Trichoderma species are cosmopolitan soil fungi found in several substrates, including decaying wood and fungal fruiting bodies, establishing beneficial interactions with plants (Carreras-Villaseñor et al., 2012; Kubicek et al., 2019; Vos et al., 2015), and negative ones with other microorganisms (Druzhinina et al., 2006; Guzman-Guzman et al., 2019; Harman et al., 2004; Sood et al., 2020). Trichoderma species are well-known mycoparasites as they can degrade, grow on other fungi, and even penetrate them, a distinctive feature that has been exploited in agriculture for their use as biocontrol agents of different phytopathogens, including Botrytis cinerea (Naglot et al., 2015; Rahman et al., 2009).

Trichoderma's mycoparasitic behavior starts with the secretion of low levels of cell wall degrading enzymes (CWDE) (Druzhinina et al., 2011; Seidl et al., 2009; Vos et al., 2015) that dismantle the fungal prey’s hyphae. Release of small molecules derived from the host can function as molecular signals in Trichoderma, allowing prey recognition (Zeilinger et al., 2005), redirecting hyphal growth, and initiating coiling around the host mycelium, followed by hyphal penetration. Indeed, the synergistic effect of enhanced CWDE and toxic secondary metabolite (SM) production allows Trichoderma’s hyphae to penetrate the prey’s lumen, achieving its killing (Belanger et al., 1995; Druzhinina et al., 2011; Sharma, 2011; Steyaert et al., 2003).

Trichoderma species present developmental responses to different stresses, of which conidiation induced by light and mechanical damage has been extensively studied (Casas-Flores and Herrera-Estrella, 2013). Physical damage of mycelia in Trichoderma atroviride triggers a developmental injury response, leading to conidia production in the injured area through the generation of reactive oxygen species (ROS) and activation of MAPK signaling pathways (Hernández-Oñate et al., 2012; Medina-Castellanos et al., 2014). Additionally, light is also perceived as a stress signal, triggering conidiation: a light pulse (LP) given to a dark-grown T. atroviride generates several biochemical changes driving the formation of a green ring of conidia at the edge of the colony (Casas-Flores et al., 2004; Casas-Flores et al., 2006; Galun and Gressel, 1966; Schmoll et al., 2010). In T. atroviride, two genes called blue light regulators 1 and 2 (blr1 and blr2) were identified as responsible for light-induced conidiation. The BLR proteins are homologs of the transcription factors WC-1 and WC-2, which in Neurospora crassa form the white collar complex (WCC), responsible for light and circadian regulation in this fungus (Casas-Flores et al., 2004; Froehlich et al., 2002; Montenegro-Montero et al., 2015). Importantly, light can be directly perceived thanks to the presence of a light-oxygen-voltage (LOV) domain in WC-1/BLR1 (Corrochano, 2019). Furthermore, BLRs have been described as critical for the effect of light on growth, oxidative stress responses, and light-controlled gene expression (Cervantes-Badillo et al., 2013; Esquivel-Naranjo et al., 2016; Friedl et al., 2008a; Garcia-Esquivel et al., 2016). Studies on BLR1 and BLR2 and the presence of a frq homolog that is induced by light, and whose expression depends on BLR1 (Garcia-Esquivel et al., 2016; Steyaert et al., 2010a), suggest the presence of a functional clock and underlying circadian regulation in this fungus. However, to date, neither circadian oscillations of tafrq/TaFRQ levels nor circadian phenotypes have been reported in T. atroviride; meanwhile, various studies have failed at detecting overt or molecular circadian rhythms in several fungi across fungal clades (Cascant-Lopez et al., 2020; Hevia et al., 2016; Larrondo and Canessa, 2019).

Circadian rhythms have emerged as an adaptation to Earth’s rotation around its axis, allowing organisms to keep track of time, anticipating and adapting to periodic and predictable environmental changes through the temporal coordination of gene expression, metabolic pathways, physiological responses, and even behaviors (Bell-Pedersen et al., 2005; Hurley et al., 2016). Recent findings have shown that interactions between organisms may also be subjected to circadian regulation (reviewed in Hevia et al., 2016; Larrondo and Canessa, 2019). And while several reports have highlighted the importance of the plant clock in plant–pathogen interactions, there has been scarce information from the pathogen perspective. Yet, it has been reported that the interaction between the phytopathogenic fungus B. cinerea and Arabidopsis thaliana largely depends on the fungal functional circadian clock, with virulence reaching higher levels at nighttime (Hevia et al., 2015; Ingle et al., 2015).

The ascomycete N. crassa is a salient model in chronobiology due to its robust circadian phenotype of daily spore production at dawn and a wealth of molecular resources (Baker et al., 2012). Its central oscillator is based on a negative transcriptional–translational feedback loop (TTFL), a shared core architecture across phyla (Dunlap, 1999; Dunlap and Loros, 2017; Loros, 2020). In N. crassa, the WCC activates the expression of the TTFL-negative element, frequency (frq), which leads to increasing levels of the FRQ protein, that starts recruiting kinases and negatively regulates its own expression by phosphorylating and inactivating the WCC. FRQ itself is progressively modified until reaching its maximum phosphorylation state at nighttime, losing its affinity for WCC and allowing a new transcription/translation cycle to start. Both frq mRNA and FRQ protein levels oscillate during this process with a period close to 22.5 hr (Larrondo et al., 2015; Montenegro-Montero et al., 2015). Albeit wc-1, wc-2, and frq homologs are present in several fungi, the description of overt clock phenotypes, as the molecular characterization of circadian rhythms in fungal species besides N. crassa, has been limited (Montenegro-Montero et al., 2015; Salichos and Rokas, 2010). Indeed, despite different studies addressing the latter, there are seldom examples of characterized circadian oscillators in other fungi and few studies exploring fungal circadian phenomena and clock components. Among these is B. cinerea, where rhythms in BcFRQ1 levels were confirmed, along with BcFRQ1’s key role in conferring the daily difference on virulence potential. Remarkably, the evidence also suggests that in B. cinerea BcFRQ1 plays an extra-circadian role in developmental decisions as Δbcfrq1 displays altered developmental phenotypes observed even under conditions where the clock function is abolished, such as constant light, and in contrast to the phenotype observed in the absence of BcWCL1 (Hevia et al., 2015).

To deepen the role of circadian regulation in organismal interactions, and simultaneously evaluate the presence of a functional circadian clock in T. atroviride, we sought to assess the daily oscillations of TaFRQ expression under free-running conditions while assessing canonical clock properties and the clock’s ability to respond to environmental stimuli. By generating clock mutant strains, we evaluated the impact of core clock components in T. atroviride secondary metabolism, developmental programs, and on its mycoparasitic capabilities against different B. cinerea strains, including those deficient in circadian regulation. Overall, our results reveal the presence of a functional circadian clock in T. atroviride, a role of core clock components in the modulation of mycoparasitic interactions, and extra-circadian functions for TaFRQ.

While putative core clock components encoding genes can be found in fungi distributed between several orders (Montenegro-Montero et al., 2015; Salichos and Rokas, 2010), phenotypic description of fungal circadian phenomena has been less common, and the molecular characterization of those rhythms has been even scarcer. One of the few examples (besides N. crassa) is the characterization of the B. cinerea core clock and its effect on fungal virulence (Hevia et al., 2015). In our efforts to further understand the role of circadian clocks in fungal–fungal interactions, herein we provide evidence of a functional clock in the mycoparasitic fungus T. atroviride. TaFRQ^LUC reporter strains revealed circadian oscillations with a free-running period of ~26 hr, which could be entrained by LD cycles and reset by light and temperature pulses, consistent with what has been described in Neurospora and other organisms for bona fide circadian systems (Liu et al., 1998). These oscillations showed temperature compensation between 20 and 25°C, where, notably, the best rhythms in terms of amplitude were observed at 22°C, but they were lost at 28°C. While such a narrow range of temperatures allowing clear visualization of these molecular rhythms may be puzzling, it might also be associated with the environment in which Trichoderma is usually found – soil – in which temperature shifts are overall buffered (Rodriguez-Romero et al., 2010).

In the attempt to evaluate clock-controlled genes (ccg), we did not observe oscillations of additional luciferase transcriptional reporters of putative T. atroviride ccg promoters. This could be due to (i) the lack of circadian transcription of the assayed ccgs, (ii) the selected promoters fail to capture elements necessary for their circadian control, or (iii) the assay conditions were not appropriate to visualize their rhythmic expression (see below). Future analysis of the transcript levels of several ccgs will be performed under different culture conditions to confirm whether their expression is actually circadian in particular settings.

Interestingly, we found evidence of strong functional circadian conservation between Neurospora and Trichoderma. Firstly, conservation of clock cis-regulatory motifs, as the c-box sequence from the former is recognized in the latter fungus. As the DNA binding domains of WC-1 and BLR1 share a 97.2% aa identity, we hypothesize that the BRL1/BLR2 complex can recognize the _Ncc-box element driving circadian oscillations and that a similar c-box sequence could be found in the tafrq promoter. Indeed, the high conservation of WC-1 and BLR1 DNA binding domains (Casas-Flores et al., 2004; Cervantes-Badillo et al., 2013; Froehlich et al., 2002) indicates that they are expected to recognize a similar motif considering general rules of eukaryotic transcription factor sequence specificity (Weirauch et al., 2014). Two putative light response elements in the tafrq’s promoter with a putative GATA box consensus sequence at proximal and distal locations (−355 to –319 and –1180 to –1171 from the TSS, respectively) have been postulated (Cervantes-Badillo et al., 2013), of which one of them could be acting as a putative _Tacbox sequence.

