Topo2

Gene Racer kit (Invitrogen, CA, USA) was used to amplify 5’ and 3’ ends of RZE1 and ZNF2 RNA. Full length transcript of RZE1 was amplified using the primers indicated in S5 Table from RNA extracted from cells grown on V8 for 24h following the manufacturer’s instructions. All the 3 sets of 5’ and 3’ RACE primers yielded the same start and stop termini. The amplified bands were separated on 0.8% agarose gel and extracted using the gel purification kit (Invitrogen), cloned in TOPO 2.1 (Invitrogen) and sequenced.

Based on N. lugens genome and transcriptome [35 (link), 36 (link)], one RanGTPase homology (a single copy gene) was identified and the sequence was confirmed by reverse transcription polymerase chain reaction (RT-PCR) using primers listed in Table 1. The PCR product was gel purified, ligated into the vector TOPO2.1 (Invitrogen, Carlsbad, CA) and transformed into Escherichia coli DH5α competent cells (Novagen, Darmstadt, Germany). Ten recombinant plasmids from several independent subclones were fully sequenced on the Applied Biosystems 3730 automated sequencer (Applied Biosystems, Foster City, USA) from both directions. The resulting sequence (NlRan) was submitted to GenBank (KT313028).
The theoretical isoelectric point and molecular weight of the deduced NlRan protein were calculated using ExPASy [37 ]. Homologues from Drosophila melanogaster, Aedes aegypti, Apis mellifera, Harpegnathos saltator, Riptortus pedestris, Helicoverpa armigera, Bombyx mori, Acyrthosiphon pisum and Pediculus humanus corporis were aligned with NlRan using ClustalW2 [38 (link)].

Based on the published N. lugens genomic and transcriptomic data (Xue et al., 2014 (link); Wan et al., 2015 (link)), NlHR3 and NlFTZ-F1 homologies were identified and their sequences were confirmed by the reverse transcription polymerase chain reaction (RT-PCR) using primers, as shown in Supplementary Table S1. The PCR product was gel purified, ligated into the vector TOPO2.1 (Invitrogen, Carlsbad, CA), and transformed into Escherichia coli DH5α competent cells (Novagen, Darmstadt, Germany). A total of 10 recombinant plasmids from several independent subclones were fully sequenced on the Applied Biosystems 3730 automated sequencer (Foster City, CA) from both directions. The newly described transcript variants of NlHR3 and NlFTZ-F1 were submitted to GenBank.
ClustalW2 was used to perform a homologous sequence alignment of HR3 and FTZ-F1 proteins from Nilaparvata lugens, Drosophila melanogaster, Tribolium castaneum, Bombyx mori, Apis mellifera, Aedes aegypti, Pediculus humanus corporis, Blattella germanica, and Acyrthosiphon pisum (Larkin et al., 2007 (link)). The conserved domains were predicted by using the Simple Modular Architectural Research Tool (SMART; http://smart.embl-heidelberg.de/) and InterPro: protein sequence analysis and classification (http://www.ebi.ac.uk/interpro/).