Characterization of small GTPases Cdc42 and Rac and the relationship between Cdc42 and actin cytoskeleton in vegetative and ectomycorrhizal hyphae ofSuillus bovinus

This work reports the isolation and molecular characterization of CDC42 and RAC1 cDNAs from the ectomycorrhiza forming filamentous homobasidiomycete S uillus bovinus. Previously, no RAC gene was described from filamentous fung i and no CDC42 gene was described from homobasidiomycetes. Southern hybridi zation with SbCDC42 and SbRAC1 cDNAs indicated that the S. bovinus genome c ontains only one CDC42 and one RAC1 gene. The predicted amino acid sequence of SbRac1p is 77% identical with the Rac1B protein of chick, whereas SbCdc 42p is most identical with Schizosaccharomyces pombe Cdc42p, showing 88% id entity, In the predicted amino acid sequences of SbRac1p and SbCdc42p, the five guanine nucleotide binding regions, switch I and II, and the effector domain are highly identical to those known in other small GTPases. These do main structures suggest that in S. bovinus, SbRac1p and SbCdc42p function a s molecular switches regulating the organization of actin cytoskeleton, sim ilar to yeasts and mammals, SbRAC1 and SbCDC42 were expressed in vegetative and ectomycorrhizal hyphae, and SbCdc42p was detected in ectomycorrhiza-fo rming hyphae if growth and differentiation of the symbiotic hyphae took pla ce. Cdc42p and actin were localized at the tips of S. bovinus vegetative hy phae. Similar to yeast, in filamentous fungi Cdc42p may be necessary to mai ntain the actin cytoskeleton at hyphal tips, making the polarized growth of the hyphae possible. In developing ectomycorrhiza, Cdc42p and actin were v isualized in association with plasma membrane in swollen cells typical to t he symbiotic hyphae. The role of Cdc42p and actin in regulation of the grow th pattern and morphogenesis of ectomycorrhizal hyphae is discussed.