We were able to generate ~26 hr rhythms in a Neurospora Δfrq strain complemented with the tafrq coding region, under the control of the Neurospora endogenous regulatory sequences. Comparison of the tafrq and ncfrq sequences informs a 54 and 50% of identity at the DNA and protein levels, respectively (using Clustal Omega multiple sequence alignment), which provides sufficient functional conservation to allow rescuing of the core clock function in a N. crassa arrhythmic strain. Most importantly, the fact that tafrq complements the function of the endogenous frq in Neurospora strongly validates the role of TaFRQ as a core clock circadian component. While we did not conduct some classic experiments such as the ones designed by Aronson et al., 1994, we present compelling evidence of the existence of such a functional circadian TTFL. Thus, in addition to the complementation itself, it is the fact that TaFRQ is indeed acting as a negative element in Neurospora, which can be clearly inferred by the overall decrease of WCC activity (as seen by the reduction in luciferase expression when compared to the recipient strain). Importantly, as we restored the core clock in Δfrq, the resulting oscillations reflected the endogenous ~26 hr period originally observed in T. atroviride, instead of the Neurospora WT rhythm of ~22 hr, implying that period determinants are encoded in the TaFRQ protein sequence and that they are ‘read’ by the rest of the circadian machinery in a proper manner, despite the phylogenetic distance of both genera. Thus, this not only further validates TaFRQ as a bona fide core clock component, but it also highlights how its sequence determinants modulate period. Additionally, we also confirmed that TaFRQ is phosphorylated and degraded as seen over a 12 hr time course, after a light to dark transfer, in accordance to Neurospora FRQ behavior. Recent studies have shown that the quality of FRQ regarding phosphorylation status rather than its stability or half-life is important for period length (Larrondo et al., 2015). Baker et al., 2009 partially characterized FRQ’s phosphocode in N. crassa, of which some mutations in these sites were experimentally shown to affect clock properties. In TaFRQ, 82.6% (19/23) of such sites are preserved, with the divergent ones all residing in the C-terminal portion of the protein, after the PEST-2 domain (Figure 2—figure supplement 3). The high conservation of known period-affecting phosphorylation sites could partially explain the ability of TaFRQ to function, whereas differential phosphorylation of TaFRQ compared to NcFRQ could potentially explain the longer period. Further analysis of TaFRQ characteristics and phosphorylation sites will shed additional light on the commonalities and peculiarities of the circadian clocks in these two fungi.

One of our study’s striking findings was the strong influence of culture media composition in allowing the visualization of molecular circadian oscillations. Indeed, rhythms in TaFRQ^LUC, or in _Ncc-box-luc levels, were only observed in GYEC media and then further improved when plant-derived material was included. While the impact of nutrient availability in certain aspects of circadian rhythms has been observed from mammals to N. crassa (Bae et al., 2019; Oosterman et al., 2015), our results indicate an even bigger dependence on culture conditions in allowing molecular visualization of the Trichoderma clock. Thus, although the quality and other aspects of frq and clock-controlled genes can vary, depending on media characteristics in Neurospora (Díaz and Larrondo, 2020; Dunlap and Loros, 2017; Hurley et al., 2014; Larrondo et al., 2015; Olivares-Yañez et al., 2016) we were surprised to observe such a dramatic effect in Trichoderma: that is, an almost nutritional-conditional rhythmicity dependent on media-derived cues. Besides the puzzling and yet uncharacterized mechanism underlying this nutritional conditionality, we believe that this may serve as a cautionary tale for other attempts to characterize circadian rhythms in different fungi. If we had limited our analysis to a small set of media or higher temperatures, we would have been prone to conclude that there were no rhythms in Trichoderma TaFRQ levels. Yet, hitting the right conditions allowed us to confirm their existence and therefore the presence of a circadian oscillations in this organism. It is also provoking and an exciting lesson to consider that the addition of plant-derived material in a fungus that naturally interacts with plants has such a strong effect on visualizing these rhythms (see below).

In T. atroviride, the use of different carbon sources in culture media allows, in some cases, observation of photostimulation of growth and conidiation in the dark, processes in which BLR1 plays a critical role in carbon-source selectivity (Friedl et al., 2008a; Friedl et al., 2008b). Also, C:N ratios are relevant environmental factors influencing conidiation in Trichoderma (Steyaert et al., 2010b). Additionally, organic nitrogen sources in culture media have been described as important for fungal mycoparasitic behavior (Barnett, 1963). Also, nitrogen sources are known to affect growth in Trichoderma species (Danielson and Davey, 1973; Nolan and Nolan, 1972). Due to the importance of nutrients in such processes, we hypothesize that the high amounts of organic nitrogen found in GYEC media (derived from casamino acids and yeast extract, absent in the other tested media) to some extent may mimic the composition of Trichoderma’s natural niche, in which different nitrogen sources derived from its preys are available.

Moreover, the addition of peas could better resemble the nutrients or chemical features found in Trichoderma’s interaction with plants, including different sugars (Druzhinina et al., 2011; Steyaert et al., 2010b). The latter also raises fundamental questions about the importance of plant-derived signals in Trichoderma’s clock robustness and its role in the outcome of plant–fungal interactions. A complex temporal communication could be occurring during their interaction in the soil, affecting clock synchronization in Trichoderma. Indeed, the plant circadian clock could alter the composition and function of the rhizosphere microbiome as it has been observed in the rhizosphere of WT and long/short period mutants of A. thaliana (Hubbard et al., 2018). Rhythmically structured metabolic exchanges between the organisms in the community in collective benefit could be occurring (Sartor et al., 2019), although it still remains to be studied in this context.

These findings reinforce the importance of defined nutrient/temperature conditions to monitor the clockworks and help future fungal circadian studies visualize otherwise weak oscillations, like those observed in Pyronema confluens and Ophiocordyceps kimflemingiae (de Bekker et al., 2017; Traeger and Nowrousian, 2015). This could also help monitor frq molecular oscillations in fungi where previous attempts have failed, like in Verticillium dahliae, Aureobasidium pullulans, and Magnaporthe oryzae (Cascant-Lopez et al., 2020; Deng et al., 2015; Franco et al., 2017).

While frq in N. crassa does not have a described role outside the pacemaker (Aronson et al., 1994), in B. cinerea, it exhibits extra-circadian roles associated with developmental programs (Hevia et al., 2015). In T. atroviride, we observed that the core clock components play a role in development, secondary metabolism, and mycoparasitism. Thus, TaFRQ is critical for conidia formation after an LP and mechanical damage, showing a dramatic reduction of conidiation in the latter. In fungi, little is known about FRQ function besides N. crassa and B. cinerea. A recent study of frq in the phytopathogen V. dahliae found no significant phenotypic differences in the deletion strain regarding sporulation and microsclerotia formation in DD or LD (Cascant-Lopez et al., 2020). Similarly, in the entomopathogenic fungi Metarhizium robertsii, FRQ is necessary for ring formation under LD 12:12, although neither the WT nor the mutant strain displayed such rings under DD conditions. Besides this, M. robertsii FRQ is dispensable in almost all evaluated phenotypes (growth and conidiation in different stress conditions), including virulence against Galleria mellonella (Peng et al., 2022). Meanwhile, studies in Beauveria bassiana (in which two frq homologs exists) and M. oryzae strain Guy11 have shown that their frq knockouts exhibit diminished conidiation and virulence against G. mellonella (Tong et al., 2021) and rice seedlings (Shi et al., 2019), respectively. These findings pose new questions regarding the role in developmental decisions that FRQ may have acquired (or lost) across the fungal kingdom, raising questions about the extra-circadian role of clock negative elements in other organisms, including animals.

We found altered developmental responses and enhanced production of diffusible and volatile compounds in Δtafrq, suggesting a general repressor regulatory role of TaFRQ over secondary metabolism. Interestingly, the antifungal compound 6-PP, a key feature of Trichoderma species, was dramatically reduced in Δtafrq even in LL. The clock function is expected to be abolished in LL, suggesting, therefore, a potential TaFRQ extra-circadian activator role in 6-PP production.

SMs have been related to developmental programs in fungi and could have a role in response to damage in T. atroviride (Atriztán-Hernández et al., 2019; Schmoll et al., 2010; Tisch and Schmoll, 2010). Reports show that after injury and fungivory a group of VOCs, including 6-PP, increase in TaWT but not in the MAPK mutant Δtmk3 (Atriztán-Hernández et al., 2019), which shows diminished conidiation after an injury as observed in the Δtafrq mutant (Medina-Castellanos et al., 2014). Additionally, it was previously suggested that oxylipin could be involved in signaling after damage (Medina-Castellanos et al., 2014). We propose that the reduction of injury-induced conidiation in Δtafrq could be related to its dysregulated diffusible and VOC production profiles, in which further studies identifying those compounds could shed light on their role in injury-induced conidiation. On the contrary, Δblr1 showed repressed production of several compounds (diffusible and VOCs), suggesting a role of light perception in secondary metabolism regulation. This is consistent with the light-induced production of several compounds in TaWT.

