ExoRNAi , a new tool to probe plant gene function exposes contrasting roles 1 for sugar exudation in host - finding by plant pathogens

12 Here we present a simple and rapid new method for RNAi-induced knockdown of genes in 13 tomato seedlings, through treatment with an aqueous solution of double-stranded RNA 14 (exoRNAi). The exoRNAi method is used to assess the involvement of tomato Sugar 15 Transporter Protein genes, stp1 and stp2 on the root exudation of glucose, fructose and 16 xylose; monosaccharide constituents of tomato root exudate. Plant parasitic nematodes 17 (PPNs) are responsible for an estimated 12.3% loss in crop production globally, which 18 equates to financial losses of approximately £100 billion annually. Our data show that 19 infective juveniles of the promiscuous PPN, Meloidogyne incognita are attracted to glucose 20 and fructose, but not xylose. Glucose and fructose also agonise serotonergic stylet thrusting 21 in M. incognita infective juveniles; a key parasitic behaviour necessary for invasion and 22 parasitism of host plants. In contrast, infective juveniles of the selective Solanaceae PPN, 23 Globodera pallida are not attracted to tested monosaccharides, nor do the 24 monosaccharides stimulate stylet thrusting. We demonstrate that knockdown of both 25 SlSTP1 and SlSTP2 in tomato seedlings by the exoRNAi method is robust and specific, and 26 that corresponding reductions of glucose and fructose, but not xylose, in collected exudate, 27 correlate directly with reduced infectivity and stylet thrusting of M. incognita. Knockdown 28 of SlSTP1 or SlSTP2 have no impact on the infectivity or stylet thrusting of G. pallida. The 29 exoRNAi platform can underpin future efforts to understand the early stages of plant-30 pathogen interactions in tomato, and potentially other crop plants.