Phylogenetic and gene expression analyses
Multiple sequence alignment was performed using ClustalW software (Kyoto
University Bioinformatics Center, Kyoto, Japan) utilizing CDS ofNtSUS1-7 and StSUS3 to build a phylogenetic tree by
FastTree tool (Price, Dehal, and Arkin 2009). Total RNA from leaves and
guard cells of 45 days-old plants were isolated and the cDNA synthesized
using SV Total RNA Isolation System and M-MLV Reverse Transcriptase
(Promega Corporation, Madison, Wisconsin, EUA). The primer for protein
phosphatase 2A (PP2A ) was designed by aligning the coding
sequence (CDS) obtained from NCBI database using the muscle tool in Mega
X software (FW 5’-CACTTCAGTCAATTGATAACGTC-3’; REV
5’-GCAAAATCCTACCAAAGAGGG-3’). The primers used to investigateNtSUS expression were obtained from previous work (Wang et al.,
2015) (FW 5’- CACATTGATCCATACCACGGGGAT-3’; REV
5’-ACAGCAGCCAGTGTCAACAACCGA-3’). In order to estimate SUSsilencing, qRT-PCR was performed by using GoTaq® qPCR Master Mix
(Promega Corporation, Madison, Wisconsin, EUA) accordingly to the
manufacturer instructions. Relative transcripts expression was
calculated by the 2-ΔΔCt method, in which PP2Awas used as internal control and WT was used as calibrator (Livak &
Schmittgen, 2001).