Interestingly, Δblr1 produced higher amounts of 6-PP in DD and even in LL, whereas in TaWT, 6-PP production is inhibited by light. This suggests a repressor role of light and BLR1 in the production of 6-PP and other molecules, as seen for 1679.08 m/z, and that other photoreceptors could activate its production in the absence of BLR1 (Garcia-Esquivel et al., 2016). A light-repression effect on 6-PP production was also observed in T. atroviride P1 and IMI 206040 strains when 6-PP production was analyzed under LD and DD conditions (Speckbacher et al., 2020a; Contreras-Cornejo et al., 2022) and when grown in different light wavelengths (Moreno-Ruiz et al., 2020). We also observed differences in the SM profiles between LL and LP conditions, indicating that the duration of light stimuli differently activates/represses the production of several compounds. Whereas a short light exposure could be perceived as a strong environmental cue triggering the production of different metabolites, constant exposure to light could lead to a prolonged stress response marked by a different subset of metabolites. Light regulation of SMs has been reported in several fungi (Tisch and Schmoll, 2010) as the involvement of WC-1 homologs in Cercospora zeae-maydis and Fusarium graminearum as repressors of cercosporin and trichothecene production, respectively (Kim et al., 2014; Kim et al., 2011). Nevertheless, the involvement of TaFRQ in the production of SMs in T. atroviride (or FRQ of any other fungus) has not been reported to date.

In concordance with the circadian oscillations of TaFRQ^LUC, we observed rhythmic levels of volatile SMs in T. atroviride under constant conditions (DD) but not in Δtafrq, suggesting circadian control of the production of these volatile compounds. Our experimental approach showed that these molecules exhibited rhythmic levels. Nevertheless, it did not provide their molecular identity. We expect future work to dive into their chemical identification and their specific role in T. atroviride’s lifestyle.

Overall, our results reinforce the importance of environmental perception (including time) in Trichoderma’s biology, particularly in the production of SMs involved in several processes from development to mycoparasitism. On the contrary, in Neurospora how these daily rhythms impact metabolite levels has not been described so far, despite ample efforts that have systematically shown the clock’s influence on its transcriptome and proteome (Hurley et al., 2014; Hurley et al., 2018; Sancar et al., 2015), suggesting temporal compartmentalization of metabolism at large, with catabolic and anabolic reactions concentrated at dawn and dusk, respectively (Hurley et al., 2016). Thus, we present a proof of concept that a fungal circadian clock can also impact the production of SMs. These studies and future comprehensive analyses of circadian metabolic profiles in classic models such as Neurospora and other fungi will help to bridge the gap on how clock regulation impacts a fungus’s daily life, from physiology to organismal interactions.

Our study also provides new insights into the mycoparasitic interaction between T. atroviride and B. cinerea and how its outcome is regulated by core clock components and LD conditions. Δblr1 showed the strongest mycoparasitic behavior; in contrast, Δtafrq did not show significant differences to TaWT in most interactions, except a slightly weaker mycoparasitic behavior against Δbcwcl1 and Δbcfrq1 in LL. Trichoderma species' antagonistic capacity is correlated with their ability to produce 6-PP (Reino et al., 2008). The Δblr1 strain has higher amounts of this molecule, whereas, in Δtafrq, its production is severely dampened, consistent with their different mycoparasitic abilities. Trichoderma’s antagonist capacity was reduced in LL and enhanced in darkness, which could be due to differential metabolite profiles found in LL and DD. Similar inhibitory effects of light in mycoparasitism in T. atroviride have been reported against Fusarium oxysporum (Moreno-Ruiz et al., 2020; Speckbacher et al., 2020b), along with differential VOC production upon illumination during confrontation assays. High amounts of 2-heptanone are produced during the confrontation against F. oxysporum in a light-dependent manner that negatively correlates with Trichoderma antagonism, which proposes that 2-heptanone production was a result of a stress response elicited by F. oxysporum in the light.

Finally, mycoparasitic interactions under a circadian paradigm revealed an enhanced overgrowth of TaWT and Δtafrq over B05.10 and Δbcwcl1 when interactions started at dawn under LD cycles in contrast to a reduced overgrowth at dusk. We have previously observed differential interactions in the context of B. cinerea and A. thaliana, where fungal virulence was enhanced at dusk (Hevia et al., 2015). The differential effect on the Trichoderma–Botrytis dynamic was lost when Δblr1 was used against all B. cinerea strains. A similar phenomenon was observed by Hevia et al., 2015, when A. thaliana was infected with Δbcwcl1, which did not show circadian behavior. Disruption of BLR1 abrogates both light and circadian responses, so the effect observed for Δblr1 could also be due to a light-driven phenomenon (besides a circadian effect) because the absence of the blue-light perception overrides the differential effect observed in TaWT. Yet, it is noteworthy that Δtafrq yields result comparable to what is seen in TaWT, suggesting that – paradoxically – the observed time-of-the-day effect does not appear to strongly depend on the T. atroviride’s clock.

On the other hand, under LD cycles, the loss of BcWCL1 does not considerably affect these fungal dynamics, whereas, in Δbcfrq1, the differential outcome of the daily interaction disappears. Lack of BcFRQ1 implies that temporal perception in B. cinerea is absent, in concordance with the loss of the time-of-the-day observed phenotype, something that is not seen when Trichoderma lacks TaFRQ. This suggests that the B. cinerea clock is critical in the outcome of the interaction of tested organisms – both plants and fungi – suggesting a complex mechanism in which light and time perception interact, giving rise to the observed phenotypes. Even though we lack a precise molecular understanding underlying this phenomenon (and integrating the role of the different clock/light-perception components), this work constitutes the first description of an interplay of core and light regulation in a mycoparasitic interaction. In nature, organisms are usually exposed to light/dark transitions. Therefore, the differential outcome depending on the time (or the light/dark cycles) at which confrontations are established could impact biocontrol strategies in agriculture, optimizing the application of Trichoderma in crops.

All data generated and analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1 and 2 and Table 1.

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Decision letter after peer review:

Thank you for submitting your article "Circadian oscillations in Trichoderma atroviride and the role of core clock components in secondary metabolism, development, and mycoparasitism against the phytopathogen Botrytis cinerea" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Detlef Weigel as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Kevin Fuller (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The reviewers have made several constructive suggestions that you should carefully consider and address in a point-by-point reply. In particular, please pay particular attention to the following revisions:

1) Please perform experiments that demonstrate entrainment (as suggested by Reviewer #1).

2) Please perform the interaction assays across a range of environmental conditions (nutritional, temperature) to determine if the contributions of the clock proteins (in either organism) change accordingly (as suggested by Reviewer #2).

3) Please re-complement your mutants (as suggested by Reviewer #3).

Reviewer #1:

The goal of this study was to establish that the fungus Trichoderma atroviride, and important biocontrol agent, has a circadian clock, and to test if this clock plays a role in development, secondary metabolite production, and mycoparasitism. The authors clearly establish rhythms in the organism that are dependent on TcFRQ, a homolog of the core clock component FRQ in N. crassa, and show that TcFRQ and BRL1, the homolog of the blue light photoreceptor and core clock component WC-1 in N. crassa, play differing roles in development, metabolite levels, and mycoparasitism in laboratory conditions. However, the authors do not fully establish that the rhythms are controlled by a circadian clock , nor that TcFRQ is a core clock component. While aspects of this work will be of interest to circadian and fungal biologists, the not all of the aims were met, and the data do not fully support the conclusions.

Strengths: The circadian clock in the fungus N. crassa serves as an important model for understanding the mechanisms that keep time in eukaryotes, and how this clock controls daily rhythms in gene expression. However, little is known about clocks in other fungi, despite conservation of core clock components found in N. crassa, including fungal pathogens and biocontrol agents like Trichoderma. Thus, the strength of this work is in that it goes a long way to establish a clock in T. atroviride that is directly, or indirectly involved in controlling development, metabolism, and mycoparasitism. Importantly, this work opens up the opportunity to determine how the clock impacts fungal-fungal interactions, with the potential of using this to improve biocontrol.

Weaknesses: There are several weaknesses. The first is that while the authors established conditions to demonstrate two canonical properties needed to establish a circadian clock, a free running rhythm that is close to 24 h and temperature compensation, the authors did not fully test one of the cardinal properties of a circadian clock, entrainment. Second, the authors do not establish that TcFRQ is a core component of the clock oscillator. Third, the authors made conclusions from a strain that is supposed to overexpress TcFRQ from an actin promoter but, there is no data demonstrating that TcFRQ is actually overexpressed. Fourth, the rationale for concluding that TcFRQ has an extra-clock role, as opposed to a clock role, in development and metabolism is not clear Finally, a red safety light was used in all circadian experiments in constant dark; however, there is no data to support that T. atroviride is not responsive to red light.

