In fungi, ambient pH acts as a key regulator of development and virulence. The vascular wilt pathogenFusarium oxysporumuses host alkalinization to promote infection of plant hosts through activation of the invasive growth mitogen-activated protein kinase (MAPK) Fmk1. The molecular events underlying pH-driven MAPK regulation are unknown. Using the ratiometric GFP-based pH sensor pHluorin, we find that bothF. oxysporumandSaccharomyces cerevisiaerespond to extracellular alkalinization or acidification with a transitory shift in cytosolic pH (pHc) and rapid changes in phosphorylation levels of the three fungal MAPKs Fmk1, Mpk1/Slt2 (cell wall integrity) and Hog1 (hyperosmotic stress). Pharmacological inhibition of the essential plasma membrane H+-ATPase Pma1, which leads to pHcacidification, is sufficient to trigger reprogramming of MAPK phosphorylation even in the absence of an extracellular pH shift. Screening of a subset ofS. cerevisiaemutants identified the sphingolipid-regulated AGC kinase Ypk1/2 as a key upstream component of pHc-modulated MAPK responses. We further show that acidification of pHcinF. oxysporumleads to an increase of the long chain base (LCB) sphingolipid dihydrosphingosine (dhSph) and that exogenous addition of dhSph activates Mpk1 phosphorylation. Our results reveal a pivotal role of pHcin the regulation of MAPK signaling and suggest new ways to control fungal growth and pathogenicity.

Fungal phytopathogens cause devastating losses in global agriculture. All plant-infecting fungi use conserved MAPK signaling pathways to successfully locate, enter, and colonize their hosts.

Like many hemibiotrophic plant pathogens, the root-infecting vascular wilt fungus Fusarium oxysporum induces an increase in the pH of the surrounding host tissue. How alkalinization promotes fungal infection is not fully understood, but recent studies point towards the role of cytosolic pH (pHc) and mitogen-activated protein kinase (MAPK) signaling. In fungi, pHc is mainly controlled by the essential plasma membrane H+-ATPase Pma1. Here we created mutants of F. oxysporum lacking casein kinase 1 (Ck1), a known negative regulator of Pma1. We found that the ck1Δ mutants have constitutively high Pma1 activity and exhibit reduced alkalinization of the surrounding medium as well as decreased hyphal growth and conidiation. Importantly, the ck1Δ mutants exhibit defects in hyphal chemotropism towards plant roots and in pathogenicity on tomato plants. Thus, Ck1 is a key regulator of the development and virulence of F. oxysporum.

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