparison to non-transgenic control plants. Expression of Xa21 gene was normalized with banana 25S ribosomal gene (internal control) and non-transgenic plant served as a calibrator. Relative expression was determined from replicate measurements in two independent biological replicates and three technical replicates. Data are mean ±SD.

Figure 6

Figure showing comparison of plant growth characteristics in transgenic and non-transgenic banana plants. a) Plant height, b) Pseudostem girth, c) Number of functional leaves and d) Total leaf area of transgenic lines in comparison to non-transgenic control plants. All values were mean ± standard deviation of three replicates of independent transgenic line and non-transgenic control plant. Plant growth characteristics were recorded at 90 days of planting in pots in glasshouse.