Figure 1 While these data support the existence of a circadian clock in this organism based on free running rhythms and temperature compensation, it is important to provide additional data to demonstrate that the rhythm is entrained, one of the 3 criteria established in the field.

a. To demonstrate circadian entrainment, as opposed to a direct response to the environmental cues, specifically light, they need to examine different length LD cycles to demonstrate that the clock entrains to the different cycles. In addition, to demonstrate that an acute light or temperature pulse is resetting the clock, as opposed to masking, a a phase response curve is needed to show advances and delays. It is not clear what phase the light pulse was given in Figure 1D.

b. In the Supplement to Figure 2, several genes are arrhythmic under conditions in which TAFRQLUC is rhythmic. This is an important control to show, for example, that there is not a rhythm in ATP levels that might be driving the rhythm in the TAFRQLUC reporter. I would like to see this mentioned earlier in relation to Figure 1.

c. It would be helpful to know circadian time in Figure 1, and of interest to know if the peak time is similar to N. crassa FRQ.

Figure 2 demonstrates that the cis-acting promoter element of N. crassa frq that bound by the positive element, the WCC, is necessary for frq mRNA rhythms and functioning of the feedback loop, is sufficient to drive rhythms in luciferase in T. atroviride in DD, and is responsive to light and temperature pulses. This experiment suggests that there is conservation of the c-box, and raises the possibility that TAFRQ functions similarly in the T. atroviride clock as a negative element; however, this experiment raises more questions than it answers, and therefore seems premature. There are several instances in the paper where TaFRQ is called a core clock component, but there are no data to support this claim.

a. Is there a similar sequence present in the promoter of TAFRQ? If so, what happens if this sequence is mutated?

b. What happens to the rhythm, levels, of TAFRQ in a Blr1 deletion or mutated strain?

c. Additional experiments are needed to show that Blr1 binds to this sequence and to fully describe TAFRQ role in the clock mechanism.

In Supplementary file 1, 2A, B and C, they examine if TaFRQ has a role in development, and this is supported by the data presented by comparing WT versus TaFRQ deletion strains. They show that conidia formation is delayed in the mutant compared to WT, and decreased following light treatment or injury. In addition, they also examined a construct aimed at overexpressing TaFRQ (OE::TaFRQ) using the actin promoter. No differences were observed between WT and OE::TaFRQ; however, it is difficult to make any conclusions here because there were no experiments demonstrating that TaFRQ was actually overexpressed in this strain.

Figure 3 examines the ability of T. atroviride to overgrow B. cinerea under different lighting conditions and in mutants as an indicator of mycoparasitic behavior. The key findings were that constant light inhibits overgrowth, and that mutations in blr1 in T. atroviride enhances overgrowth in constant darkness. However, it is not clear how these data and those that follow establish and extra-clock role for TaFRQ in these processes.

Figure 4 looks at dawn and dusk confrontations between T. atroviride and B. cinereai in LD cycles. They found that WT T. atroviride and TaFRQ deletion strains had overgrowth that was enhanced at dawn, whereas deletion of TaBLR1 did not show a time of day difference. However, when frq was deleted in B. cinerea, all T. atroviride strains overgrew. These data supported that homologs of N. crassa clock components in T. atroviride and B. cinereai play differing roles in time of day specific interactions, and this information could prove useful in biocontrol measures in the future.

Figure 5 examines if T. atroviride FRQ and BLR1 mutants alter secondary metabolism by examining diffusible and volatile compounds in DD and LL, and following a light pulse. They found several differences, but were unable to correlate these with the overgrowth experiments in Figures 3 and 4. A circadian metabolic profile demonstrated that some compounds were rhythmic in WT and arrhythmic in taFRQ deletion cells. These data provide evidence to support a role for a clock in controlling metabolism in T. atroviride.

Discussion: The paper lacks definitive experiments to show that taFRQ is a core clock component, and therefore, it is not clear how these data support a extra-circadian role for taFRQ. In addition, aspects of the discussion are very lengthy, especially the section describing how this work could foster looking for clocks in other fungi.

Methods:

a. It is critical to show that T. atroviride is not sensitive to red light given red safety lights were used in circadian experiments.

b. The constructs used to examine potential rhythms in known fungal ccgs used the promoter, but not the 3' ends of the genes. The authors may want to consider adding this in the future.

Supplemental Figures

1.4 In part A, the light pulse did not seem to improve the rhythm similar to what was shown in Figure 1. What the light pulse given at the same time of day?

1.5. When was the light pulse given? These data should be plotted.

2.1. The data in A appear different from Figure 2A, although it appears to be the same experiment.

File 1. This figure is difficult to follow.

File 2: It is not clear if the plot in B represents 3 or 7 days after a light pulse.

Reviewer #2:

In this manuscript, Henríquez-Urrutia and colleagues establish the existence of a core circadian rhythm in the mycoparasitic fungus Trichoderma atroviride. Using both translational and transcriptional reporters for a core clock component, FRQ/frq, the authors demonstrate that the rhythm meets all the requirements of bona fide circadian oscillator, e.g. entrainment and temperature compensation. Interestingly and importantly, they demonstrate that the robustness of the oscillation is largely dependent on the nutritional environment; specifically, the rhythm was only discernable under growth conditions that likely resemble the natural environment of the fungus. This information will be of keen interest to the chronobiology community, particularly those that attempting to identify a clock in their microbe of interest. Moreover, the authors demonstrate that two processes are under control of the Trichoderma clock, or at least under the regulation of clock components: (1) secondary metabolism, and (2) the myoparasitic interaction with another fungus, Botrytis. Although the connection between those two phenomena in this study is unclear, together they demonstrate the importance of biological clocks in regulating fungal behaviors of interest. Understanding how the time-of-day influences fungal parasitism, for example, could inform the optimization of fungi as biocontrol agents in the agricultural setting. In conclusion, the work provided here is novel, the conclusions are supported by the data, the ideas are well-marshalled. Consequently, I believe this would be would be a good fit for eLife and its readership. However, the manuscript may be strengthened by an expanded discussion or additional experiments as outlined below.

1) The authors do an excellent job in defining how environmental parameters (e.g. nutrients and temperature), influence the robustness of Trichoderma core oscillation. It is therefore surprising that the Trichoderma/Botrytis interaction assays were not analyzed across a similar variety of conditions. For example, the authors performed the interaction assays on PDA media at 20C, the data from which support the conclusion the Trichoderma FRQ does not significantly influence its parasitism of Botrytis. However, what would these results look like on media in which the Trichoderma oscillator is more robust, i.e. on GYEC media at 22C? Conversely, how would these changing conditions influence the Botrtyis clock proteins and their role in the interaction? The inclusion of such data may strengthen the overall discussion of the fungal clock operating within stringent environmental conditions.

2) For the interaction assay, it is clear the inoculating the plates at dawn results in greater Trichoderma overgrowth compared to inoculation at dusk. However, the authors state in the discussion (pg 33, line 15), that "overgrowth is increased when the "interactions started at dawn…". Does the inoculation time really represent the time at which the fungal interaction take place? Surely there must be a lag that reflects the growth of the organisms towards each other. Perhaps the interaction begins 12 h after inoculation, i.e. at dusk. Relatedly, how does growth rate of the various mutant strains (of either species) under the various light conditions influence the time in the solar/circadian day in which the initial interaction takes place?

3) Much attention is given to circadian influence on Trichoderma secondary metabolism, which is indeed very interesting. However, the relationship between this and the Botrytis interaction results are fairly weak. Can the authors demonstrate that culture supernatants of Trichoderma taken at different times across the circadian day, or in DD v LL, have an differential impact on Botrytis growth or viability? Maybe this is an oversimplified expectation. In any case , it is noted that Botrytis proteins have the greatest impact on the interaction, which would suggest that the circadian regulation of Trichoderma metabolites may not be particularly important. Do the authors think the relative susceptibility of Botrytis to Trichoderma metabolites, or some other stressor, is regulated by the Bc clock proteins? In other words, some discussion about the fungal-fungal interaction from the Botrytis perspective may be welcomed.

4) Overall, the 'Introduction' and 'Discussion' sections are nicely written and easy to read. By contrast, the 'Results' section contains some phrasing and grammatical issues that should be revised.

This is a very interesting study and my major "concerns" are outlined above. I may have misunderstood key aspects of the experiments, in which case a clarifying statement in the rebuttal may suffice. However, I do think that the authors should clarify some things in the manuscript as outlined here.

1) Please describe the assay conditions more clearly throughout the manuscript. The assay medium for Figure 3, for example, is not mentioned in the results or figure legend. I can't even see a definitive mention of the medium in the methods. In fact, the figure 4 legend is the only place where PDA is explicitly stated. Similarly, it is unclear what media was used for the secondary metabolite profiles. Based on the language in the methods, it seems that the light experiments were performed on PDA and the circadian time course was on GYEC… is that correct? If so, why use different media? This conditions be clearly stated in the figure legends in any case.

2) To what extent does the relative growth rate of the two organisms influence the interaction? For example, if a particular B. cinerea (Bc) mutant were slow growing, would this make the apparent overgrowth of T. atroviride (Ta) seem larger? The methods state that the readout is based on percent overgrowth, but this isn't really obvious in most of the pictures. To me, and perhaps to many readers, the readout (dotted line) seems to reflect the point of contact between the two organisms, sort of like a mating assay, which would presumably be growth rate dependent.

