Non-nematode-derived double-stranded RNAs induce profound phenotypic changes in Meloidogyne incognita and Globodera pallida infective juveniles

Abstract

Nine non-nematode-derived double-stranded RNAs (dsRNAs), designed for use as controls in RNA interference (RNAi) screens of neuropeptide targets, were found to induce aberrant phenotypes and an unexpected inhibitory effect on motility of root knot nematode Meloidogyne incognita J2s following 24 h soaks in 0.1 mg/ml dsRNA; a simple soaking procedure which we have found to elicit profound knockdown of neuronal targets in Globodera pallida J2s. We have established that this inhibitory phenomenon is both time- and concentration-dependent, as shorter 4 h soaks in 0.1 mg/ml dsRNA had no negative impact on M. incognita J2 stage worms, yet a 10-fold increase in concentration to 1 mg/ml for the same 4 h time period had an even greater qualitative and quantitative impact on worm phenotype and motility. Further, a 10-fold increase of J2s soaked in 0.1 mg/ml dsRNA did not significantly alter the observed phenotypic aberration, which suggests that dsRNA uptake of the soaked J2s is not saturated under these conditions. This phenomenon was not initially observed in potato cyst nematode G. pallida J2s, which displayed no aberrant phenotype, or diminution of migratory activity in response to the same 0.1 mg/ml dsRNA 24 h soaks. However, a 10-fold increase in dsRNA to 1 mg/ml was found to elicit comparable irregularity of phenotype and inhibition of motility in G. pallida, to that initially observed in M. incognita following a 24 h soak in 0.1 mg/ml dsRNA. Again, a 10-fold increase in the number of G. pallida J2s soaked in the same volume of 1 mg/ml dsRNA preparation did not significantly affect the observed phenotypic deviation. We do not observe any global impact on transcript abundance in either M. incognita or G. pallida J2s following 0.1 mg/ml dsRNA soaks, as revealed by reverse transcriptase-PCR and quantitative PCR data. This study aims to raise awareness of a phenomenon which we observe consistently and which we believe signifies a more expansive deficiency in our knowledge and understanding of the variables inherent to RNAi-based investigation.

Introduction

Plant parasitic nematodes (PPNs) impose a major economic burden on agriculture, horticulture, forestry and amenity sectors worldwide; current best estimates suggest combined losses in excess of $US 125 billion annually (Bird and Kaloshian, 2003, Chitwood, 2003). The vast majority of these losses are due to parasitism by sedentary species, in particular root knot (Meloidogyne spp.) and cyst (Heterodera and Globodera spp.) nematodes which induce complex feeding sites in the root vascular tissue (Gheysen and Fenoll, 2002, Bird, 2004, Caillaud et al., 2008), leading to reduced water and solute transport both locally and systemically, stunted growth, chlorosis and reduced crop yield (Williamson and Gleason, 2003). Conventionally, nematicides have been used as part of an integrated approach to the management of PPNs. However, as concerns grow over the environmental implications associated with sustained use of some nematicides, many have been forced into withdrawal, leaving major shortcomings in our ability to successfully limit yield loss. Therefore, efforts to identify novel approaches to PPN control are imperative.

RNA interference (RNAi), the conserved phenomenon of gene silencing mediated by double-stranded RNA (dsRNA), represents a promising molecular tool with potential applications in both the functional genomics and control of PPNs. The ability to specifically knock down a selected mRNA transcript allows the investigation of gene function and interaction, in addition to the validation of putative control targets through loss-of-function phenotype analysis. The study of gene function through RNAi is well documented for many PPNs, including both the potato cyst nematode Globodera pallida (Urwin et al., 2002, Kimber et al., 2007) and the root knot nematode Meloidogyne incognita (Bakhetia et al., 2005, Rosso et al., 2005, Shingles et al., 2007), two economically important phytoparasitic species. In addition to these reverse-genetics applications, in planta RNAi has shown potential as a method of PPN control, with six published accounts of such an approach in cyst and root knot nematodes (Huang et al., 2006, Steeves et al., 2006, Yadav et al., 2006, Fairbairn et al., 2007, Valentine et al., 2007, Sindhu et al., 2008).

Initial study of RNAi in PPNs revealed a process by which specific and robust gene silencing could be performed routinely, supporting the utility of RNAi in the development of innovative control strategies through the exposition of unique molecular traits, which could be targeted by novel nematicides, or through in planta expression of nematode-specific dsRNA and the silencing of genes essential to the induction and maintenance of successful parasitism. However, subsequent gene silencing efforts have exposed pronounced discrepancies in the efficacy of application and reproducibility among different free-living and parasitic nematode species (Geldhof et al., 2006, Geldhof et al., 2007); observations in direct contrast to the early promise and sustained application of RNAi in PPNs.

Methodological disparities can make direct comparison of results between and even within different laboratories difficult, especially when applying similar protocols to different targets in biologically diverse parasites. However, technical discrepancy should perhaps be expected at such an early stage in the investigation of RNAi mechanisms in nematode parasites, when based upon the current paucity of relevant modelling strategies. Whilst the resultant diversity of investigative approaches has proffered an immediate and informative return, a better understanding of the variables inherent to RNAi is required if progress is to continue towards unequivocal gene function analyses and therapeutic potential. To this end, we present for consideration an unexpected and previously undocumented phenomenon discovered during RNAi screens of FMRFamide-like peptide (FLP) transcripts in both M. incognita and G. pallida J2 infective stage nematodes.

FLPs are members of the largest known family of neuropeptides in nematodes, which play crucial and diverse roles in the modulation of behaviour, motor and sensory functions, feeding and reproduction (Maule et al., 2002, McVeigh et al., 2006). Following FLP knockdown by RNAi, we subject the worms to a migration assay and routinely observe strong inhibition of migratory ability in G. pallida, compared with worms soaked in non-nematode control dsRNA or water only (Kimber et al., 2007). We believe that FLPergic processes present a promising target for the development of novel in planta controls. The data presented within this current study emerged from efforts to apply FLP silencing protocols successfully employed in G. pallida to the southern root knot nematode M. incognita.

For reasons not easily explained, the control of RNAi-based investigation remains notoriously inconsistent and whilst the majority of recent publications in the field have employed some form of non-nematode dsRNA control, the parameters with which specificity is examined vary greatly. As independent gene target and protocol combinations will be perceived a priori, to impact on worm biology in diverse and often disparate ways, the design and rationale behind experimental controls will often be tailored accordingly. However, in attempting to increase the rigour with which we examined the specificity of gene knockdown, it was discovered that both M. incognita and G. pallida infective stage J2s present abnormal phenotypes and behaviour on exposure to relatively low amounts of non-nematode dsRNAs, which were originally intended for use as negative controls. Preliminary study of gene silencing application in the root knot nematode Meloidogyne minor, an emerging pathogen of temperate grass and arable crops (Turner and Fleming, 2005), reveals a similar phenomenon. We believe these findings may have serious implications for the procedures used to exploit RNAi as a reverse-genetics tool in these PPNs.