Abstract
Radopholus similis is a migratory plant-parasitic nematode that causes severe damage to many agricultural and horticultural crops. Calreticulin (CRT) is a Ca2+-binding multifunctional protein that plays key roles in the parasitism, immune evasion, reproduction and pathogenesis of many animal parasites and plant nematodes. Therefore, CRT is a promising target for controlling R. similis. In this study, we obtained the full-length sequence of the CRT gene from R. similis (Rs-crt), which is 1,527-bp long and includes a 1,206-bp ORF that encodes 401 amino acids. Rs-CRT and Mi-CRT from Meloidogyne incognita showed the highest similarity and were grouped on the same branch of the phylogenetic tree. Rs-crt is a multi-copy gene that is expressed in the oesophageal glands and gonads of females, the gonads of males, the intestines of juveniles and the eggs of R. similis. The highest Rs-crt expression was detected in females, followed by juveniles, eggs and males. The reproductive capability and pathogenicity of R. similis were significantly reduced after treatment with Rs-crt dsRNA for 36 h. Using plant-mediated RNAi, we confirmed that Rs-crt expression was significantly inhibited in the nematodes, and resistance to R. similis was significantly improved in transgenic tomato plants. Plant-mediated RNAi-induced silencing of Rs-crt could be effectively transmitted to the F2 generation of R. similis; however, the silencing effect of Rs-crt induced by in vitro RNAi was no longer detectable in F1 and F2 nematodes. Thus, Rs-crt is essential for the reproduction and pathogenicity of R. similis.
Highlights
The burrowing nematode, Radopholus similis [(Cobb, 1893) Thorne, 1949] is a migratory plant parasitic nematode, that has been listed as a quarantine plant pest in many countries and regions [1, 2]
The Rs-crt cDNA was composed of a 52-bp 50-untranslated region (50-untranslated regions (UTR)), a 1,206-bp ORF, and a 269-bp 30-UTR containing a typical polyadenylation signal (ATTAAA) (S2 Fig)
We confirmed that Rs-crt plays key roles in the reproduction and pathogenicity of R. similis using in vitro and plant-mediated RNA interference (RNAi)
Summary
The burrowing nematode, Radopholus similis [(Cobb, 1893) Thorne, 1949] is a migratory plant parasitic nematode, that has been listed as a quarantine plant pest in many countries and regions [1, 2]. RNAi mediated by dsRNA soaking (in vitro RNAi) has been developed as an effective tool for studying gene function in many plant nematodes and other organisms [8,9,10,11,12,13]. The silencing effect of in vitro RNAi on some genes in plant-parasitic nematodes is highly time-limited [11, 14]. Most previous studies have focused on sedentary plant endoparasitic nematodes [21,22,23,24,25,26], and there is limited information available regarding the use of plant-mediated RNAi to control migratory plant parasitic nematodes
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