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The fungal plant pathogen Slafractonia leguminicola produces two mycotoxins that affect animals: slaframine, which causes slobbers, and swainsonine, which causes locoism. Slafractonia leguminicola contains the swainsonine-associated orthologous gene clusters, “SWN”, which include a multifunctional swnK gene (NRPS-PKS hybrid), swnH1 and swnH2 (nonheme iron dioxygenase genes), swnN and swnR (reductase genes), and swnT (transmembrane transporter). In addition to these genes, two paralogs of swnK, swnK1 (paralog1) and swnk2 (paralog2), are found in S. leguminicola. cDNAs from total mRNA were isolated from the S. leguminicola mycelia grown in the culture plates as well as from leaves inoculated with the fungal mycelia at different time points, and expression pattern of the SWN genes were analyzed using RT-qPCR. The concentrations of swainsonine and slaframine production from this fungus at different time points were also examined using liquid chromatography–mass spectrometry. The timing of gene expression was similar in cultured fungus and inoculated leaves and agreed with our proposed biosynthetic pathway. Substantially more swainsonine was produced than slaframine during time course studies. 

Slafractonia leguminicola infects red clover and other legumes, causing black patch disease. This pathogenic fungus also produces two mycotoxins, slaframine and swainsonine, that are toxic to livestock grazing on clover hay or pasture infested with S. leguminicola. Swainsonine toxicosis causes locoism, while slaframine causes slobbers syndrome. The mechanism of toxin secretion by S. leguminicola is poorly understood. The aim of this research was to investigate the role of a putative transmembrane transporter, SwnT, in mycotoxin transport. The swnT gene was silenced by RNA interference using the silencing vector Psilent1, which included inverted repeat transgenes of swnT. This resulted in a significant reduction of swnT transcript levels compared with the controls. Silencing caused a decline in the active efflux of toxins from the mycelia to the media, as shown by LC–MS analysis. Transformants in which swnT was silenced showed higher concentrations of both toxins in the mycelia compared with the concentrations in the media. These transformants exhibited a visibly distinct phenotype with much thicker and shorter mycelia than in the wild type. These transformants were also unable to infect detached clover leaves, unlike the controls, suggesting that SwnT function may play an important role in pathogenesis in addition to mycotoxin transport. This research demonstrates the importance of this transporter to the secretion of mycotoxins for this phytopathogenic fungus. 

Swainsonine is a cytotoxic alkaloid produced by fungi. Genome sequence analyses revealed that these fungi share an orthologous gene cluster, SWN, necessary for swainsonine biosynthesis. To investigate the SWN cluster, the gene sequences and intergenic regions were assessed in organisms containing swnK, which is conserved across all fungi that produce swainsonine. The orders of fungi which contained orthologous swainsonine genes included Pleosporales, Onygenales, Hypocreales, Chaetothyriales, Xylariales, Capnodiales, Microthyriales, Caliciales, Patellariales, Eurotiales, and a species of the Leotiomycetes. SwnK and swnH2 genes were conserved across all fungi containing the SWN cluster; in contrast, swnT and swnA were found in a limited number of fungi containing the SWN cluster. The phylogenetic data suggest that in some orders that the SWN cluster was gained once from a common ancestor while in other orders it was likely gained several times from one or more common ancestors. The data also show that rearrangements and inversions of the SWN cluster happened within a genus as species diverged. Analysis of the intergenic regions revealed different combinations and inversions of open reading frames, as well as absence of genes. These results provide evidence of a complex evolutionary history of the SWN cluster in fungi.