High-throughput MS/MS was used to identify proteins secreted by Fusarium graminearum (Gibberella zeae) during growth on 13 media in vitro and in planta during infection of wheat heads. In vitro secreted proteins were collected from the culture filtrates, and in planta proteins were collected by vacuum infiltration. A total of 289 proteins (229 in vitro and 120 in planta) were identified with high statistical confidence. Forty-nine of the in planta proteins were not found in any of the in vitro conditions. The majority (91-100%) of the in vitro proteins had predicted signal peptides, but only 56% of the in planta proteins. At least 13 of the nonsecreted proteins found only in planta were single-copy housekeeping enzymes, including enolase, triose phosphate isomerase, phosphoglucomutase, calmodulin, aconitase, and malate dehydrogenase. The presence of these proteins in the in planta but not in vitro secretome might indicate that significant fungal lysis occurs during pathogenesis. On the other hand, several of the proteins lacking signal peptides that were found in planta have been reported to be potent immunogens secreted by animal pathogenic fungi, and therefore could be important in the interaction between F. graminearum and its host plants.

Based on the analysis of its genome sequence, the ectomycorrhizal (ECM) basidiomycetous fungus Laccaria bicolor was shown to be lacking many of the major classes of secreted enzymes that depolymerize plant cell wall polysaccharides. To test whether this is also a feature of other ECM fungi, we searched a survey genome database of Amanita bisporigera with the proteins found in the secretome of Trichoderma reesei (syn. Hypocrea jecorina), a biochemically well-characterized industrial fungus. Additional proteins were also used as queries to compensate for major groups of cell-wall-degrading enzymes lacking in the secretome of T. reesei and to substantiate conclusions drawn from the T. reesei collection. By MS/MS-based "shotgun" proteomics, 80 proteins were identified in culture filtrates of T. reesei strain RUTC30 grown on corn cell walls and in a commercial "cellulase" preparation, Spezyme CP. The two T. reesei enzyme preparations were qualitatively and quantitatively similar, the most striking difference being the lack of at least five major peptidases from the commercial enzyme mixture. Based on our analysis of A. bisporigera, this ECM fungus is deficient in many major classes of cell-wall-degrading enzymes, including both glycosyl hydrolases and carbohydrate esterases. By comparison, the genomes of the saprophytic basidiomycetes Coprinopsis cinerea and Galerina marginata (using a genome survey sequence approximately equivalent in depth to that of A. bisporigera) have, like T. reesei, a much more complete complement of cell-wall-degrading enzymes.

The class IV homeodomain leucine zipper transcription factor GLABRA2 (GL2) acts in a complex regulatory circuit that regulates the differentiation of trichomes in Arabidopsis thaliana. We describe a genetic interaction with HOMEODOMAIN GLABROUS11 (HDG11), previously identified as a negative regulator of trichome branching. gl2 hdg11 double mutants display enhanced trichome cell-type differentiation defects. Transgenic expression of HDG11 using the GL2 promoter partially suppresses gl2 trichome phenotypes. Vice versa, expression of GL2 under the control of its native promoter partially complements hdg11 ectopic branching. Since gl2 hdg11 and gl2 myb23 double mutants and the triple mutant display similar trichome differentiation defects, we investigated a connection to the R2R3-MYB transcription factor MYB23. We show that MYB23 transcript levels are significantly reduced in shoots from gl2 mutants and that GL2 can drive the expression of a MYB23-promoter fusion to green fluorescent protein. Yeast one-hybrid, chromatin immunoprecipitation, and in planta reporter gene experiments indicate that an L1-box in the MYB23 promoter acts as a GL2 binding site. Taken together, our findings reveal a functional redundancy between GL2 and HDG11, two homeodomain leucine zipper transcription factors previously thought to mediate opposing functions in trichome morphogenesis. A model is proposed in which GL2 transcript levels are maintained through a positive feedback loop involving GL2 activation of MYB23.

Summary Glucosylceramides (GlcCer), glucose-conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryote cells. Yet, the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi-organ eukaryotes. To address this, we examined Arabidopsis lines lacking or deficient in GlcCer by insertional disruption or by RNAi suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis. Null mutants for GCS (designated “gcs-1”) were viable as seedlings, albeit strongly reduced in size, and failed to develop beyond the seedling stage. Heterozygous plants harboring the insertion allele exhibited reduced transmission through the male gametophyte. Undifferentiated calli generated from gcs-1 seedlings and lacking GlcCer proliferated in a manner similar to calli from wild-type plants. However, gcs-1 calli, in contrast to wild-type calli, were unable to develop organs on differentiation media. Consistent with a role for GlcCer in organ-specific cell differentiation, calli from gcs-1 mutants formed roots and leaves on media supplemented with the glucosylated sphingosine glucopsychosine, which was readily converted to GlcCer independent of GCS. Underlying these phenotypes, gcs-1 cells had altered Golgi morphology and fewer cisternae per Golgi apparatus relative to wild-type cells, indicative of protein trafficking defects. Despite seedling lethality in the null mutant, GCS RNAi suppression lines with ≤2% of wild-type GlcCer levels were viable and fertile. Collectively, these results indicate that GlcCer are essential for cell-type differentiation and organogenesis, and plant cells produce GlcCer amounts in excess of that required for normal development.

Two fungal-secreted α-fucosidases and their genes were characterized. FoFCO1 was purified from culture filtrates of Fusarium oxysporum strain 0685 grown on L-fucose and its encoding gene identified in the sequenced genome of strain 4287. FoFCO1 was active on p-nitrophenyl-α-fucoside (pNP-Fuc), but did not defucosylate a nonasaccharide (XXFG) fragment of pea xyloglucan. A putative α-fucosidase gene (FgFCO1) from Fusarium graminearum was expressed in Pichia pastoris. FgFCO1 was ~1,800 times less active on pNP-Fuc than FoFCO1, but was able to defucosylate the XXFG nonasaccharide. Although FgFCO1 and FoFCO1 both belong to Glycosyl Hydrolase family 29, they share <25 % overall amino acid identity. Alignment of all available fungal orthologs of FoFCO1 and FgFCO1 indicated that these two proteins belong to two subfamilies of fungal GH29 α-fucosidases. Fungal orthologs of subfamily 1 (to which FoFCO1 belongs) are taxonomically more widely distributed than subfamily 2 (FgFCO1), but neither was universally present in the sequenced fungal genomes. Trichoderma reesei and most species of Aspergillus lack genes for either GH29 subfamily.