Strain y2hgold

Y2H was performed according to the Clontech yeast protocol. Briefly, plasmids expressing viral and host proteins were co-transformed into yeast cells (strain Y2H Gold, Clontech catalog number: 630498). Then, selective culture mediums lacking tryptophan and leucine (SD/-Trp-Leu) or tryptophan, leucine, histidine, and adenine (SD/-Trp-Leu-His-Ade) were plated with yeast cells mentioned above to confirm the right transformation or analyze the interactions. BiFC and subcellular localization experiments were performed as previously described (Li et al. 2018a (link)). After 36–72 h infiltration, 1–2 cm² leaf sections were excised for examining fluorescence in epidermal cells by confocal microscopy (Carl Zeiss 980, German), equipped with a 63 × water-corrected objective in multitrack mode. CFP was excited at 458 nm and captured at 470–500 nm, YFP was excited at 514 nm and captured at 565–585 nm, and RFP was excited at 543 nm and captured at 590–630 nm. In general, at least 20 cells were examined for each experiment. The sequential scanning mode was applied for the co-imaging of different fluorescent proteins. Images were collected and analyzed using ZEN 2 (Carl Zeiss Microscope GmbH2011) imaging software.