In order to identify novel genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans that show hypersensitivity to the chitin-binding agent Calcofluor White (CFW). Affected loci are designated cal loci. The phenotype of one of these alleles, calI11, also includes shortened hyphal compartments and increased density of branching in the absence of CFW, as well as reduced staining of cell walls by the lectin FITC-Concanavalin A (ConA), which has strong binding affinity for mannosyl residues. We have identified two A. nidulans genes (AN8848.3 and AN9298.3, designated gmtA and gmtB, respectively) that complement all aspects of the phenotype. Both genes show strong sequence similarity to GDPmannose transporters (GMTs) of Saccharomyces and other yeasts. Sequencing of gmtA from the calI11 mutant strain reveals a G to C mutation at position 943, resulting in a predicted alanine to proline substitution at amino acid position 315 within a region that is highly conserved among other fungi. No mutations were observed in the mutant strain's allele of gmtB. Meiotic mapping demonstrated a recombination frequency of under 1 % between the calI locus and the phenA locus (located~9.5 kb from AN8848.3), confirming that gmtA and calI are identical. A GmtA-GFP chimera exhibits a punctate distribution pattern, consistent with that shown by putative Golgi markers in A. nidulans. However, this distribution did not overlap with that of the putative Golgi equivalent marker CopA-monomeric red fluorescent protein (mRFP), which may indicate that the physically separated Golgi-equivalent organelles of A. nidulans represent physiologically distinct counterparts of the stacked cisternae of plants and animals. These findings demonstrate that gmtA and gmtB play roles in cell wall metabolism in A. nidulans similar to those previously reported for GMTs in yeasts.

In order to identify genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans. We have identified two distinct genes (GMT1, GMT2) in one such mutant (R205) that are homologous to the yeast VRG4 GDP‐mannose transporter genes. GDP‐mannose transporters carry nucleotide sugars from the cytosol across the Golgi apparatus membrane in various eukaryotic organisms including plants and a variety of fungi. We have observed punctate patterns of fluorescence of GMT1 and GMT2 GFP chimeras, indicative of Golgi localization in A. nidulans. Cloned GMT1 and GMT2 complement hypersensitivity to the chitin binding agent calcofluor white in strain R205, and ameliorate hyper‐branching and septation abnormalities R205 displays under normal growth conditions. GMT1 in R205 contains a genetic mutation that causes an A315P amino acid substitution, and the amino acid sequence of GMT2 in R205 is identical to wild type. However, both GMT1 and GMT2 appear to be essential. These findings suggest that GMT1 and GMT2 are involved in cell wall maintenance in A. nidulans.Funded by Research Corporation and the Merck/AAAS Undergraduate Science Research Program