Abstract
Burrowing nematodes (Radopholus similis) cause severe harm in many agronomic and horticultural crops and are very difficult to manage. Cathepsin S is one of the most important cysteine proteinases and plays key roles in nematodes and many other parasites. To evaluate the effect of in planta RNAi on the control of this nematode, a specific fragment from the protease gene, cathepsin S (Rs-cps), was cloned into the binary vector pFGC5941 in the forward and reverse orientations to construct recombinant plant RNAi vectors. Transgenic Nicotiana benthamiana plants expressing Rs-cps dsRNA were obtained and studied. The transcript abundance of Rs-cps dsRNA appeared to be diverse in the different transgenic lines. Moreover, the bioassay results revealed that Rs-cps transgenic N. benthamiana plants were resistant to R. similis and the transcription level of Rs-cps in R. similis was drastically decreased. In addition, the reproduction and hatching rate of R. similis isolated from the Rs-cps transgenic plants were also significantly reduced. Our results suggest that Rs-cps is essential for the reproduction and pathogenicity of R. similis. This is the first study to employ in planta RNAi approach to target the Rs-cps gene for the control of plant parasitic nematodes.
Highlights
Radopholus similis is a migratory endoparasitic nematode that is known to be a destructive pest of bananas, peppers, coffee, citrus crops and many other agronomic and horticultural crops[1,2,3,4]
The transgenic seedlings germinated from transformed N. benthamiana calli were selected for kanamycin resistance, and the seedlings were transferred into the rooting medium when they grew to approximately 2 cm
The results showed that these Rs-cps transgenic plants had one to four insertion loci (Plants No 4 and had a single insertion locus, plants No 6 and had two insertion loci, plants No 7 and 12 had three insertion loci, and plant No 3 had four insertion loci), but no Rs-cps hybridization bands were observed with genomic DNA from the egfp transgenic plants and wild-type N. benthamiana plants (Fig. 2C)
Summary
Radopholus similis is a migratory endoparasitic nematode that is known to be a destructive pest of bananas, peppers, coffee, citrus crops and many other agronomic and horticultural crops[1,2,3,4]. It is important to explore effective approaches for controlling the nematode One such strategy involves the application of plant-mediated RNA interference (in planta RNAi), which confers resistance to plants engineered to express specific dsRNA to target and silence specific genes involved in the reproduction, development, parasitism and pathogenesis of nematodes. We demonstrated that the cathepsin S gene of R. similis (Rs-cps) plays important roles in reproduction and pathogenesis using in vitro RNAi16. Despite reports of in vitro RNAi studies, cps genes of plant parasitic nematodes have not yet been targeted using the in planta RNAi approach. The reproduction and hatching of R. similis isolated from Rs-cps transgenic N. benthamiana plants were significantly inhibited This is the first study to employ in planta RNAi targeting of the Rs-cps gene to control the plant parasitic nematode
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