Dnaman version 6

Gene prediction and structure analysis were carried out using the GRAMENE database (http://www.gramene.org). Homologs of TCD3 were identified by BLASTP analysis against the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov) and subjected to multiple sequence alignment using DNAMAN version 6.0 (Lynnon Biosoft, USA). The signal peptide was predicted with SignalP version 2.0^{47 (link)}. The phylogenetic tree was constructed using MEGA version 6 software (http://www.megasoftware.net).

The PCR amplification products were sent to the Bioengineering Co. Ltd. laboratory (Shanghai, China) for sequencing. Single nucleotide polymorphisms (SNPs) located in the 5′ flanking region of the HSP70 gene were detected by analyzing the polymorphic loci of the target sequence in the DNA samples of 196 Holstein cows using the DNAMAN version 6.0 (Lynnon Biosoft, San Ramon, CA, USA) and Chromas2.0 (Technelysium, South Brisbane, Australia) software.

The DNA and amino acid sequences of OsLIL3 and its homologues were acquired from GenBank (http://www.ncbi.nlm.nih.gov) by BLAST. The prediction of chloroplast transit peptide was carried out by TargetP and ChloroP (http://www.cbs.dtu.dk/services/TargetP/;http://www.cbs.dtu.dk/services/ChloroP/) [31 (link), 32 (link)]. Multiple sequence alignment and phylogenetic analysis were conducted using DNAMAN version 6.0 (Lynnon Biosoft).

Sequence gaps between clones were determined by RT-PCR using specific primers designed based on the obtained cDNA sequences (Table S1). The 5′ and 3′ terminal sequences of the viral RNA were determined by rapid amplification of cDNA ends (RACE) with a kit (GeneRacer™ Core kit, Cat no. 45-0168, Lot no. 1362098. Invitrogen, Carlsbad, USA) following the manufacturer′s instructions. The authenticity of the siRNA-assembled viral genome sequences was validated by Sanger sequencing of the RT-PCR products covering the entire genome. The amplified PCR products were cloned into the pMD18-T vector (TaKaRa, Dalian, China) and transformed into competent cells of Escherichia coli DH5α. Sequencing was performed at Sangon Biotech (Shanghai) Co., Ltd, China, and each nucleotide was determined from at least three independent overlapping clones. The obtained clone sequences were assembled together using DNAMAN version 6.0 (Lynnon Biosoft Corporation, USA, http://www.lynon.com/).

AxyPrepTM DNA gel extraction kit (Axygen, CA, USA) was used to purify amplified products from the agarose gel. The purified PCR harvests were ligated to pMD19-T (Simple) vector (TaKaRa, Shiga, Japan) following the manufactures instruction and subsequently transformed into the competent cell of Escherichia coli strain MC1022 (a gift from Dr. Salah Bouzoubaa, University of Strasbourg, Strasbourg, France) as described earlier by Sambrook et al. [53 ]. Recombinant plasmids were confirmed by PCR and validated by successive sequencing of three positive clones of each plasmid (Genscript Biological Science, Nanjing, China) from each region of Bangladesh. Gene sequences from this study were scrutinized by DNAMAN version 6.0 (LynnonBiosoft, QC, Canada). Homology of the expected gene sequences was determined by the BLASTn server of NCBI.

The open-reading-frame sequence of NbWRKY40 (Niben101Scf04944g05002.1) was downloaded from NCBI¹. Homologs from other species were identified using an NCBI database sequence matching tool (blastn), and sequences with high similarity were downloaded. DNAMAN version 6.0 (Lynnon Biosoft, Quebec, Canada) was used to generate a multiple sequence alignment. MEGA 7.0 was used to perform a phylogenetic analysis. The neighbor-joining method was used to construct a phylogenetic tree with bootstrap values of 1000 (Kumar et al., 2016 (link)).
For NbWRKY40 gene cloning, total RNA was extracted from N. benthamiana seedlings using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) and then treated with DNase (Invitrogen, Carlsbad, CA, United States). The full-length NbWRKY40 sequence was amplified using reverse-transcription PCR (RT-PCR), specific primers (Supplementary Table 1), and super-fidelity DNA polymerase (Phanta Max, Vazyme Biotech Co., Ltd., Nanjing, China). The purified PCR product was then inserted into a plasmid vector pGEM-T easy and transformed into E. coli (pGEM-T Easy vector; Promega, Madison, WI, United States), and the full NbWRKY40 was sequenced from positive clones.