The UPR-regulated Phu1 protein mediates ER homeostasis, stress resistance and pathogenic growth of Ustilago maydis

The unfolded protein response (UPR) is a conserved signaling pathway that ensures endoplasmic reticulum (ER) homeostasis under ER-stress inducing conditions. In the corn smut fungus Ustilago maydis, the UPR is specifically activated during biotrophic growth and remains active during the whole infection process, requiring a tight regulation to prevent apoptosis and to ensure proper protein folding, processing, and secretion of effector proteins. The physical interaction between the central UPR regulator Cib1 (Clp1 interacting bZIP 1) and the key developmental regulator Clp1 (clampless 1) leads to complex formation of both proteins, modulating UPR target gene expression. Following this, Cib1 phosphorylation is reduced, stability of both proteins is increased and the expression of UPR target genes is modulated. In this study, phu1 (potential homolog of UDP-galactose transporter 1 (HUT1)) was identified as a direct Cib1 target gene and crucial component for filamentous and pathogenic growth of U. maydis. Deletion of phu1 leads to a nearly complete loss of virulence, increased ER and cell wall stress susceptibility and severely impaired fungal proliferation in planta. Moreover, Δphu1 strains display a constitutively active UPR, which negatively affects the expression of mating type genes and consequently interferes with the pathogenic development of U. maydis. Importantly, mitogen-activated protein kinase (MAPK) mediated signaling within the pheromone response and cell wall integrity (CWI) pathways is not affected in the deletion mutant. Conditional expression of phu1 during growth in planta revealed its importance during early stages of infection, up to three days post inoculation. Complementation strains expressing the Homo sapiens SLC35B1 and Saccharomyces cerevisiae HUT1 orthologs are fully virulent and stress-resistant, indicating that Phu1 likely fulfills a conserved function across species. SLC35B1 encodes an ATP/ADP exchanger localized in the ER membrane, which provides ATP to a plethora of ATP consuming processes in the ER, including the essential ER chaperone BiP (binding immunoglobulin protein). Accordingly, absence of phu1 likely results in ATP depletion within the ER, impaired chaperone activity and defective protein folding and modification. Furthermore, impaired posttranslational modifications potentially affect proteins like chitin deacetylases (CDAs), resulting in abnormal or mislocated deposition of newly synthesized chitin in the cell walls of Δphu1 strains. The accumulation of unfolded or misfolded proteins triggers chronic UPR activation which critically depends on Phu1. As a consequence, loss of phu1 may lead to UPR hyperactivation and subsequent apoptosis. Thus, Phu1 appears to be essential for maintaining ER function and cellular homeostasis in U. maydis and its loss leads to multilayered defects including uncontrolled UPR activation, reduced mating gene expression and misregulated chitin synthesis, all together preventing successful host infection.