SummaryRho-type GTPases regulate polarized growth in yeast by reorganization of the actin cytoskeleton and through signalling pathways that control the expression of cell wall biosynthetic genes. We report the cloning and functional analysis of rho1 from Fusarium oxysporum, a soilborne fungal pathogen causing vascular wilt on plants and opportunistic infections in humans. F. oxysporum strains carrying either a Drho1 loss-of-function mutation or a rho1 G14V gain-offunction allele were viable, but displayed a severely restricted colony phenotype which was partially relieved by the osmotic stabilizer sorbitol, indicating structural alterations in the cell wall. Consistent with this hypothesis, Drho1 strains showed increased resistance to cell wall-degrading enzymes and staining with Calcofluor white, as well as changes in chitin and glucan synthase gene expression and enzymatic activity. Re-introduction of a functional rho1 allele into the Drho1 mutant fully restored the wild-type phenotype. The Drho1 strain had dramatically reduced virulence on tomato plants, but was as virulent as the wild type on immunodepressed mice. Thus, Rho1 plays a key role during fungal infection of plants, but not of mammalian hosts.

In eucaryotic cells, the delivery of a secreted protein to the plasma membrane via vesicles must include transport, recognition, and fusion events. Proteins exposed on the cytoplasmic face of the secretory vesicles play a role in these events; these include the GTP-binding proteins, which are crucial components in this process. Fractions enriched with vesicles carrying glucose oxidase (GOX) activity from Fusarium oxysporum f. sp. lycopersici, a soilborne fungal pathogen causing vascular wilt on tomato plants, were obtained using two successive sucrose gradients, the first a linear-log and the second an isopycnic gradient. In this study, we used the following Fusarium strains: a wild-type and a strain carrying a Δrho1 loss-of-function mutation (presenting dramatically reduced virulence). By ADP-ribosylation with C3 exotoxin, and Western blot analysis with specific antibodies, we identified the small GTPases Rho1, Rho4, Cdc42 and Rab8, and a heterotrimeric Gα protein associated with vesicles carrying GOX activity. This was done for both strains, with the exception of Rho1, which was absent in the mutant strain; in addition, the levels of the Cdc42 protein were observed to be higher in the Δrho1 strain. These data indicate that three Rho proteins, Rho1, Rho4, and Cdc42, are present in secretory vesicles carrying GOX activity in F. oxysporum, and that Rho1 is not essential for the transport and secretion of, at least, cargo proteins carried in secretory vesicles, or Cdc42/Rho4 can fulfill its role in these events.