– To perhaps clarify this issue, the authors might consider is showing the each strain (Ta and Bc) by itself on a plate under each of the conditions (e.g. DD, LL, LD, etc). This would allow the reader to better visualize the morphology and growth rate of each organism in isolation, and consequently interpret the interaction experiments better.

3) Pg. 13, line 12: "After 72 h of cultivation in LL, conidia in Δtafrq exhibited a dark-green color compared to the TaWT and the OE::tafrq strains (Supplementary file 2A) which displayed the characteristic T. atroviride green coloration."

– This sentence is a bit confusing. So "green" is normal, and "dark green" is abnormal? It may be helpful for the unfamiliar reader if the authors briefly describe basic Trichoderma colony morphology, i.e. what color are the condia and underlying hyphae.

4) Some specific areas in the 'Results' section that need revision include, but are not limited to, the following:

– Pg 13, line 8: "This distinctive phenotype…"

– Pg. 14, Line 15.

– Page 18, Line 2: "Regarding…"

5) Pg. 7, Line 14: "ostensibly" doesn't seem like the correct word here.

Reviewer #3:

The most obvious change during the course of a day is the dark-light cycle. Associated to light changes are changes in harmful UV light, in temperature, in humidity, the possibility for finding food or the orientation in the environment. Circadian clocks help organisms to adapt to these predictable periodic changes before they actually occur. E.g. for humans it was good to wake up during dusk and be alert before their predators are active. For microorganisms, like filamentous fungi, environmental parameters may be important for instance if they produce spores for distribution. It may be advantageous to produce them early in the morning, when the humidity is high, because spores are often produced at the surface of substrates and hence the mycelium is at risk for desiccation. In the case of plant-pathogenic fungi it may be advantageous to spread the spores at times where plants are more susceptible for a fungal attack. This had been shown before by the same research group in the case of Botrytis cinerea. The current paper is a continuation of the work on organismic interactions and highlights the role of a circadian clock in the mycoparasitic relationship between Trichoderma atroviride and the grey mold B. cinerea. They identified a circadian clock in T. atroviride and show that it influences spore and secondary metabolite production. In the mycoparasitic interaction the clock of the prey is important for the success of the intruder rather than the other way around. The downstream components of the circadian clock, which are important for the outcome of the mycoparasitic interaction, remain to be discovered.

The paper has many important implications. The molecular mechanism of circadian clocks has been studied extensively in Neurospora crassa, but only in a few other fungi so far. Many fungi seem to lack one of the central components of the N. crassa clock, namely the negative element frequency. Therefore, it is important to collect more examples for circadian mechanisms in fungi. In T. atroviride the clock mechanism appears to be conserved in comparison to N. crassa. A second important aspect is that T. atroviride is used as a biocontrol agent and hence the work may help to optimize the application of the fungus in the field. Last but not least, the paper shows that secondary metabolism is largely influenced by the clock, an aspect so far largely neglected. Given that filamentous fungi are important producers of secondary metabolites, such as penicillin or lovastatin, circadian clocks should be considered when optimizing production processes.

The first two chapters describe the characterization of the circadian clock of T. atroviride. The authors show that it can be entrained by light-dark cycles, that it can be reset by a light or temperature pulse, and it is temperature compensated in a range between 20 and 26{degree sign}C. The mechanism of clock functioning appears to be similar to the one in N. crassa with a conserved cis element in the promoter of the frequency gene. Interestingly, addition of pea powder significantly enhanced the amplitude of the clock-dependent reporter expression. Is this a specific effect of pea or do other plants or fruits serve the same function? This is especially important because this aspect is also highlighted in the Discussion.

I find it very surprising, but interesting at the same time, that several tested and known clock-controlled genes apparently are not clock-controlled in T. atroviride. The authors should look at some regulators of the pathways they identified as being under clock control. The candidates could be further analyzed for the identified cis element in their promoters.

Next, they tested a function of the frq gene outside the clock function, because such an additional function had been described before in B. cinerea. They found that indeed conidiation is delayed in a frq-deletion strain, independent of ligh-dark cycles. To verify these results the deletion strain should be re-complemented with a wild-type copy.

In the following chapters they focus on the mycoparasitic interaction and use frq and wc mutant strains of both fungi in confrontation assays. They first found that light inhibits the parasitic capacity of T. atroviride and that the WC-1 orthologue is required for the aggressive behavior of T. atroviride in the dark. Again in this series of experiments a T. atroviride frq re-complemented strain is missing. In light-dark cycles it turned out that the B. cinerea Frq protein is important for the outcome of the interaction. This shows that the interaction is clock-controlled, but on the prey side.

In the last chapter they analyzed the production of different secondary metabolites and found that the production is under light control. The WC orthologue and Frq are involved in the regulation, independent of their functions in the circadian clock. However, the authors also show that the production of some compounds is clock controlled, and most required Frq. I guess for specialists the source data tables are useful. I cannot evaluate them.

Essential revisions:

The reviewers have made several constructive suggestions that you should carefully consider and address in a point-by-point reply. In particular, please pay particular attention to the following revisions:

We appreciate the editor's comments, summarizing the primary considerations as well as the different comments raised by all reviewers. We provide a point-by-point rebuttal, and hope that now the manuscript meets all criteria for publication.

1) Please perform experiments that demonstrate entrainment (as suggested by Reviewer #1).

While we understand that Reviewer 1 requested this type of experiments to be fully convinced of the presence of a bona fide circadian clock, due to technical issues and additional complications associated with the extended pandemic, we could not perform those suggested experiments. Indeed, experimental work was severely hampered at our universities during most of 2021, as access was extremely restricted. Nevertheless, we generated new data that actually go towards that direction, providing additional proof that this is a proper circadian system (see response #1 to Reviewer 1). We truly appreciate the reviewer’s comments as they allowed us to strengthen the dataset regarding this point. We are convinced that the new results not only do that, but also provide new interesting observations included in the manuscript.

2) Please perform the interaction assays across a range of environmental conditions (nutritional, temperature) to determine if the contributions of the clock proteins (in either organism) change accordingly (as suggested by Reviewer #2).

In the new text we now provide an important technical aspect of the confrontation experiments, commenting that only PDA media provides proper conditions to clearly visualize overgrowth or Trichoderma mycelia over Botrytis, as in other media (such as GYEC) Botrytis behaves oddly in terms of mycelia consistence and coloration. Importantly, the main purpose of these experiments was not to systematically test the effect of different conditions on the interaction but instead, to provide a proof of concept that two variables – light (Figure 3), and time (Figure 4) – have a relevant effect modulating the outcome of the interaction. We anticipate that future work in our lab (and the labs of others) will examine the additional effects of other variables in combination with the abovementioned ones.

3) Please re-complement your mutants (as suggested by Reviewer #3).

We thank the reviewer for raising this observation. Accordingly, we have now included complementation experiments as depicted in (new figures: Figure 3—figure supplement 3 and 4) confirming that, as expected, one of the hallmark phenotypes of the mutant is reversed in the complemented strain. We have included information of the new experiments in the Materials and methods section.

Reviewer #1:

The goal of this study was to establish that the fungus Trichoderma atroviride, and important biocontrol agent, has a circadian clock, and to test if this clock plays a role in development, secondary metabolite production, and mycoparasitism. The authors clearly establish rhythms in the organism that are dependent on TcFRQ, a homolog of the core clock component FRQ in N. crassa, and show that TcFRQ and BRL1, the homolog of the blue light photoreceptor and core clock component WC-1 in N. crassa, play differing roles in development, metabolite levels, and mycoparasitism in laboratory conditions. However, the authors do not fully establish that the rhythms are controlled by a circadian clock , nor that TcFRQ is a core clock component. While aspects of this work will be of interest to circadian and fungal biologists, the not all of the aims were met, and the data do not fully support the conclusions.

We sincerely appreciate the positive reception of our work. The reviewer's primary concern is related to the absence of data showing that TaFRQ is indeed a clockcore component. After careful examination, we have pondered our results and performed additional experiments that should help dissipate any potential doubts about that main conclusion. Thus, to fully support the latter, and to clearly establish that a circadian clock controls the observed rhythms, we now have included relevant additional information describing that TaFRQ is indeed a core clock component based on functional complementation experiments (aka classic old school approach) in Neurospora crassa. Accordingly, as highlighted in the new version of the MS, we do not only demonstrate that T. atroviride's clock free runs and compensates for temperature, but also that the tafrq gene is capable of functionally complementing a frq null strain of Neurospora (new figures: Figure 2 and figure supplement 2, and pages 12-13). Most notably, the latter experiment also shows that the “long” free running rhythms characterized in T. atroviride can be "transplanted” along with the Tafrq sequence to Neurospora. We think that these data fully support our conclusions, including extra-circadian functions of TaFRQ (see below).

Strengths: The circadian clock in the fungus N. crassa serves as an important model for understanding the mechanisms that keep time in eukaryotes, and how this clock controls daily rhythms in gene expression. However, little is known about clocks in other fungi, despite conservation of core clock components found in N. crassa, including fungal pathogens and biocontrol agents like Trichoderma. Thus, the strength of this work is in that it goes a long way to establish a clock in T. atroviride that is directly, or indirectly involved in controlling development, metabolism, and mycoparasitism. Importantly, this work opens up the opportunity to determine how the clock impacts fungal-fungal interactions, with the potential of using this to improve biocontrol.

