Extended Data Fig. 1: Complementation of the stp phenotype with wild type and HA-tagged alleles and characterization of stp deletion strains.

a, List of putative effector genes deleted at the onset of this study. The observed virulence phenotype is indicated. * indicates that stp1 was originally deleted as part of the virulence gene cluster 5b¹⁸ and subsequently shown to be responsible for the loss of virulence¹⁹. b, Seven-day-old maize seedlings were infected with the indicated U. maydis deletion strains, the respective single copy complemented strains (c), as well as deletion strains complemented with the respective single copy HA fusion protein. 12 d.p.i. disease symptoms were scored. The dashed lines separate sets of independent experiments. All data represent mean of n = 3 biologically independent experiments. Total numbers of infected plants are indicated above the respective columns. c, Maize epidermal cells 1 day post infection with the indicated stp deletion strains in the SG200AN1 or SG200AM1 background. Shown are confocal z-stack maximum projections of GFP (green) and bright-field (grey) channel overlays. Fluorescent hyphae on the plant surface are traced in white, untraced hyphae are intracellular. Arrows indicate appressoria. Scale bars, 5 μm. d-e, Anti-HA western blot of HA-specific immunoprecipitations from total maize leaf extracts 3 d.p.i. with the indicated U. maydis strains. f, Anti-HA western blot of the total extract (T) and eluate after HA-specific immunoprecipitation (IP) obtained from leaves 3 d.p.i. with the strain SG200Δstp5 HA-stp5. The HA-Stp5 protein, indicated by an asterisk, was only detectable after denaturing SDS-PAGE in total extracts (T) but the HA-tag was not accessible to IP under native conditions (IP). The heavy and light chain (hc, lc) of the HA-antibody used for IP appear here and not in (d) and (e) because in comparison to (d) and (e) the eluate from 10 times more beads was loaded. g, Relative expression levels of stp genes during biotrophic development of U. maydis were revealed by RNA sequencing analysis of RNA samples collected from FB1 × FB2 infected maize plants (dataset available via ref. ³ and GEO accession number GSE103876). The vertical axis indicates normalized counts from DESeq2 analysis. The horizontal axis indicates the infection stages: 0.5, 1, 2, 4, 6, 8 and 12 d.p.i. Data represent mean ± s.d. of n = 3 biological replicates. Colour codes for the different genes are indicated on the right. h, Filamentous growth of the spotted indicated strains was visible on PD-charcoal plates grown for 48 h at room temperature. i, Stress sensitivity of stp deletion strains. Serial 10-fold dilutions of SG200 and the indicated deletion strains were spotted on complete medium supplemented with 2% (w/v) glucose in the absence of stressors (CM) or in the presence of the indicated stressors. The plates were incubated for 48 h at 28 °C.

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