Domestication signatures in the non-conventional yeast Lachancea cidri.

Evaluating domestication signatures beyond model organisms is essential for a thorough understanding of the genotype-phenotype relationship in wild and human-related environments. Structural variations (SVs) can significantly impact phenotypes playing an important role in the physiological adaptation of species to different niches, including during domestication. A detailed characterization of the fitness consequences of these genomic rearrangements, however, is still limited in non-model systems, largely due to the paucity of direct comparisons between domesticated and wild isolates. Here, we used a combination of sequencing strategies to explore major genomic rearrangements in a Lachancea cidri yeast strain isolated from cider (CBS2950) and compared them to those in eight wild isolates from primary forests. Genomic analysis revealed dozens of SVs, including a large reciprocal translocation (~16 kb and 500 kb) present in the cider strain, but absent from all wild strains. Interestingly, the number of SVs was higher relative to single-nucleotide polymorphisms in the cider strain, suggesting a significant role in the strain's phenotypic variation. The set of SVs identified directly impacts dozens of genes and likely underpins the greater fermentation performance in the L. cidri CBS2950. In addition, the large reciprocal translocation affects a proline permease (PUT4) regulatory region, resulting in higher PUT4 transcript levels, which agrees with higher ethanol tolerance, improved cell growth when using proline, and higher amino acid consumption during fermentation. These results suggest that SVs are responsible for the rapid physiological adaptation of yeast to a human-related environment and demonstrate the key contribution of SVs in adaptive fermentative traits in non-model species.IMPORTANCEThe exploration of domestication signatures associated with human-related environments has predominantly focused on studies conducted on model organisms, such as Saccharomyces cerevisiae, overlooking the potential for comparisons across other non-Saccharomyces species. In our research, employing a combination of long- and short-read data, we found domestication signatures in Lachancea cidri, a non-model species recently isolated from fermentative environments in cider in France. The significance of our study lies in the identification of large array of major genomic rearrangements in a cider strain compared to wild isolates, which underly several fermentative traits. These domestication signatures result from structural variants, which are likely responsible for the phenotypic differences between strains, providing a rapid path of adaptation to human-related environments.

Developing a Temperature-Inducible Transcriptional Rheostat in Neurospora crassa.

Heat shock protein (HSP)-encoding genes (hsp), part of the highly conserved heat shock response (HSR), are known to be induced by thermal stress in several organisms. In Neurospora crassa, three hsp genes, hsp30, hsp70, and hsp80, have been characterized; however, the role of defined cis elements in their responses to discrete changes in temperature remains largely unexplored. To fill this gap, while also aiming to obtain a reliable fungal heat shock-inducible system, we analyzed different sections of each hsp promoter by assessing the expression of real-time transcriptional reporters. Whereas all three promoters and their resected versions were acutely induced by high temperatures, only hsp30 displayed a broad range of expression and high tunability, amply exceeding other inducible promoter systems existing in Neurospora, such as quinic acid- or light-inducible ones. As proof of concept, we employed one of these promoters to control the expression of clr-2, which encodes the master regulator of Neurospora cellulolytic capabilities. The resulting strain fails to grow on cellulose at 25°C, whereas it grows robustly if heat shock pulses are delivered daily. Additionally, we designed two hsp30 synthetic promoters and characterized them, as well as the native promoters, using a gradient of high temperatures, yielding a wide range of responses to thermal stimuli. Thus, Neurospora hsp30-based promoters represent a new set of modular elements that can be used as transcriptional rheostats to adjust the expression of a gene of interest or for the implementation of regulated circuitries for synthetic biology and biotechnological strategies. IMPORTANCE A timely and dynamic response to strong temperature fluctuations is paramount for organismal biology. At the same time, inducible promoters are a powerful tool for fungal biotechnological and synthetic biology endeavors. In this work, we analyzed the activity of several N. crassa heat shock protein (hsp) promoters at a wide range of temperatures, observing that hsp30 exhibits remarkable sensitivity and a dynamic range of expression as we charted the response of this promoter to subtle increases in temperature, and also as we built and analyzed synthetic promoters based on hsp30 cis elements. As proof of concept, we tested the ability of hsp30 to provide tight control of a central process, cellulose degradation. While this study provides an unprecedented description of the regulation of the N. crassa hsp genes, it also contributes a noteworthy addition to the molecular toolset of transcriptional controllers in filamentous fungi.

A global search for novel transcription factors impacting the Neurospora crassa circadian clock.

Eukaryotic circadian oscillators share a common circuit architecture, a negative feedback loop in which a positive element activates the transcription of a negative one that then represses the action of the former, inhibiting its own expression. While studies in mammals and insects have revealed additional transcriptional inputs modulating the expression of core clock components, this has been less characterized in the model Neurospora crassa, where the participation of other transcriptional components impacting circadian clock dynamics remains rather unexplored. Thus, we sought to identify additional transcriptional regulators modulating the N. crassa clock, following a reverse genetic screen based on luminescent circadian reporters and a collection of transcription factors (TFs) knockouts, successfully covering close to 60% of them. Besides the canonical core clock components WC-1 and -2, none of the tested transcriptional regulators proved to be essential for rhythmicity. Nevertheless, we identified a set of 23 TFs that when absent lead to discrete, but significant, changes in circadian period. While the current level of analysis does not provide mechanistic information about how these new players modulate circadian parameters, the results of this screen reveal that an important number of light and clock-regulated TFs, involved in a plethora of processes, are capable of modulating the clockworks. This partial reverse genetic clock screen also exemplifies how the N. crassa knockout collection continues to serve as an expedite platform to address broad biological questions.

Characterization of PIR1, a GATA family transcription factor involved in iron responses in the white-rot fungus Phanerochaete chrysosporium.

Iron, although toxic in excess, is an essential element for biological systems. Therefore, its homeostasis is of critical importance and tight mechanisms participate in its acquisition by microbial organisms. Lately, the relevance of this metal for biomass conversion by wood-degrading fungi has been gaining increasing attention. Iron plays a critical role as cofactor of key enzymes such as lignin and manganese peroxidases in lignin-degrading white-rot fungi, while Fe(II) also serves a pivotal role in Fenton reactions that are central in cellulose depolymerization by brown-rotters. It has been hypothesized that multicopper oxidases with ferroxidase activity might participate in controlling the bioavailability of iron in the hyphal proximity, fine-tuning Fenton chemistry and balancing lignin versus cellulose degradation. In order to further explore the dynamics of iron regulation in the well known white-rot fungus Phanerochaete chrysosporium, we analyzed the mRNA levels of the multicopper oxidases genes (mcos) in response to iron supplementation, confirming down-regulation of their expression in response to this metal. To gain a better understanding on the transcriptional mechanisms mediating this effect, we searched for a gene encoding a GATA-type transcription factor with homology to URBS1, the major transcriptional regulator of iron homeostasis in Ustilago maydis. Due to the limitation of experimental tools in P. chrysosporium, the alleged Phanerochaete iron regulator (PIR1) was studied by complementation of a Neurospora SRE/URBS1-deficient strain, where phenotypic and molecular characteristics of this transcriptional regulator could be easily assessed. In addition, using a genome-wide in silico strategy, we searched for putative cis-acting iron-responsive elements in P. chrysosporium. Some of the identified genes showed reduced transcript levels after 30 min in the presence of the metal, consistent with an SRE/URBS1-mediated mechanism, and suggesting a broad effect of iron on the regulation of several cellular processes.