Isolation of a Novel Gene (HSGc11) Whose Expression is Apparently Limited to the Hyphal Stage of Microbotryum violaceum

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Abstract
Microbotryum violaceum, Basidiomycota, infects species in the Caryophyllaceae (Pinks). Three morphological stages are found during the life cycle of M violaceum: haploid sporidial cells (of opposite mating type), dikaryotic hyphae, and diploid teliospores. The conjugated haploid cells and the dikaryotic hyphae are the only forms that are infectious to the host plant. Gene expression during hyphal development of this fungus was examined by mRNA differential display Using differential display, we isolated one fragment (c11) that represented a transcript expressed in the dikaryotic hyphal stage but not in the conjugated or haploid stages. The differential expression of the corresponding gene was confirmed by Northern blotting. This partial cDNA was cloned and sequenced. Rapid Amplification of cDNA Ends, 5'-RACE was used to recover the 5' end of a cDNA for c11. The combined sizes of the 5'-RACE product and the 3'-end cDNA generated from differential display corresponded with the mRNA size estimated by Northern analysis Sequences for several 5'-RACE clones differed by as much as 20% from each other. Sequence analysis for three 5'-RACE clones with identical sequence, when combined with that for the c11 differential display fragment, revealed an open reading frame (HSGc11) that encodes a protein with 455 amino acids; no significant similarities were found between this protein and others in the protein database. Southern hybridization analysis indicated that this gene belongs to a gene family in M. violaceum. The gene hybridized to DNA for M. violaceum sporidial strains of both mating types. The successful cloning of the HSGc11 gene demonstrated that the combination of mRNA differential display and 5'-RACE offers a relatively efficient approach for identification and cloning of developmental genes in this and other plant pathogenic fungi