We sincerely appreciate the positive reception of our work.

Weaknesses: There are several weaknesses. The first is that while the authors established conditions to demonstrate two canonical properties needed to establish a circadian clock, a free running rhythm that is close to 24 h and temperature compensation, the authors did not fully test one of the cardinal properties of a circadian clock, entrainment. Second, the authors do not establish that TcFRQ is a core component of the clock oscillator.

We thank the reviewer's thorough review. As mentioned above, although we did not explore entrainment in this work, the new dataset (FRQ complementation) provides new and unequivocal evidence that TaFRQ is a bona fide core-clock component, and therefore of the existence of a canonical clock in Trichoderma. Additionally, our data also provides clear evidence that the T. atroviride system readily responds to temperature and light cues which, albeit it is not entrainment per se, is tightly connected with it. Thus, we can now establish that TaFRQ indeed encodes a clock-core component (new figures: Figure 2-figure supplement 2, pages 12 and 13). Having cleared the waters in terms of its clock function, we can also mention about its extra-circadian roles (see following question-answer below). This is important, as we have demonstrated an analogous observation in Botrytis cinerea, and we anticipate that more than an exception this extracircadian functions may soon emerge as a transversal and common property of clock components.

Third, the authors made conclusions from a strain that is supposed to overexpress TcFRQ from an actin promoter but, there is no data demonstrating that TcFRQ is actually overexpressed.

Again, we sincerely appreciate the reviewer’s exhaustive review. Based on his/her observations we performed RT-qPCR analysis, which confirmed overexpression, particularly under DD conditions (circa 4-fold), info which can now be found in Figure 3figure supplement 2 (new figure) and page 14, Line 21-23. Since we also observed that in LL the degree of overexpression is less marked (albeit still statistically significant), we have made that explicit in the text and in the discussion of the data.

Fourth, the rationale for concluding that TcFRQ has an extra-clock role, as opposed to a clock role, in development and metabolism is not clear.

Indeed, this may have not been clear enough in the text. Now with the new data we have unmistakably stablished that Tafrq has a canonical role as a core-clock component. In addition, it has “extra-circadian” roles, as it affects (delays) sporulation and injury response even in LL conditions. As the clock only runs in DD (and not in LL) we refer to these effects as “extra circadian” since they are not based on a running clock.

Finally, a red safety light was used in all circadian experiments in constant dark; however, there is no data to support that T. atroviride is not responsive to red light.

We apologize for the confusion: in all free-running experiments, constant darkness means the total absence of light. Red safety lights are utilized in the dark room, which houses our light-proof incubators (Percival). Thus, experiments (CCD recordings, growth in Petri plates, etc.) were conducted inside the incubators in full darkness or in the presence of white light (when indicated). Red lights were only used to manipule equipment when required, or when opening incubators. Indeed, cultures were seldom exposed to red light in the experiments conducted in this MS. Related to the general question raised by the reviewer, only a few genes (three) have been detected as red-light regulated in T. atroviride after a 5 min red light-pulse (PMID: 17074901). In any case, both light intensity and time of exposure significantly differ from occasional expose to redlight during culture manipulation (i.e., harvesting).

Figure 1 While these data support the existence of a circadian clock in this organism based on free running rhythms and temperature compensation, it is important to provide additional data to demonstrate that the rhythm is entrained, one of the 3 criteria established in the field.

a. To demonstrate circadian entrainment, as opposed to a direct response to the environmental cues, specifically light, they need to examine different length LD cycles to demonstrate that the clock entrains to the different cycles. In addition, to demonstrate that an acute light or temperature pulse is resetting the clock, as opposed to masking, a a phase response curve is needed to show advances and delays. It is not clear what phase the light pulse was given in Figure 1D.

As mentioned earlier, we were unable to properly perform entrainment experiments. Instead, we adopted a different approach to provide compelling data regarding the characterization of the Trichoderma FRQ-based clock (see complementation experiments). Future work will, undoubtedly, focus on additional circadiana of this system, particularly phase response curves. Nevertheless, we estimate the revised data set provides sufficient information to support the main conclusions of the current manuscript.

b. In the Supplement to Figure 2, several genes are arrhythmic under conditions in which TAFRQLUC is rhythmic. This is an important control to show, for example, that there is not a rhythm in ATP levels that might be driving the rhythm in the TAFRQLUC reporter. I would like to see this mentioned earlier in relation to Figure 1.

We are glad to know that the reviewer also appreciates this data. Indeed, now we mention this is page 9, lines 1-13.

c. It would be helpful to know circadian time in Figure 1, and of interest to know if the peak time is similar to N. crassa FRQ.

The data suggests that the phase of the first peak of TaFRQ expression (as FRQ^LUC) is similar, albeit slightly advanced, compared to what occurs in N. crassa (once converted to circadian time), being roughly CT 11 for the latter, and CT 8 for Trichoderma. This is now mentioned in page 7, line 18.

Figure 2 demonstrates that the cis-acting promoter element of N. crassa frq that bound by the positive element, the WCC, is necessary for frq mRNA rhythms and functioning of the feedback loop, is sufficient to drive rhythms in luciferase in T. atroviride in DD, and is responsive to light and temperature pulses. This experiment suggests that there is conservation of the c-box, and raises the possibility that TAFRQ functions similarly in the T. atroviride clock as a negative element; however, this experiment raises more questions than it answers, and therefore seems premature. There are several instances in the paper where TaFRQ is called a core clock component, but there are no data to support this claim.

Indeed, this experiment raises a lot of interesting new questions. Nevertheless, in addition to the new complementation data, it provides a general and valuable conclusion: there is conservation of core-clock mechanisms and components across two species belonging to the same class, and there is cross-class recognition of such components. Thus, we believe it nicely contributes to the story.

a. Is there a similar sequence present in the promoter of TAFRQ? If so, what happens if this sequence is mutated?

b. What happens to the rhythm, levels, of TAFRQ in a Blr1 deletion or mutated strain?

c. Additional experiments are needed to show that Blr1 binds to this sequence and to fully describe TAFRQ role in the clock mechanism.

This reviewer raises interesting observation/questions, that also intrigue us. While these formal questions are highly relevant, we deem that they are not essential in the context of the overall message of this manuscript. Indeed, we have not identified a defined "endogenous" c-box sequence in Trichoderma, nevertheless, as described in the manuscript, “Two putative light response elements in the tafrq’s promoter with a putative GATA box consensus sequence at proximal and distal locations (-355 to -319 and -1180 to -1171 from the TSS, respectively) have been postulated (Cervantes-Badillo et al., 2013), of which one of them could be acting as a putative _Tacbox sequence.”.

We did not directly determine TaFRQ levels in a BLR1 mutant strain. Importantly, transcriptomic datasets address this, confirming that, as expected, tafrq expression depends on BLR1, and is downregulated in the absence of this TF (PMID: 27020152).

This is now mentioned in Page 4, line 16.

In addition, the collective evidence of the new data showing how TaFRQ complements the Neurospora's frq null mutant, the fact that c-box of Neurospora works rhythmically in T. atroviride, and the absence of rhythms of other promoter-luc constructs in the latter fungus, all clearly reinforces the idea that we are describing a circadian clock and that TaFRQ is indeed a core clock component along with BLR1, aspects that are now commented in the discussion. Thus, while we appreciate and also share some of the questions raised by the reviewer, we believe they are beyond the current scope of the work.

In Supplementary file 1, 2A, B and C, they examine if TaFRQ has a role in development, and this is supported by the data presented by comparing WT versus TaFRQ deletion strains. They show that conidia formation is delayed in the mutant compared to WT, and decreased following light treatment or injury. In addition, they also examined a construct aimed at overexpressing TaFRQ (OE::TaFRQ) using the actin promoter. No differences were observed between WT and OE::TaFRQ; however, it is difficult to make any conclusions here because there were no experiments demonstrating that TaFRQ was actually overexpressed in this strain.

As commented above, we have now measured tafrq expression in the overexpressing strain, confirming that levels are statistically higher in the latter compared to WT, which is particularly marked in DD conditions. Indeed, when it comes to the mentioned phenotypes (conidiation, injury response) the OE strain behaves as the WT. Nevertheless, as evidenced in Figure 5, regarding metabolic profiles, the OE strain shows a pattern quite distinct compared to WT, particularly under DD, when expression levels of tafrq in OE versus WT are more pronounced.

Figure 3 examines the ability of T. atroviride to overgrow B. cinerea under different lighting conditions and in mutants as an indicator of mycoparasitic behavior. The key findings were that constant light inhibits overgrowth, and that mutations in blr1 in T. atroviride enhances overgrowth in constant darkness. However, it is not clear how these data and those that follow establish and extra-clock role for TaFRQ in these processes.

