Mutant screen for iri phenotypes and characterization of the iri1 mutant in Arabidopsis. A, Schematic diagram of the three successive selection stages of the iri mutant screen on 23,547 T-DNA insertion lines from the SALK/SAIL collection. Small populations of approximately five seedlings were screened per line (stage 1) and rescreened (stage 2) for sporulation by Hyalopoeronospora arabidopsidis WACO9 (Hpa) upon saturating the soil to a final concentrations of 0.5-mM RBH and subsequent inoculation with Hpa conidionspores (top). Putative iri lines were validated in controlled RBH-IR assays by scoring leaves from water- and RBH-treated (0.5 mM) plants into four Hpa colonization classes at 5–7 dpi (bottom; Supplemental Figure S1). Representative photographs of trypan blue-stained leaves on the bottom left indicate the Hpa colonization classes, ranging from healthy leaves (I), hyphal colonization without conidiospores (II), hyphal colonization with conidiophores (III), to extensive hyphal colonization with conidiophores and deposition of sexual oospores (IV). B, Gene model of the IRI1 gene (At5g40780) encoding LHT1. Triangles indicate two independent T-DNA insertions in the lht1-5 (iri1-1) and lht1-4 (iri1-2) mutants, respectively, to confirm the involvement of LHT1 in RBH-IR against Hpa. C, Quantification of RBH-IR against Hpa in leaves of Col-0, lht1-4 and lht1-5. Shown are frequency distributions of trypan blue-stained leaves across the four Hpa colonization classes (see A). Different letters indicate statistically significant differences between samples at 6 dpi (Fisher’s exact tests + Bonferroni correction; P < 0.05; n = 70–80 leaves). D, Quantification of arrested Hpa colonization by callose. Hpa-induced callose was analyzed in aniline blue/calcofluor-stained leaves by epifluorescence microscopy. Shown are percentages of callose-arrested and nonarrested conidiospores at 3 dpi, as detailed by Schwarzenbacher et al. (2020). Different letters indicate statistically significant differences in frequencies between samples (Fisher’s exact tests + Bonferroni correction; P < 0.05; n > 100 conidiospores).