Extended Data Fig. 5: Identification of the SCREW receptor NUT.
From: Phytocytokine signalling reopens stomata in plant immunity and water loss
a, Scheme to identify SCREW receptor candidates. Among 269 LRR-RKs in Arabidopsis, 26 members are upregulated by flg22 treatment, among which 11 members contain at least 18 LRRs in the extracellular domain. The cognate ligands of five of them remain unknown at the time of the study. b, NUT belongs to the XI subfamily of LRR-RK and is phylogenetically close to HAE and HSLs. Phylogenetic analysis of 52 LRR-RKs from the subfamily VII (orange curved line), X (purple curved line), XI (green curved line), and XII (grey curved line) is shown. Purple and red bars indicate the induction folds after elf18 and flg22 treatments, respectively. Olive green bars with numbers indicate the number of LRRs. Blue squares indicate cognate ligands of LRR-RKs. AT5G25930 (NUT) is highlighted in bold red font. The protein sequences were retrieved from NCBI (https://www.ncbi.nlm.nih.gov/) for MEGAX phylogenetic analysis using the neighbour-joining method with 1,000 bootstrap replicates. The phylogenetic tree was displayed by iTOL (https://itol.embl.de/). The expression data were from GENEVESTIGATOR V3. c, SCREWs and NUT are conserved in dicots and monocots. Protein sequences were blast-searched in NCBI using Arabidopsis SCREW1, NUT, or FLS2 as queries, and the phylogenetic analysis was performed as in (b). Red, purple, and olive curved lines indicate monocots, dicots, and other plant classes, respectively; Orange and teal bars indicate the percentage of homology of FLS2 and NUT in different plant species, respectively. Blue dots, olive stars, and red squares indicate FLS2, NUT, and SCREW homologs, respectively. Peach, lime green, grey, and brown fans denote different plant families. d, Diagram of AT5G25930 (NUT) with annotated T-DNA insertion sites in nut-1 (WiscDslox450B04) and nut-2 (SALK_207895). Solid bars indicate exons, lines for introns, and open boxes for UTRs. Arrows indicate primers used for genotyping. e, Genotyping of nut-1 and nut-2. The T-DNA insertions in the NUT coding region were PCR-amplified using genomic DNAs of WT, nut-1, or nut-2 as templates and primers shown in (d). f, RT–qPCR analysis of NUT transcripts. NUT expression levels in ten-day-old plate-grown seedlings were analysed using RT–qPCR with UBQ10 as an internal control. Data are shown as mean ± s.d. (n = 3, biologically independent samples) with one-way ANOVA followed by Tukey’s test. Exact P values are provided in the graph and Supplementary Table 3. Experiments were repeated three times with similar results (e, f).