We apologize for the confusion, as we did not intend to imply that the data in Figure 3 relates to the extra circadian role of TaFRQ. To provide more context, we were also interested to analyze whether TaFRQ had additional roles, besides its clock function, a thought inspired from our findings for Botrytis FRQ. This is a relevant observation since, in the case of Neurospora, the FRQ protein seems to have only clock-related functions, while in Botrytis, in an environmental condition where the clock is not functional (in constant light), the frq mutant shows additional extra-circadian functions related to altered development (conidiation). Now, this is better explained in the text in page 15, between lines 09-16.

Figure 4 looks at dawn and dusk confrontations between T. atroviride and B. cinereai in LD cycles. They found that WT T. atroviride and TaFRQ deletion strains had overgrowth that was enhanced at dawn, whereas deletion of TaBLR1 did not show a time of day difference. However, when frq was deleted in B. cinerea, all T. atroviride strains overgrew. These data supported that homologs of N. crassa clock components in T. atroviride and B. cinereai play differing roles in time of day specific interactions, and this information could prove useful in biocontrol measures in the future.

We appreciate the reviewer's comments as they highlight our results and potential future applications. Indeed, we are looking forward to see how our observation can be translated to applied studies.

Figure 5 examines if T. atroviride FRQ and BLR1 mutants alter secondary metabolism by examining diffusible and volatile compounds in DD and LL, and following a light pulse. They found several differences, but were unable to correlate these with the overgrowth experiments in Figures 3 and 4. A circadian metabolic profile demonstrated that some compounds were rhythmic in WT and arrhythmic in taFRQ deletion cells. These data provide evidence to support a role for a clock in controlling metabolism in T. atroviride.

Again, we appreciate the reviewer's comments as they highlight the relevance of our results.

Discussion: The paper lacks definitive experiments to show that taFRQ is a core clock component, and therefore, it is not clear how these data support a extra-circadian role for taFRQ. In addition, aspects of the discussion are very lengthy, especially the section describing how this work could foster looking for clocks in other fungi.

We acknowledge, as the reviewer pointed out, the limitation that our study had. Therefore, we took the approach of complementing an arrhythmic Neurospora strain with tafrq (see above). Such results not only provide compelling evidence of TaFRQ role in the clockworks, but also opens the door to interesting future comparative studies. Having dissipated such doubts, it may now be easier to visualize the concept of “extracircadian” roles of FRQ, as they lead to phenotypes even in a condition where clock function is abrogated (constant light). We have also modified parts of the discussion, to make it more fluid.

Methods:

a. It is critical to show that T. atroviride is not sensitive to red light given red safety lights were used in circadian experiments.

We have now clarified that low level red-lights are used only to manipulate cultures, equipment, although most of the experiments described herein were conducted in full DD (inside incubators), which were actually housed inside the dark room equipped with the safety lights. Please, refer to (above).

b. The constructs used to examine potential rhythms in known fungal ccgs used the promoter, but not the 3' ends of the genes. The authors may want to consider adding this in the future.

We thank the reviewer's suggestions. Indeed, we are considering this in other studies conducted with collaborators.

Supplemental Figures

1.4 In part A, the light pulse did not seem to improve the rhythm similar to what was shown in Figure 1. What the light pulse given at the same time of day?

We have modified the legend of figure 1.4, to better reflect that the conditions of the light pulse were not equivalent to the one in Figure 1.

1.5. When was the light pulse given? These data should be plotted.

We have also modified the figure legend to better explain when the LP was given.

2.1. The data in A appear different from Figure 2A, although it appears to be the same experiment.

We have simplified the legend of figure 2.1 (Figure 2—figure supplement 1) so it is now clear that the experiment was conducted in GYEC, whereas the one in 2A was conducted in GYEC + peas, which (as seen in previous figures) dramatically improves rhythms.

File 1. This figure is difficult to follow.

A series of modifications were made to the figure and its legend for simplification.

File 2: It is not clear if the plot in B represents 3 or 7 days after a light pulse.

This file (now called Supplementary file 3) has been improved as requested.

Reviewer #2:

In this manuscript, Henríquez-Urrutia and colleagues establish the existence of a core circadian rhythm in the mycoparasitic fungus Trichoderma atroviride. Using both translational and transcriptional reporters for a core clock component, FRQ/frq, the authors demonstrate that the rhythm meets all the requirements of bona fide circadian oscillator, e.g. entrainment and temperature compensation. Interestingly and importantly, they demonstrate that the robustness of the oscillation is largely dependent on the nutritional environment; specifically, the rhythm was only discernable under growth conditions that likely resemble the natural environment of the fungus. This information will be of keen interest to the chronobiology community, particularly those that attempting to identify a clock in their microbe of interest. Moreover, the authors demonstrate that two processes are under control of the Trichoderma clock, or at least under the regulation of clock components: (1) secondary metabolism, and (2) the myoparasitic interaction with another fungus, Botrytis. Although the connection between those two phenomena in this study is unclear, together they demonstrate the importance of biological clocks in regulating fungal behaviors of interest. Understanding how the time-of-day influences fungal parasitism, for example, could inform the optimization of fungi as biocontrol agents in the agricultural setting. In conclusion, the work provided here is novel, the conclusions are supported by the data, the ideas are well-marshalled. Consequently, I believe this would be would be a good fit for eLife and its readership. However, the manuscript may be strengthened by an expanded discussion or additional experiments as outlined below.

1) The authors do an excellent job in defining how environmental parameters (e.g. nutrients and temperature), influence the robustness of Trichoderma core oscillation. It is therefore surprising that the Trichoderma/Botrytis interaction assays were not analyzed across a similar variety of conditions. For example, the authors performed the interaction assays on PDA media at 20C, the data from which support the conclusion the the Trichoderma FRQ does not significantly influence its parasitism of Botrytis. However, what would these results look like on media in which the Trichoderma oscillator is more robust, i.e. on GYEC media at 22C? Conversely, how would these changing conditions influence the Botrtyis clock proteins and their role in the interaction? The inclusion of such data may strengthen the overall discussion of the fungal clock operating within stringent environmental conditions.

We share the curiosity expressed by the reviewer. Examination of different media modulating the Botrytis-Trichoderma interaction will be a future area of study (particularly involving conditions mimicking soil). Nevertheless, they were beyond the scope of this first report, and due to all the complications of a closed lab during almost 16 months, and people moving on to new jobs, this was further delayed. Based on the reactions of colleagues, as we first presented the data in public in 2022 (Fungal Genetics, and Botrytis meetings), we are convinced that the story, as it is, will be extremely well received and, most importantly, will inspire new angles of studies in different labs.

2) For the interaction assay, it is clear the inoculating the plates at dawn results in greater Trichoderma overgrowth compared to inoculation at dusk. However, the authors state in the discussion (pg 33, line 15), that "overgrowth is increased when the "interactions started at dawn...". Does the inoculation time really represent the time at which the fungal interaction take place? Surely there must be a lag that reflects the growth of the organisms towards each other. Perhaps the interaction begins 12 h after inoculation, i.e. at dusk. Relatedly, how does growth rate of the various mutant strains (of either species) under the various light conditions influence the time in the solar/circadian day in which the initial interaction takes place?

As the reviewer noticed, there must be a lag reflecting the growth of both fungi towards each other. Interestingly, in the case of the Botrytis-plant interaction, which also is influenced by the time of the day as reported by our group years ago, there is also a lag: the infection assays are regularly conducted employing a droplet containing conidia that first needs to develop the infecting hyphae, and later, the appressorium to penetrate the plant tissue. However, differences are observed between dawn and dusk inoculation times irrespective of this delay.

The reviewer, for sure, is concerned that this delay might be too extended in time, explained, for example, by a significant difference in growth rate between the two fungi. Therefore, we controlled for it: after two days of growth, the colony sizes of Trichoderma and Botrytis was measured, and no statistical differences were observed. Please, refer to new figure (Figure 4-figure supplement 1) and page 17, Line 9-17.

3) Much attention is given to circadian influence on Trichoderma secondary metabolism, which is indeed very interesting. However, the relationship between this and the Botrytis interaction results are fairly weak. Can the authors demonstrate that culture supernatants of Trichoderma taken at different times across the circadian day, or in DD v LL, have an differential impact on Botrytis growth or viability? Maybe this is an oversimplified expectation. In any case , it is noted that Botrytis proteins have the greatest impact on the interaction, which would suggest that the circadian regulation of Trichoderma metabolites may not be particularly important. Do the authors think the relative susceptibility of Botrytis to Trichoderma metabolites, or some other stressor, is regulated by the Bc clock proteins? In other words, some discussion about the fungal-fungal interaction from the Botrytis perspective may be welcomed.

The question raised by the reviewer is fascinating and could even have biotechnological implications in using Trichoderma for biocontrol. And although the products secreted by Trichoderma when growing alone in a liquid medium differ from those produced when growing with Botrytis in the same Petri dish, the interpretation of the data would be very complex since the secreted products (proteins, metabolites, etc.) are very stable over time. We have briefly included ideas as the ones mentioned by the reviewer, in the discussion session.

4) Overall, the 'Introduction' and 'Discussion' sections are nicely written and easy to read. By contrast, the 'Results' section contains some phrasing and grammatical issues that should be revised.

Thanks. We have revised the Results section.

