Molecular Characteristics and Efficacy of 16D10 siRNAs in Inhibiting Root-Knot Nematode Infection in Transgenic Grape Hairy Roots

Abstract

Root-knot nematodes (RKNs) infect many annual and perennial crops and are the most devastating soil-born pests in vineyards. To develop a biotech-based solution for controlling RKNs in grapes, we evaluated the efficacy of plant-derived RNA interference (RNAi) silencing of a conserved RKN effector gene, 16D10, for nematode resistance in transgenic grape hairy roots. Two hairpin-based silencing constructs, containing a stem sequence of 42 bp (pART27-42) or 271 bp (pART27-271) of the 16D10 gene, were transformed into grape hairy roots and compared for their small interfering RNA (siRNA) production and efficacy on suppression of nematode infection. Transgenic hairy root lines carrying either of the two RNAi constructs showed less susceptibility to nematode infection compared with control. Small RNA libraries from four pART27-42 and two pART27-271 hairy root lines were sequenced using an Illumina sequencing technology. The pART27-42 lines produced hundred times more 16D10-specific siRNAs than the pART27-271 lines. On average the 16D10 siRNA population had higher GC content than the 16D10 stem sequences in the RNAi constructs, supporting previous observation that plant dicer-like enzymes prefer GC-rich sequences as substrates for siRNA production. The stems of the 16D10 RNAi constructs were not equally processed into siRNAs. Several hot spots for siRNA production were found in similar positions of the hairpin stems in pART27-42 and pART27-271. Interestingly, stem sequences at the loop terminus produced more siRNAs than those at the stem base. Furthermore, the relative abundance of guide and passenger single-stranded RNAs from putative siRNA duplexes was largely correlated with their 5′ end thermodynamic strength. This study demonstrated the feasibility of using a plant-derived RNAi approach for generation of novel nematode resistance in grapes and revealed several interesting molecular characteristics of transgene siRNAs important for optimizing plant RNAi constructs.

The Impact of Plant-Parasitic Nematodes on Agriculture and Methods of Control

“…Mc16D10L expression was reduced in eggs and juveniles developed on transgenic potato which suggest a stable heritability of the construct. Decreased egg production was also observed in transgenic grape lines expressing 16D10L [160].…”

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…This allows not only experimental manipulations in the lab but also target parasitic nematodes by creating nematode resistant plants (Yang et al, 2013). However, there is a heterogeneity in the nature in the presence of non-cell autonomous RNAi.…”

RNA ‘Information Warfare’ in Pathogenic and Mutualistic Interactions

“…Considerable data have shown that plant-feeding invertebrates, such as nematodes [75][76][77] and arthropods [78][79][80], can ingest and process plant RNAs into small RNAs that are active within their cells (reviewed in [81]). In plants and fungi, natural uptake of exogenous RNA seems to be almost unknown to date, although apparently targeted silencing induced by dsRNA exogenously applied to germinating Fusarium spores under in vitro conditions has been reported [34,38].…”