Further, CgOPT1 might facilitate incorporation of metabolites or small peptides that BYL719 in vitro can be used as signalling molecules e.g., during plant infection. CgOPT1 was activated in the presence of IAA in a concentration-dependent manner. Transcription was already enhanced at 50 μM IAA and was further enhanced at higher concentrations, with saturation at 500 μM. These concentrations are much higher than the IAA levels in plants but are within the range of IAA amounts produced by C. gloeosporioides . Lack of activation by acetic acid, indole-3-ethanol
(tryptophol) or tryptamine ruled out possible activation of CgOPT1 by auxin-induced changes in pH, or as a general response to indoles. Nevertheless, at this stage it is impossible to determine whether up-regulation of CgOPT1 in the presence of IAA is a check details direct response to IAA or rather, an
indirect response to other changes that might be brought about by IAA. Further, induction by IAA does not necessarily imply that it would be involved in IAA transport, especially because C. gloeosporioides produces large quantities of IAA, so induction might be through endogenous rather than exogenous IAA. In addition to the IAA-induced transcription of CgOPT1, the gene was differentially expressed during fungal development, particularly during spore germination. CgOPT1 transcript could not be detected in resting spores, it was highly induced during germination, and then it declined during mycelium formation. This expression pattern is opposite to that of the vacuolar copper-transporting gene CgCTR2, which is necessary for the initial stages
of germination and is highly expressed in resting spores and down-regulated immediately Thiamet G after spore germination . Therefore, CgOpt1 is probably important during germ-tube formation and elongation, but is not required for the initiation of spore germination. Silencing of the gene provided additional evidence for the involvement of CgOptT1 in development as well as pathogeniCity: cgopt1-silenced mutants displayed reduced sporulation and pigmentation, and were less pathogenic than the wild-type strain. These pleiotropic effects suggest GSK1120212 datasheet association of CgOpt1 with several different processes. IAA appears to have an enhancing effect on processes such as sporulation, spore germination, and germ-tube elongation. However, the effects of IAA vary with experimental conditions, and opposite results might be obtained. In our sporulation assay, we took special care to eliminate possible interference and side effects from experimental parameters such as solvent, medium, or light. IAA was applied to filter paper, the ethanol was evaporated, and then the filter was placed between two layers of agar to avoid direct contact with the fungus. Additionally, because sporulation is enhanced by light, the experiments were conducted under both light and dark conditions.