This is a very interesting study and my major "concerns" are outlined above. I may have misunderstood key aspects of the experiments, in which case a clarifying statement in the rebuttal may suffice. However, I do think that the authors should clarify some things in the manuscript as outlined here.

1) Please describe the assay conditions more clearly throughout the manuscript. The assay medium for Figure 3, for example, is not mentioned in the results or figure legend. I can't even see a definitive mention of the medium in the methods. In fact, the figure 4 legend is the only place where PDA is explicitly stated. Similarly, it is unclear what media was used for the secondary metabolite profiles. Based on the language in the methods, it seems that the light experiments were performed on PDA and the circadian time course was on GYEC… is that correct? If so, why use different media? This conditions be clearly stated in the figure legends in any case.

Thanks for bringing this up. We have now made sure that each key figure is explicit regarding the utilized media. The need to use PDA in some experiment’s and GYEC + peas media in others is discussed within the manuscript.

2) To what extent does the relative growth rate of the two organisms influence the interaction? For example, if a particular B. cinerea (Bc) mutant were slow growing, would this make the apparent overgrowth of T. atroviride (Ta) seem larger? The methods state that the readout is based on percent overgrowth, but this isn't really obvious in most of the pictures. To me, and perhaps to many readers, the readout (dotted line) seems to reflect the point of contact between the two organisms, sort of like a mating assay, which would presumably be growth rate dependent.

– To perhaps clarify this issue, the authors might consider is showing the each strain (Ta and Bc) by itself on a plate under each of the conditions (e.g. DD, LL, LD, etc). This would allow the reader to better visualize the morphology and growth rate of each organism in isolation, and consequently interpret the interaction experiments better.

Originally, we asked ourselves questions similar to the ones raised by the reviewer. We explored them by changing the relative position of the inoculation point of one of the confronting fungi yet, we saw results similar to the ones presented in the manuscript. Please, refer to response #2 (above).

3) Pg. 13, line 12: "After 72 h of cultivation in LL, conidia in Δtafrq exhibited a dark-green color compared to the TaWT and the OE::tafrq strains (Supplementary file 2A) which displayed the characteristic T. atroviride green coloration."

– This sentence is a bit confusing. So "green" is normal, and "dark green" is abnormal? It may be helpful for the unfamiliar reader if the authors briefly describe basic Trichoderma colony morphology, i.e. what color are the condia and underlying hyphae.

Please, see modification in page 14, lines 12-15.

4) Some specific areas in the 'Results' section that need revision include, but are not limited to, the following:

– Pg 13, line 8: "This distinctive phenotype…"

– Pg. 14, Line 15.

– Page 18, Line 2: "Regarding…"

Reviews are appreciated. All these comments were resolved in the corresponding sections.

5) Pg. 7, Line 14: "ostensibly" doesn't seem like the correct word here.

Modified. Now you can read “quality of the oscillations greatly improved”.

Reviewer #3:

The first two chapters describe the characterization of the circadian clock of T. atroviride. The authors show that it can be entrained by light-dark cycles, that it can be reset by a light or temperature pulse, and it is temperature compensated in a range between 20 and 26{degree sign}C. The mechanism of clock functioning appears to be similar to the one in N. crassa with a conserved cis element in the promoter of the frequency gene. Interestingly, addition of pea powder significantly enhanced the amplitude of the clock-dependent reporter expression. Is this a specific effect of pea or do other plants or fruits serve the same function? This is especially important because this aspect is also highlighted in the Discussion.

At present, we have not done systematic experiments to evaluate other plant species because this would deviate our efforts in the lab. But indeed, this is a very exciting area to explore, and particularly towards identifying the molecular nature of the plant element(s) that seem(s) to enhance rhythms. Future plans entertain the idea of adding different extracts from plant roots, which is a more "natural" environment for T. atroviride. Since, as a group, we have been working longer with B. cinerea, we routinely use plant material to stimulate conidiation in the latter fungus, but we also do not know the component(s) that promote this enhanced conidiation in B. cinerea.

I find it very surprising, but interesting at the same time, that several tested and known clock-controlled genes apparently are not clock-controlled in T. atroviride. The authors should look at some regulators of the pathways they identified as being under clock control. The candidates could be further analyzed for the identified cis element in their promoters.

Indeed, we were surprised too by their lack of rhythmicity. It is possible that the promoter region used does not contain all the information which is discussed in the manuscript. In any case, a systematic and unbiased strategy (e.g., RNA-Seq) could help indicate, in the future, whether orthologs of known ccgs (as well as unexpected ones) are rhythmic in Trichoderma.

Next, they tested a function of the frq gene outside the clock function, because such an additional function had been described before in B. cinerea. They found that indeed conidiation is delayed in a frq-deletion strain, independent of ligh-dark cycles. To verify these results the deletion strain should be re-complemented with a wild-type copy.

We acknowledge the reviewer's suggestions. We have verified our results by performing complementation experiments: please, see new Figure 3—figure supplement 3 and 4, (Δtafrq complementation), pages 15, lines 9-15.

In the following chapters they focus on the mycoparasitic interaction and use frq and wc mutant strains of both fungi in confrontation assays. They first found that light inhibits the parasitic capacity of T. atroviride and that the WC-1 orthologue is required for the aggressive behavior of T. atroviride in the dark. Again in this series of experiments a T. atroviride frq re-complemented strain is missing. In light-dark cycles it turned out that the B. cinerea Frq protein is important for the outcome of the interaction. This shows that the interaction is clock-controlled, but on the prey side.

We appreciate the reviewer's suggestions. As the lack of frq in Trichoderma does not have a major impact in these assays, we mainly looked at the complemented strain in the context of the previous question (see above).

In the last chapter they analyzed the production of different secondary metabolites and found that the production is under light control. The WC orthologue and Frq are involved in the regulation, independent of their functions in the circadian clock. However, the authors also show that the production of some compounds is clock controlled, and most required Frq. I guess for specialists the source data tables are useful. I cannot evaluate them.

Indeed, the data provides nice evidence of rhythmic metabolites (which depend on TaFRQ). We look forward to unveiling the molecular events controlling their production as well as their identity and physiological role.

Author details

1. Marlene Henríquez-Urrutia

   1. ANID – Millennium Science Initiative Program - Millennium Institute for Integrative Biology (iBio), Santiago, Chile
   2. Pontificia Universidad Católica de Chile, Biological Sciences Faculty, Molecular Genetics and Microbiology Department, Santiago, Chile

   Contribution

   Conceptualization, Data curation, Validation, Investigation, Visualization, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

2. Rebecca Spanner

   1. ANID – Millennium Science Initiative Program - Millennium Institute for Integrative Biology (iBio), Santiago, Chile
   2. Pontificia Universidad Católica de Chile, Biological Sciences Faculty, Molecular Genetics and Microbiology Department, Santiago, Chile

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

3. Consuelo Olivares-Yánez

   1. ANID – Millennium Science Initiative Program - Millennium Institute for Integrative Biology (iBio), Santiago, Chile
   2. Centro de Biotecnología Vegetal, Facultad de Ciencias de la Vida, Universidad Andrés, Santiago, Chile

   Contribution

   Data curation, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. Aldo Seguel-Avello

   1. ANID – Millennium Science Initiative Program - Millennium Institute for Integrative Biology (iBio), Santiago, Chile
   2. Pontificia Universidad Católica de Chile, Biological Sciences Faculty, Molecular Genetics and Microbiology Department, Santiago, Chile

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

5. Rodrigo Pérez-Lara

   1. ANID – Millennium Science Initiative Program - Millennium Institute for Integrative Biology (iBio), Santiago, Chile
   2. Pontificia Universidad Católica de Chile, Biological Sciences Faculty, Molecular Genetics and Microbiology Department, Santiago, Chile

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

6. Hector Guillén-Alonso

   Department of Biotechnology and Biochemistry, Cinvestav Unidad Irapuato, Irapuato, Mexico

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

7. Robert Winkler

   Department of Biotechnology and Biochemistry, Cinvestav Unidad Irapuato, Irapuato, Mexico

   Contribution

   Conceptualization, Resources, Data curation, Supervision, Validation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

8. Alfredo Herrera-Estrella

   Laboratorio de expresión génica y desarrollo en hongos, Unidad de Genómica Avanzada-LANGEBIO, Irapuato, Mexico

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

9. Paulo Canessa

   1. ANID – Millennium Science Initiative Program - Millennium Institute for Integrative Biology (iBio), Santiago, Chile
   2. Centro de Biotecnología Vegetal, Facultad de Ciencias de la Vida, Universidad Andrés, Santiago, Chile

   Contribution

   Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Visualization, Writing - original draft, Writing – review and editing

   For correspondence

   paulo.canessa@unab.cl

   Competing interests

   No competing interests declared

10. Luis F Larrondo

    1. ANID – Millennium Science Initiative Program - Millennium Institute for Integrative Biology (iBio), Santiago, Chile
    2. Pontificia Universidad Católica de Chile, Biological Sciences Faculty, Molecular Genetics and Microbiology Department, Santiago, Chile

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing – review and editing

    For correspondence

    llarrondo@bio.puc.cl

    Competing interests

    Reviewing editor, eLife

    "This ORCID iD identifies the author of this article:" 0000-0002-8832-7109

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