Submitted to: Mycological Progress
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/4/2011
Publication Date: 4/13/2011
Citation: Roberts, R.G., Reymond, S.T., Bischoff, J.F. 2011. Differential gene expression in Alternaria gaisen exposed to dark and light. Mycological Progress. DOI: 10.1007/s11557-011-0752-3. Interpretive Summary: Many species have been described in the genus Alternaria based upon their differing appearance and biologies, but existing DNA-based analyses of genes have not supported these species. As a result the naming of these organisms, especially the plant pathogens, has become a practical problem for scientists who have to identify exotic pathogens in order to prevent their introduction into the U.S. Because the appearance of these species is determined in part by how they react to light stimulation, we grew one exotic plant pathogen, Alternaria gaisen, in both light and dark and then sampled the genes that were activated by the fungus during a specific life stage called sporulation, which is when it makes spores. We discovered that one of the genes expressed by A. gaisen after light exposure was very similar to a gene for a protein called ‘aegerolysin’, so we obtained a partial sequence of the ‘aegerolysin’ gene from a number of different species. The sequences of the ‘aegerolysin’ gene were much more informative than other genes, and an analysis of this gene from a limited number of strains showed that it could be used to separate the small-spored species of Alternaria into groups that are consistent with their biologies and appearance. Efforts are underway to obtain the entire ‘aegerolysin’ gene sequence and to evaluate its possible role in pathogen identification with a much broader sampling of species.
Technical Abstract: Character states observed during sporulation have been the basis for segregation and description of many of the small-spored species of Alternaria. Phylogenetic analyses of ITS and housekeeping genes from small-spored Alternaria spp. do not support most of the currently defined morphological species or species-groups. However, these molecular data are incongruent with a range of other species-associated physiological and pathological characters. The conidiation response of Alternaria gaisen was characterized by forward and reverse selective subtractive hybridization of cDNA produced from cultures of A. gaisen grown either in total darkness or in total darkness followed by scarification and 24h exposure to light. Differential expression of 184 transcripts was confirmed by virtual Northern analysis. Transcripts or their translation products were identified by similarity to known sequences using BLAST. Sixty-seven cloned ESTs or their hypothetically translated proteins had either no significant similarity to any GenBank accession or were similar to shotgun sequences with no known function. Multiple transcripts with similarity to ORF-1 of the AM-toxin gene were obtained from the light library. L152 is a full reading frame EST in the light library whose ORF translation has similarity to the conserved domain aegerolysin (pfam06355), a family of hemolytic proteins associated with sporulation events in bacteria and fungi. A Coca’s extract of A. gaisen mycelium was hemolytic to sheep red blood cells in an agar plate test. A set of eleven ex-type or representative isolates including A. alternata, A. gaisen, A. yaliinficiens, A. arborescens, A. tenuissima and A. brassicicola were resolved by UPGMA analysis of a partial genomic sequence (415-425 base pairs) of L152, but were not resolved by a similar analysis of ITS sequences. Based upon these preliminary results, the sequences of putative aegerolysin homologs appear to be variable and informative, and with additional analyses of a broader and more inclusive isolate set, may be proven to have useful levels of variability for phylogenetic studies.