High-throughput screening reveals mechanisms of environmental control of germination in a fungal thermophile

Thermothelomyces thermophilus is a filamentous fungus isolated from self-heating compost. Unlike most of the fungal kingdom, this species exhibits a growth optimum at 45{degrees}C and is intolerant of temperatures below 30{degrees}C. To investigate genetic contributors to temperature-dependent fitness in this system, we implemented a large-scale insertional mutagenesis approach. We generated thousands of T. thermophilus mutants and cultured them at temperature extremes in standard medium. Phenotyping-by-sequencing identified dozens of disrupted loci representing candidate determinants of thermophilic life history, including several annotated in metal transport. We then validated a subset of screen hits with a directed, single-gene knockout paradigm. The results revealed a temperature-dependent regulatory logic for germination, the developmental decision by which a fungal spore initiates growth. Surprisingly, most mutants germinated far better at 50{degrees}C than the wild-type in standard medium and showed markedly slower germination at lower temperatures, consistent with altered germination regulation rather than enhanced intrinsic heat tolerance. We hypothesized that T. thermophilus has evolved sophisticated regulatory machinery to block germination at high temperature unless environmental conditions are favorable. As a proof of concept, we surveyed media conditions and established that elevated zinc dampened germination of wild-type T. thermophilus at 50{degrees}C but promoted it at lower temperature; mutation experiments made clear that such sensitivity was mediated in part by the zinc transporter zip. We interpret these results under a model in which T. thermophilus integrates temperature and nutrient availability to control the transition from spore dormancy to vegetative growth, a developmental decision that shapes fitness outcomes across temperatures. SignificanceFungal thermophiles thrive at temperatures that represent the upper limits of eukaryotic life. The regulatory and developmental mechanisms that shape their temperature-dependent fitness remain poorly understood. In this work, we elucidate how Thermothelomyces thermophilus integrates temperature cues with other environmental inputs during germination, a key life-cycle stage for dispersal. Our findings highlight germination regulation as an important contributor to fitness at elevated temperatures in a thermophilic eukaryote. These insights are of basic biological interest and provide a foundation for rational strategies to modulate temperature-dependent performance in industrial strains, with applications for high-temperature bioprocessing.