Conservation of the molecular mechanisms controlling appressorium-mediated penetration during evolution was assessed through a functional study of the ClPLS1 gene from Colletotrichum lindemuthianum orthologous to the MgPLS1 from Magnaporthe grisea, involved in penetration peg development. These two plant-pathogenic Pyrenomycetes differentiate appressoria to penetrate into plant tissues. We showed that ClPLS1 is a functional homologue of MgPLS1 in M. grisea. Loss of ClPLS1 function had no effect on vegetative growth, conidiation or on appressorium differentiation and maturation. However, Clpls1::hph mutants are non-pathogenic on either intact or wounded bean leaves, as a result of a defect in the formation and/or positioning of the penetration pore and consequently in the formation of the penetration peg. These observations suggest that the fungal tetraspanins control a conserved appressorial function that could be required for the correct localization of the site where the penetration peg emerges.