Intensilight c hgf1

HEK293 cells were purchased from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Cells were transfected with (1) the combination of pX459ver2-LDLR gRNA#1, #2, or #3 with pCAG-EGxxFP-LDLR exon 4; (2) the combination of pX459ver2-LDLR gRNA#4 or #5 with pCAG-EGxxFP-LDLR exon 2; and (3) the combination of pX459ver2-LDLR gRNA#6 with pCAG-EGxxFP-LDLR exon 3. After the transfection using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA), cells were incubated for 2 days in Dulbecco’s Modified Eagle’s Medium (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). Then, the cells were observed under a fluorescent microscope Eclipse TS100 equipped with Intensilight C-HGF1 (Nikon, Tokyo, Japan).

Primary leaves of plants were inoculated with B. hordei at 5 DAS (see below), and then at 7 DAS the leaves of infected and equivalent uninfected plants were used for DAPI staining (adapted from Chazotte, 2011 ). Microscopy observations and disease scoring in the infected leaves were according to an adapted protocol outlined by Lambertucci et al. (2019) (link). The infected leaf samples were cleared with ethanol (80% v/v) and acetic acid (20% v/v) solution overnight at 4 °C in the dark, and then rinsed with phosphate buffered saline (PBS) for 30 mins. DAPI solution (2 ng µl^–1) was added to leaf tissues for 20 min and then they were de-stained for 30 min with PBS. The DAPI-stained leaves were also stained with 2 μg ml^–1 of propidium iodide (PI ; adapted from Scheler et al., 2016 (link)). The leaves with their adaxial side up were placed on glass slides with PBS and sealed with nail varnish. Nuclei were imaged using a Nikon Eclipse Ni-E Upright microscope with preselected filters for DAPI (358/461 nm) and TxRed (535/617 nm) with a Nikon Intensilight C-HGF1 UV light source. i-stack images were taken of uninfected epidermal cells and infected nuclei (near the haustoria) and analysed using ImageJ software.