The Cell Wall Integrity MAPK pathway controls actin cytoskeleton assembly during fungal somatic cell fusion.

Somatic cell fusion in Neurospora crassa depends on two MAP kinases, MAK-1 and MAK-2, but their precise contributions to directed growth have remained unclear. Using analog-sensitive alleles and live-cell imaging, we found that MAK-1 activity is required for RAC-1 activation and polarized actin assembly, while MAK-2 controls the positioning of RAC-1 and actin at the fusion tips. Inhibition of MAK-1 disrupted cell communication, prevented actin focus formation, and blocked membrane recruitment of MAK-2 and SO. In contrast, MAK-2 inhibition caused SO and actin to shift to alternative cortical sites, showing that MAK-2 regulates their spatial orientation rather than activation. Actin disruption with latrunculin A eliminated MAK-2 and SO recruitment, confirming its essential role in their localization. Importantly, MAK-1 controlled actin polymerization only during tropic growth, not during general hyphal extension, and acted upstream of RAC-1. These findings establish complementary roles for MAK-1 and MAK-2 in directing RAC-1-dependent actin dynamics during fungal cell fusion. Summary statementMAK-1 activates the Rho GTPase RAC-1 to drive actin polymerization, while MAK-2 orients this RAC-1/actin complex toward the fusion partner, defining complementary MAPK roles in somatic cell fusion.