Fusarium head blight epidemics of wheat and barley cause heavy economic losses to farmers due to yield decreases and production of mycotoxin that renders the grain useless for flour and malt products. No highly resistant cultivars are available at present. Hyphae of germinating fungal spores use different paths of infection: After germination at the extruded tip of an ovary, the hyphae travel along the epicarp in the space between the lemma and palea. Infection of the developing kernel proceeds through the epicarp, successively destroying the layers of the fruit coat and finally the starch and protein accumulating endosperm. Hyphae reaching the rachis proceed to apically located developing kernels. Using a constitutively green fluorescence protein-expressing Fusarium wild-type strain, and its knockout mutant, preventing trichothecene synthesis, we demonstrate that trichothecenes are not a virulence factor during infection through the fruit coat. In the absence of trichothecenes, the fungus is blocked by the development of heavy cell wall thickenings in the rachis node of Nandu wheat, a defense inhibited by the mycotoxin. In barley hyphae of both wild-type and the trichothecene knockout mutant, are inhibited at the rachis node and rachilla, limiting infection of adjacent florets through the phloem and along the surface of the rachis. Effective resistance to Fusarium head blight requires expression of genes that combat these different pathways of infection.

Intracellular and secreted proteases fulfill multiple functions in microorganisms. In pathogenic microorganisms extracellular proteases may be adapted to interactions with host cells. Here we describe two cell surface-associated aspartic proteases, Sap9 and Sap10, which have structural similarities to yapsins of Saccharomyces cerevisiae and are produced by the human pathogenic yeast Candida albicans. Sap9 and Sap10 are glycosylphosphatidylinositol-anchored and located in the cell membrane or the cell wall. Both proteases are glycosylated, cleave at dibasic or basic processing sites similar to yapsins and Kex2-like proteases, and have functions in cell surface integrity and cell separation during budding. Overexpression of SAP9 in mutants lacking KEX2 or SAP10, or of SAP10 in mutants lacking KEX2 or SAP9, only partially restored these phenotypes, suggesting distinct target proteins of fungal origin for each of the three proteases. In addition, deletion of SAP9 and SAP10 modified the adhesion properties of C. albicans to epithelial cells and caused attenuated epithelial cell damage during experimental oral infection suggesting a unique role for these proteases in both cellular processes and host-pathogen interactions.Proteases possess multiple functions in nature ranging from regulation of subtle cellular processes by activating distinct proproteins to nonspecific degradation of proteins for recycling of biomolecules. Several pathogenic microorganisms have adapted this biochemical property to fulfill a number of specialized functions during the infective process. The substrate specificities of these proteases may be very narrow, as in the case of bacterial toxins responsible for botulism or anthrax. In contrast, facultative pathogens may secrete proteases that have more general and much broader effects and play important roles in both saprophytic growth and infection.The yeast Saccharomyces cerevisiae has served as a eukaryotic model organism to study functions of regulatory proteases including the Kex2 protein, a proprotein processing subtilisin-like serine protease of the late Golgi compartment (1-5). Soon after the discovery of Kex2, numerous Kex2-like processing systems were identified in other eukaryotic species, including humans (6), plants (7), the fission yeast Schizosacchoromyces pombe (8), and the plant pathogenic fungus Ustilago maydis (9). Characteristic of target proteins of Kex2-like proteases are N-terminal processing sites with dibasic amino acids, in particular Lys-Arg. Although Kex2 was shown to be the major protease for processing at dibasic residues, alternative pathways exist in S. cerevisiae (10). Gene products of YPS1 (yapsin 1) and YPS2 (yapsin 2), two closely related glycosylphosphatidylinositol (GPI) 2 -anchored aspartic proteases, were able to cleave the ␣-factor pheromone precursor (a well known Kex2 substrate) at Lys-Arg sites (10 -12), and overexpression of YPS1 and YPS2 partially compensated for the loss of Kex2 (10, 13). However, the biological roles of yapsins are unknown.The human...

The ability to change between yeast and hyphal cells (dimorphism) is known to be a virulence property of the human pathogen Candida albicans. The pathogenesis of disseminated candidosis involves adhesion and penetration of hyphal cells from a colonized mucosal site to internal organs. Parenchymal organs, such as the liver and pancreas, are invaded by C. albicans wild-type hyphal cells between 4 and 24 h after intraperitoneal (i.p.) infection of mice. In contrast, a hypha-deficient mutant lacking the transcription factor Efg1 was not able to invade or damage these organs. To investigate whether this was due to the inability to undergo the dimorphic transition or due to the lack of hypha-associated factors, we investigated the role of secreted aspartic proteinases during tissue invasion and their association with the different morphologies of C. albicans. Wild-type cells expressed a distinct pattern of SAP genes during i.p. infections. Within the first 72 h after infection, SAP1, SAP2, SAP4, SAP5, SAP6, and SAP9 were the most commonly expressed proteinase genes. Sap1 to Sap3 antigens were found on yeast and hyphal cells, while Sap4 to Sap6 antigens were predominantly found on hyphal cells in close contact with host cells, in particular, eosinophilic leukocytes. Mutants lacking EFG1 had either noticeably reduced or higher expressed levels of SAP4 to SAP6 transcripts in vitro depending on the culture conditions. During infection, efg1 mutants had a strongly reduced ability to produce hyphae, which was associated with reduced levels of SAP4 to SAP6 transcripts. Mutants lacking SAP1 to SAP3 had invasive properties indistinguishable from those of wild-type cells. In contrast, a triple mutant lacking SAP4 to SAP6 showed strongly reduced invasiveness but still produced hyphal cells. When the tissue damage of liver and pancreas caused by single sap4, sap5, and sap6 and double sap4 and -6, sap5 and -6, and sap4 and -5 double mutants was compared to the damage caused by wild-type cells, all mutants which lacked functional SAP6 showed significantly reduced tissue damage. These data demonstrate that strains which produce hyphal cells but lack hypha-associated proteinases, particularly that encoded by SAP6, are less invasive. In addition, it can be concluded that the reduced virulence of hypha-deficient mutants is not only due to the inability to form hyphae but also due to modified expression of the SAP genes normally associated with the hyphal morphology.