Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4

Abstract

Banana (Musa spp.) is a staple food for more than 400 million people. Over 40% of world production and virtually all the export trade is based on Cavendish banana. However, Cavendish banana is under threat from a virulent fungus, Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) for which no acceptable resistant replacement has been identified. Here we report the identification of transgenic Cavendish with resistance to TR4. In our 3-year field trial, two lines of transgenic Cavendish, one transformed with RGA2, a gene isolated from a TR4-resistant diploid banana, and the other with a nematode-derived gene, Ced9, remain disease free. Transgene expression in the RGA2 lines is strongly correlated with resistance. Endogenous RGA2 homologs are also present in Cavendish but are expressed tenfold lower than that in our most resistant transgenic line. The expression of these homologs can potentially be elevated through gene editing, to provide non-transgenic resistance.

Fig. 1

Transgene expression cassettes and Southern analysis of selected transgenic lines. a RGA2 and b Ced9 expression cassettes. LB, left border; RB, right border. Determination of transgene copy number in c RGA2 and d Ced9 transgenic banana lines by Southern blot analysis. Genomic DNA from WT, RGA2 and Ced9 lines was digested with HindIII and XmaI, respectively. DNA molecular weight marker II (Roche) reference is indicated on the right hand side

Fig. 2

Characteristic symptoms of Foc TR4 in susceptible and resistant banana. External symptoms and reddish-brown internal vascular discoloration of Foc TR4 in infected WT Cavendish a and b compared with resistant transgenic lines RGA2-3 c and d, and Ced9-21 e and f

Fig. 3

Disease incidence and gene expression analysis. a, b Levels of Foc TR4 infection in selected transgenic and WT banana plants throughout the 3-year field trial. a WT and RGA2 lines, and b WT and Ced9 lines. Wet seasons (November–April) are indicated. The number of biological replicates (n) of each independent line at the start of the trial is provided in Supplementary Table 1. Data points are percentage of biological replicates infected at time of assessment. *0.01 < p < 0.05, **0.001 < p < 0.01 at trial end (Tukey's HSD test). c, d Analysis of RGA2 mRNA expression levels in transgenic and WT banana plants. c Analysis of transgene (RGA2-Nos) expression levels using primers designed to amplify a 96 bp fragment spanning the RGA2 transgene/Nos terminator junction. d Analysis of RGA2 transgene and endogenous mRNA expression levels using primers designed to amplify a 92 bp fragment of both the RGA2 transgene and RGA2 endogenous sequences. All values are normalized expression levels expressed relative to line RGA2-3. WT GN; TR4-susceptible M. acuminata ssp. malaccensis (Mal-S) and TR4-resistant M. acuminata ssp. malaccensis (Mal-R). A single biological replicate was analyzed with three technical replicates (n = 3). Data are mean ± SEM

Fig. 4

Protein sequence alignment of the RGA2 transgene sequence from TR4-resistant M. acuminata ssp. malaccensis (Mal-R) and the three consensus RGA2 homologous sequences from WT GN. Amino acid differences are highlighted