Analysis of unconventional plant defence suppression by the signal peptide peptidase Spp1 in the Ustilago maydis - maize interaction

In the smut fungus Ustilago maydis, the signal peptide peptidase (SPP) Spp1 is essential for plant defence suppression and establishment of a compatible biotrophic interaction with its host plant maize. SPPs are endoplasmic reticulum (ER) resident intramembrane proteases, that are known to cleave remnant signal peptides as well as transmembrane domain (TMD) containing proteins, releasing them from the membrane for degradation or to exert a biological function. Catalytic activity is broadly conserved and crucial for the virulence function in U. maydis, however Spp1 substrates have not been identified. Under axenic culture conditions, Spp1 is dispensable, whereas its transcriptional induction upon plant infection is directly regulated by the unfolded protein response (UPR), which is tightly interconnected with biotrophic growth in U. maydis (Pinter et al., 2019). In this study, the plant defence responses to U. maydis inoculation were dissected using transcriptomics, revealing a broad but distinct response upon inoculation with Spp1 mutant strains. Transcriptome analysis of the mutant strains during in planta growth revealed a growth delay compared to WT strains and significantly reduced appressoria penetration efficiency of ∆spp1 strains. Spp1 expression needs to be dynamically regulated, with high transcript levels required throughout the whole biotrophic phase. Deletion of the UPR elements (UPREs) in its promoter abolished ER-stress and plant specific spp1 induction, but basal levels were sufficient for in planta proliferation suggesting cell type specific regulation by a so far unidentified transcriptional regulator. Moreover, the presence of UPREs in spp1 ortholog promoters and their plant specific induction seems to be conserved specifically in biotrophic fungi, hinting at a function of Spp1 in virulence exclusively of biotrophs. This was supported by the dispensability of Spp1 in the hemibiotroph V. dahliae and the requirement of Spp1 for virulence in the biotroph S. reilianum. Finally, potential substrates and interacting proteins of Spp1 were identified using a Co-IP approach from inoculated plant tissue, identifying the SCSdomain containing protein UMAG_11696 as an important Spp1-associated virulence factor and the Rsp3 effector that requires Spp1 for proper processing, which was previously shown to be required for its virulence function.