Dokument

Summary:
Secreted effectors play crucial roles during the establishment of the biotrophic interaction between Ustilago maydis and maize. In a previous study (Schipper, 2009) it had been demonstrated that a deletion of the stp1 effector gene resulted in a complete loss of virulence symptoms in maize infection and that such mutants elicited a hypersensitive response. This distinguishes stp1 from most other secreted effectors that are either dispensable for pathogenicity or have only a minor effect on virulence. This study focuses on the functional analysis of Stp1. A mutational analysis showed that the conserved N- and C-terminal domains of Stp1 can be separately expressed but are both required for Stp1 protein function. The long central variable domain was demonstrated to be dispensable yet may promote fungal growth in vascular bundles. stp1 homologs from closely related smut fungi of U. maydis could replace stp1 in U. maydis, indicating a conserved function. Stp1Δ136-432 lacking the central domain could be purified to homogeneity and was stable, while the isolated C-terminal domain, Stp1433-515, was unstable after purification. This could suggest that N- and Cterminal domains of Stp1 stabilize each other. Stp1-HA expressed by U. maydis was detected in the nucleus of plant cells by immunolocalization suggesting that Stp1 may suppress plant defense responses by affecting the transcription of respective genes. Both cytoplasmic and apoplastic maize proteins were identified as interaction partners of Stp1 by yeast two-hybrid assays using Stp1Δ136-432 as bait, suggesting that Stp1 may be an effector with both apoplastic and cytoplasmic functions The C-terminus of Stp1 as well as Stp1Δ136-432 could inhibit the activity of a maize extracellular cysteine protease, Sip3, which was identified as one of the apoplastic interaction partners. The interactions between Stp1 and the cytoplasmic interactors Sip9, a cell number regulator 8, Sip16, a CCR4-NOT transcription complex subunit, Sip19, a serine/threonine-protein kinase and Sip21, a VIP2 protein were verified with full-length cDNA but await to be confirmed by other techniques. RNA-Seq analysis demonstrated that several early defense response genes are not induced by stp1 mutants expressing the N-terminus of Stp1 while stp1 mutants expressing the Cterminus of Stp1 triggered even stronger plant defense responses than stp1 mutants during colonization. This suggests that N- and C-terminal domains of Stp1 have distinct functions.

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