In a previous report, we showed that C. gloeosporioides produces auxin both in culture and in planta [16, 17]. This raised the possibility of auxin involvement in the regulation of fungal development and pathogeniCity, and of the existence of auxin-responsive genes regulating fungal responses to IAA. As a first step towards identifying the putative IAA-responsive fungal genes, we constructed a SSH library using mycelia from auxin-containing medium as the tester. Under culture conditions, over 95% of the IAA that is produced by C. gloeosporioides is secreted into the medium . We therefore used a relatively high IAA concentration (500 μM), assuming that the endogenous concentrations would be at least 10-fold lower. We also added 500 μM IAM, the intermediate product of IAA production in C. gloeosporioides . The SSH yielded limited information on putative IAA-induced genes since only three clones showed consistent induction by IAA. Thus, although putative IAA-induced genes were identified, the results from the SSH approach do not support a massive change in gene transcription by IAA. However, the number of genes that could be tested by SSH was limited and more conclusive results might be obtained through robust transcript analysis using microarrays when such tools become available in C. gloeosporioides.
CgOPT1 exhibited consistent induction by IAA and was therefore further analyzed. Characterization of the gene as a putative OPT was strongly supported by its overall homology to other OPTs, as well as by the presence of the conserved SPYxEVRxxVxxxDDP sequence and 14 transmembrane domains, which are common to all OPTs [18, 21, 22]. Further analyses, including complementation of yeast mutants, are needed to determine that CgOPT1 is indeed an oligopeptide transporter and to find substrate specifiCity.
In S. cerevisiae, there are two genetically and physiologically distinct proton-coupled peptide transporter systems: the PTR (peptide transport) and the OPT (oligopeptide transport) protein families. Members of the PTR and OPT families differ in function and they do not share significant sequence homology (see Fig. 1C). PTR proteins are common in all organisms and transport di- or tripeptides. OPT proteins are found only in plants and fungi and transport 4- and 5-amino-acid peptides [22, 23]. Metabolically, the transport of small oligopeptides is important as an amino acid, carbon, and nitrogen source . Therefore, silencing of CgOPT1 would be expected to reduce the uptake of nutrients from proteinaceous sources, which might have an effect on growth and development. Further, CgOPT1 might facilitate incorporation of metabolites or small peptides that 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 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 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 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. Under both conditions, we observed consistent and significant enhancement of sporulation in the wild type by IAA (Fig. 6). This procedure therefore provided a reliable assay for the determination of responses to IAA in the wild type and cgopt1-silenced mutants.
The cgopt1-silenced mutants exhibited reduced sporulation compared to the wild type when grown in the light. This difference was not observed in the dark, where both the wild type and mutants produced reduced, but equal numbers of spores (Fig. 6A). Thus, CgOpt1 is probably associated only with light-dependent sporulation, and is not required for light-independent sporulation. However, IAA had no effect on sporulation in the mutants, unlike the significant enhancement of sporulation observed in the wild-type strain. These results suggest that IAA and light enhance sporulation through different pathways, and that CgOpt1 is associated with the IAA-dependent pathway, but not the light-dependent one. In addition, morphological differences were observed between the wild type and cgopt1 mutants when grown in liquid culture, and the addition of IAA induced morphological changes in the wild type, but had almost no effect on the mutants (Fig. 7). Thus both sporulation and pellet morphology, which differ between the wild type and cgopt1-silenced mutants, are affected by IAA in the wild type but not in the cgopt1 mutants. These results suggest that CgOpt1 might be associated with developmental pathways that are also affected by IAA. The abolishment of a response to IAA in the cgopt1 mutants is surprising and further research is needed to determine the connection between CgOPT1 and IAA.