Response to Environmental Stresses, Cell-wall Integrity, and Virulence Are Orchestrated Through the Calcineurin Pathway in Ustilago hordei

In eukaryotes, several biological processes are regulated through calcium signaling. Calcineurin is a calcium-calmodulin-regulated serine/threonine phosphatase consisting of catalytic subunit A and regulatory subunit B. Phosphatase activity resides in the catalytic subunit, which activates by dephosphorylation downstream components such as transcription factor Crz1. The importance of this pathway to respond to environmental stress has been explored in several fungal pathogens. The basidiomycete Ustilago hordei causes covered smut of barley. We addressed the role of the Ca2+-calcineurin activated pathway by deleting UhCna1 and UhCnb1. These genes were not essential in U. hordei but the corresponding mutants displayed a variety of phenotypes when applying environmental stress such as sensitivity to pH, temperature, H2O2, mono- and divalent cations; and to genotoxic, acid, or oxidative stresses. Cell-wall integrity was compromised and mutants displayed altered cell morphologies. Mating was delayed but not abolished, and combined sensitivities likely explained a severely reduced virulence toward barley plants. Expression analyses revealed that response to salt stress involved the induction of membrane ATPase genes UhEna1 and UhEna2, which were regulated through the calcineurin pathway. Upregulation of UhFKS1, a 1,3-beta-D-glucan synthase gene, correlated with the increased amount of 1,3-beta-D-glucan in the calcineurin mutants grown under salt stress.