Biotechnol Lett (2015) 37:2083–2090 DOI 10.1007/s10529-015-1880-7 ORIGINAL RESEARCH PAPER Inoculation of Nicotiana tabacum with recombinant potato virus X induces RNA interference in the solenopsis mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) Arif Muhammad Khan . Muhammad Ashfaq . Azhar Abbas Khan . Akhtar Rasool . Javed Iqbal . Shahid Mansoor Received: 23 February 2015 / Accepted: 2 June 2015 / Published online: 19 June 2015 Ó Springer Science+Business Media Dordrecht 2015 Abstract further showed a reduction in mRNA levels of the Objectives The chitin synthase 1 (CHS1) gene in target gene in mealybugs feeding on treated plants. Phenacoccus solenopsis (PsCHS1) was evaluated as a Mortality in parent adults and emerging nymphs (21 potential target of RNA interference (RNAi) by using and 29 %) exposed to the treated plants was signifi- Potato virus X (PVX) as a vector (recombinant PVX) cantly higher (P \ 0.05) than those exposed to for expressing RNAi triggering elements in Nicotiana uninoculated (-ve control) or inoculated with non- tabacum L. recombinant PVX (PVX-control). The number of Results RT-PCR analysis confirmed the expression surviving adults and the combined number of adults of PsCHS1 in N. tabacum inoculated with recombi- and nymphs (47 and 60 %) was significantly nant-PVX–PsCHS1 (treated). RT- and multiplex-PCR (P \ 0.05) lower on the treated plants than the -ve (76 %) or PVX (74 %) control. The visual observa- tions verified the physical deformities in mealybugs Electronic supplementary material The online version of exposed to the treated plants. this article (doi:10.1007/s10529-015-1880-7) contains supple- mentary material, which is available to authorized users. Conclusion chitin synthase 1 is a potential RNAi target in P. solenopsis and the recombinant PVX can A. M. Khan Á M. Ashfaq Á A. Rasool Á S. Mansoor be used as a tool to evaluate candidate RNAi triggering National Institute for Biotechnology and Genetic elements in plants. Engineering, Faisalabad, Pakistan A. M. Khan Keywords Mealy bug Á Phenacoccus solenopsis Á Department of Biotechnology, University of Sargodha, Potato virus X Á Recombinant virus Á RNA Sargodha, Pakistan interference Á Solenopsis mealybug M. Ashfaq (&) Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada Introduction e-mail: [email protected] A. A. Khan Solenopsis mealybug, Phenacoccus solenopsis Tins- College of Agriculture, Bahauddin Zakariya University, ley (Hemiptera: Pseudococcidae), is a highly poly- Bahadur Campus Layyah, Multan, Pakistan phagous insect recorded on more than 50 plant families (Hodgson et al. 2008). Cotton (Gossypium J. Iqbal School of Life Sciences, Beijing Institute of Technology, hirsutum L.) is a preferred host for this insect as both Beijing, People’s Republic of China adults and nymphs infest all parts of the plant causing 123 2084 Biotechnol Lett (2015) 37:2083–2090 damage by sucking phloem sap and egesting sugary study provides useful information on the potential material. RNAi targets in solenopsis mealybug and PVX as a RNAi has emerged as a new tool for insect control tool to perform transient RNAi analysis. with its applications in field (Hunter et al. 2010). RNAi-based plant protection against chewing insects is well documented (Chu et al. 2014) while its success Materials and methods against sucking insects is being debated (Christiaens and Smagghe 2014). Absence of a RNA-dependent Mealybug culture RNA polymerase system that is required to amplify the RNAi effects by continuous transcription of RNA Phenacoccus solenopsis adults were collected from molecules (Sijen et al. 2001) in most insects (Price and pesticide-free cotton fields of the National Institute for Gatehouse 2008), makes the use of RNAi for pest Biotechnology and Genetic Engineering, Faisalabad. control even more challenging. Production of dsRNA Adults were used to initiate a P. solenopsis colony on (in vivo) has been successful using vectors that can tobacco (N. tabacum) plants and maintained in express dsRNA via stable transformation (Baum et al. glasshouse under controlled conditions at 27 ± 3 °C. 2007). Virus-induced gene silencing (VIGS) is an Second instar nymphs were used for the bioassay efficient and simple substitute to reduce the endoge- studies for the evaluation of RNAi effects. nous gene expression in plants (Lu et al. 2003) and researchers have exploited VIGS via recombinant RNA extractions viruses to induce RNAi in plant feeding organisms (Kumar et al. 2012). Various plant viruses have been RNA was extracted from whole insects and/or insect effectively used for expression of transgenes in plants; body parts using TRIzol as described previously (Khan as for example, Tobacco mosaic virus (TMV) (Khan et al. 2013). RNA was precipitated by adding two et al. 2013), Tobacco rattle virus (Senthil-Kumar and volumes 95 % ethanol and 0.1 vol 3 M sodium acetate. Mysore 2014), Citrus tristeza virus (Hajeri et al. 2014) RNA pellet was washed with 75 % (v/v) ethanol, re- and Potato virus X (PVX) (Angell and Baulcombe suspended in DEPC-treated SDW and stored at -80 °C. 1999). RNA/DNA was quantified by ethidium bromide/1 % There is often a high variability in results due to (w/v) agarose gel and re-quantified by NanoDrop differing dsRNA application procedures, target genes Spectrophotometer ND-1000 (NanoDrop, USA). and target insects (Uryu et al. 2013). The practical dsRNA delivery for phloem-feeding hemipterans, like cDNA synthesis and PCR amplification mealybugs, is likely to be via their host plants (Christiaens and Smagghe 2014). As for example, First strand cDNA was synthesized using superscript Wuriyanghan and Falk (2013) employed tomatillo cDNA synthesis kit (Invitrogen) with oligo(dT)-primer (Physalis philadelphica), tomato (Solanum lycoper- using 500 ng RNA following manufacturer’s instruc- sicum) and tobacco (Nicotiana tabacum) to infest with tions. Briefly, RNA and oligo(dT) primer were mixed TMV for a successful RNAi of b-actin and and incubated at 65 °C for 5 min before adding 10 ll V-ATPase in Bactericera cockerelli. Screening of reaction mixture (0.1 M DTT 2 ll, 25 mM MgCl2 4 ll, genes vital for mealybug development can provide 10X RT buffer 2 ll, RNase out (40 U/ll) 1 lland useful information to control this insect through gene SuperScript II 1 ll). The reaction was incubated at suppression and interference. Moreover, development 42 °C for 50 min followed by 70 °C for 10 min and the of reliable transient assay to express mealybug genes cDNA was stored at -20 °C. A 507 bp fragment of in plants can help pinpointing potential target genes in CHS1 was amplified using degenerate primers from this pest for the development of transgenic plant hosts. Ashfaq et al. (2007) with a slight modification in PCR Present study was focused on identification, expres- conditions (Supplementary Table 1). These primers are sion, and analysis of a gene involved in the develop- based on conserved sequences of CHS1 from diverse ment of P. solenopsis. Furthermore, suppression of insect orders (Coleoptera, Diptera, Hymenoptera, Lepi- this gene in P. solenopsis using RNAi was evaluated doptera) and have amplified this gene from another by PVX, a virus only transmitted mechanically. The mealybug, Planococcus citri (Khan et al. 2013). 123 Biotechnol Lett (2015) 37:2083–2090 2085 RNAi target sequence selection plasmid carrying single colony of A. tumefaciens was inoculated into 3 ml LB culture in the presence of a A partial fragment of CHS1 was isolated from P. suitable antibiotic (rifampicin, kanamycin or tetracy- solenopsis cDNA (200 ng/reaction) using primers and cline). The culture was grown at 28 °C for 48 h with PCR conditions and outlined in Supplementary vigorous shaking. 600 ll of the culture was inoculated Table 1. Amplified PCR products were gel purified into 30 ml LB media (containing 30 lleachof using Spin Prep Gel DNA kit (Novagen) following kanamycin, rifampicin and tetracycline), 6 llacetosy- manufacturer’s protocol. Amplicons were ligated into ringone and 300 ll 1 M MES and allowed to grow until pTZ57R/T TA cloning vector and cloned into E. coli an OD600 = 1. Cells were centrifuged at *40009g for using standard molecular techniques, and the positive 10 min and pellet was re-suspended in 10 mM MES, clones were sequenced commercially (Macrogen Inc., 10 mM MgCl2, and 100 lM acetosyringone to an South Korea). Sequence accuracy of the genes was OD600 = 1.0. The culture was placed in dark at room verified and gene-specific primers were designed temperature for 2–3 h (or overnight) before infiltration. (adding endonuclease recognition sites) (Supplemen- Agrobacterium suspension was infiltrated onto the tary Table 1) for the re-amplification of the target gene lower side of a healthy leaf (infiltrated 2-3 leaves/plant) region. Vector sequences were removed and the edited (treated plants) using a needleless disposable syringe sequences were reconfirmed for the identity of isolated (Wuriyanghan et al. 2011). PVX-control plants were gene using basic local alignment search tool (BLAST) inoculated with non-recombinant PVX while -ve at NCBI (http://blast.be-md.ncbi.nlm.nih.gov/Blast. control plants were not inoculated. cgi). After confirming the sequence fidelity for the gene, primers were designed using Snapdragon dsRNA Detection of interfering RNAs in plants design (http://www.flyrnai.org/cgi-bin/RNAi_find_ primers.pl) (Supplementary Table 1) to amplify a After the PVX-specific symptoms were visible in the 419 bp gene fragment divergent among insect species in PVX–PsCHS1inoculated (treated) plants, the presence order to reduce the off-target RNAi effects. The partial of transgenic mRNA in plants was confirmed through fragment of P. solenopsis CHS1 (PsCHS1) was isolated RT-PCR using plant RNA and the primers developed by PCR amplification and used for further studies. from the target P. solenopsis gene sequences (Sup- plementary Table 1). Plant RNA was extracted from Transient expression of siRNA/dsRNA in plants the symptomatic leaves using TRIzol.