Extended Data Fig. 2: Differential polyadenylation of the stp6 mRNA produces in addition to Stp6 a short Stp6s protein lacking the putative transmembrane domain.

a, Scheme of the stp6 genomic region. The two polyadenylation sites (PAS) are indicated. Displayed in black are mapped RNA sequencing reads generated from FB1×FB2 infected maize plants 24 hours post infection (dataset available via ref. ³ and GEO accession number GSE103876). The stp6s and stp6 mRNAs derived from the stp6 genomic region are displayed as red lines. Yellow arrows indicate the stp6s and stp6 stop codons. Note that the stp6s stop codon is located in the first intron of stp6. Binding sites of oligonucleotides used in 3’RACE experiments and nested PCRs are indicated. The differences in abundance result from efficient polyadenylation after nucleotide position 1652 downstream of a stop codon conserved in all orthologues (leading to a 511 amino acid Stp6s (Stp6small) protein that lacks a transmembrane domain. Occasionally the first polyA signal is skipped and polyadenylation occurs after nucleotide 3661. Splicing of this mRNA eliminates the stop codon of stp6s, leading to a 953 amino acid Stp6 protein predicted to have a single transmembrane domain (Supplementary Fig. 7). b-c, DNA gels of products from 3’RACE PCR (left) and the corresponding nested PCR (right) with the indicated primer pairs. AUAP : PolyA adaptor primer provided by the 3’RACE kit. Black arrows indicate PolyA containing fragments of stp6s (b) and stp6 (c) that were sequenced. d, The stop codon of stp6s is conserved in orthologues genes. Nucleotide sequence alignment of the region containing the stop codon of stp6s from 11 sequenced smut fungi. MEPE: Melanopsichium pennsylvanicum; UESC: Ustilago esculenta; SPSC: Sporisorium scitamineum; SPRZ: Sporisorium reilianum f. sp. zeae; SPRS: Sporisorium reilianum f. sp. sorghi; UTCP: Ustilago trichophora; UTRI: Ustilago tritici; UMAG: U. maydis; UBRO: Ustilago bromivora; UHOR: Ustilago hordei (host: barley); UHOO: Ustilago hordei (host: oats). e, stp6 cDNA complements the stp6 deletion phenotype while stp6s is unable to complement. Seven-day-old maize seedlings were infected with the U. maydis stp6 deletion strain complemented with either stp6s, the stp6 cDNA or the stp6 genomic region with HA-tags inserted directly upstream of the stp6s stop codon and the stp6 stop codon (stp6s-HA stp6-HA). 12 d.p.i. disease symptoms were scored. The dashed lines indicate separate experiments. Data represent mean of n = 3 biologically independent experiments. Total numbers of infected plants are indicated above the respective columns. f, Detection of Stp6s-HA and Stp6-HA after immunoprecipitation. Plants were infected with strains SG200Δstp6-stp6s-HA (a stp6 deletion strain complemented with the stp6 genomic region with the HA-tag inserted directly upstream of the Stp6s stop codon), SG200Δstp6-stp6-HA (carrying the HA-tag at the C-terminus of Stp6) as well as the SG200Δstp6-stp6s-HA-stp6-HA described in e. Extracts from leaves were generated at 3 d.p.i. and subjected to HA-specific immunoprecipitation. Shown is an anti-HA western blot. Lanes are labeled with the expected HA-tagged proteins expressed. The filled arrow indicates Stp6-HA, the open arrow indicates Stp6s-HA. g, Stp6 is expressed during colonization. Plants were infected with SG200Δstp2-stp2-HA. Extracts from leaves were prepared at 3 d.p.i., subjected to co-IP/MS. Shown is a screenshot from Scaffold (v4.6.2, Proteome Software) of the Stp6 amino acid sequence. Highlighted in yellow are Stp6 peptides identified. The red square marks the end of Stp6s. Peptides identified from the region downstream of Stp6s indicate that the full-length Stp6 protein is expressed and is part of the complex.

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