Rational design of biosafe crop resistance to a range of nematodes using RNA interference

Double stranded RNA (dsRNA) molecules targeting two genes have been identified that suppress economically important parasitic nematode species of banana. Proteasomal Alpha Subunit 4 (pas-4) and Actin-4 (act-4) were identified from a survey of sequence databases and cloned sequences for genes conserved across four pests of banana, Radopholus similis, Pratylenchus coffeae, Meloidogyne incognita and Helicotylenchus multicinctus. These four species were targeted with dsRNAs containing exact 21 nucleotide matches to the conserved regions. Potential off-target effects were limited by comparison to Caenorhabditis, Drosophila, rat, rice and Arabidopsis genomes. In vitro act-4 dsRNA treatment of R. similis suppressed target gene expression by 2.3 fold, nematode locomotion by 66 ± 4% and nematode multiplication on carrot discs by 49 ± 5%. The best transgenic carrot hairy root lines expressing act-4 or pas-4 dsRNA reduced transcript message abundance of target genes in R. similis by 7.9 fold and 4 fold and nematode multiplication by 94 ± 2% and 69 ± 3%, respectively. The same act-4 and pas-4 lines reduced P. coffeae target transcripts by 1.7 and 2 fold and multiplication by 50 ± 6% and 73 ± 8%. Multiplication of M. incognita on the pas-4 lines was reduced by 97 ± 1% and 99 ± 1% while target transcript abundance was suppressed 4.9 and 5.6 fold. There was no detectable RNAi effect on non-target nematodes exposed to dsRNAs targeting parasitic nematodes. This work defines a framework for development of a range of non-protein defences to provide broad resistance to pests and pathogens of crops.