responses to light have been described and,
therefore, a list of promoters can be drawn
from genes with strong induction and kinetic
responses. A recent review summarizes such
studies, highlighting the top 10 most lightinduced candidates in several fungi (Fuller
et al. 2018): while in many organisms vvd
homologs are part of the list, other genes such
as con-6 or grg-1 appear as interesting candidates that could drive strong expression upon
light in a WCC-dependent manner.
The industrial workhorse Trichoderma reesei also has been used as a biological platform
to control the expression of genes of interest by
an artificial optogenetic system following the
logic of the previously mentioned “LightOn”
approach (Wang et al. 2014). The switch
allowed to control the expression of different
reporter genes such as dsred2 and gus, obtaining positive signals after 36 h of light stimulation. Subsequently, the system was improved
by using a light-switchable bidirectional promoter version based on a “light-on” and
“light-off” divergent promoter (Zhang et al.
2016a, b). The optimization also included testing different transactivation domains (selecting
VP16) as well as different flexible linkers
between VVD and the latter. The bidirectional
expression system evaluated the expression of
an alkaline cellulase encoding gene and rfp,
achieving 70-fold of expression comparing
light versus dark conditions. Both the duration
of light stimuli and light intensities were important in dosing expression levels. Nevertheless,
at high irradiance (above 137 W) a negative
effect was observed, which could be related to
cellular stress and toxicity associated with an
excess of light. The authors also tried different
work cycles, providing light pulses of different
duration and frequency, concluding that continuous light is not necessary to get high levels
of expression. In order to obtain a light-off
promoter, a light-inducible promoter was used
along with a constitutive promoter with operators between the TATA-box and the gene of
interest, allowing the expression of two different reporters in a light/dark dependent manner,
respectively (Zhang et al. 2016a, b). Another
approach developed by this group is a onestep method that combines self-excisable
marker rescue and the control of the nonhomologous end joining (NHEJ) pathway in
such industrial eukaryotic microorganisms.
Using Agrobacterium tumefaciens-mediated
transformation and a Cre-LoxP system induced
by light, this team achieved simultaneous excision of a selectable marker gene and the cre
gene by Cre recombinase, in T. reesei, N. crassa
and Aspergillus niger (Zhang et al. 2016a, b).
In the context of harnessing potential applications of light in basidiomycetes, recent studies in Pleurotus ostreatus have shown that the
promoter of a lectin encoding gene could be
used in this organism to provide lightcontrolled gene expression. Indeed, by putting
egfp under its control, the authors observed an
increase of fluorescence signal in the presence
of light compared to dark, a result that was also
supported by measurement of gfp mRNA levels
(Yin et al. 2019). Interestingly, such an effect
occurred only in the presence of white light, but
not blue light, a result that is difficult to interpret based on the little information associated
with the promoter under study (Yin et al. 2019).
C. Expanding Optogenetic Toolbox by
Unexplored Fungal Photoreceptors
Curiously, only VVD/VVD and VVD/WC-1
interactions from N. crassa have been used to
implement fungal light-controlled systems so
far. In fact, WC-1 homodimerization via LOV
domains in response to blue light has not been
tested for optogenetic applications yet. This
only being an example, the kingdom mycota
could be considered an underexploited and
rather unexplored source of novel building
blocks for optogenetic systems. In fact, photoreceptors of different nature have been identified in many fungal species, including other
ascomycetes as well as basidiomycetes, zygomycetes, and chytrids (Herrera-Estrella and
Horwitz 2007; Corrochano 2007, 2019; Idnurm
et al. 2010; Fuller et al. 2016). Even in N. crassa
more is to be uncovered: for instance, this
fungus also expresses a cryptochrome (cry)
(Galagan et al. 2003) and an opsin (nop-1)
(Bieszke et al. 1999a, b), whose roles are still
rather obscure. Despite the fact that both pro310
V. Rojas et al.
therefore, a list of promoters can be drawn
from genes with strong induction and kinetic
responses. A recent review summarizes such
studies, highlighting the top 10 most lightinduced candidates in several fungi (Fuller
et al. 2018): while in many organisms vvd
homologs are part of the list, other genes such
as con-6 or grg-1 appear as interesting candidates that could drive strong expression upon
light in a WCC-dependent manner.
The industrial workhorse Trichoderma reesei also has been used as a biological platform
to control the expression of genes of interest by
an artificial optogenetic system following the
logic of the previously mentioned “LightOn”
approach (Wang et al. 2014). The switch
allowed to control the expression of different
reporter genes such as dsred2 and gus, obtaining positive signals after 36 h of light stimulation. Subsequently, the system was improved
by using a light-switchable bidirectional promoter version based on a “light-on” and
“light-off” divergent promoter (Zhang et al.
2016a, b). The optimization also included testing different transactivation domains (selecting
VP16) as well as different flexible linkers
between VVD and the latter. The bidirectional
expression system evaluated the expression of
an alkaline cellulase encoding gene and rfp,
achieving 70-fold of expression comparing
light versus dark conditions. Both the duration
of light stimuli and light intensities were important in dosing expression levels. Nevertheless,
at high irradiance (above 137 W) a negative
effect was observed, which could be related to
cellular stress and toxicity associated with an
excess of light. The authors also tried different
work cycles, providing light pulses of different
duration and frequency, concluding that continuous light is not necessary to get high levels
of expression. In order to obtain a light-off
promoter, a light-inducible promoter was used
along with a constitutive promoter with operators between the TATA-box and the gene of
interest, allowing the expression of two different reporters in a light/dark dependent manner,
respectively (Zhang et al. 2016a, b). Another
approach developed by this group is a onestep method that combines self-excisable
marker rescue and the control of the nonhomologous end joining (NHEJ) pathway in
such industrial eukaryotic microorganisms.
Using Agrobacterium tumefaciens-mediated
transformation and a Cre-LoxP system induced
by light, this team achieved simultaneous excision of a selectable marker gene and the cre
gene by Cre recombinase, in T. reesei, N. crassa
and Aspergillus niger (Zhang et al. 2016a, b).
In the context of harnessing potential applications of light in basidiomycetes, recent studies in Pleurotus ostreatus have shown that the
promoter of a lectin encoding gene could be
used in this organism to provide lightcontrolled gene expression. Indeed, by putting
egfp under its control, the authors observed an
increase of fluorescence signal in the presence
of light compared to dark, a result that was also
supported by measurement of gfp mRNA levels
(Yin et al. 2019). Interestingly, such an effect
occurred only in the presence of white light, but
not blue light, a result that is difficult to interpret based on the little information associated
with the promoter under study (Yin et al. 2019).
C. Expanding Optogenetic Toolbox by
Unexplored Fungal Photoreceptors
Curiously, only VVD/VVD and VVD/WC-1
interactions from N. crassa have been used to
implement fungal light-controlled systems so
far. In fact, WC-1 homodimerization via LOV
domains in response to blue light has not been
tested for optogenetic applications yet. This
only being an example, the kingdom mycota
could be considered an underexploited and
rather unexplored source of novel building
blocks for optogenetic systems. In fact, photoreceptors of different nature have been identified in many fungal species, including other
ascomycetes as well as basidiomycetes, zygomycetes, and chytrids (Herrera-Estrella and
Horwitz 2007; Corrochano 2007, 2019; Idnurm
et al. 2010; Fuller et al. 2016). Even in N. crassa
more is to be uncovered: for instance, this
fungus also expresses a cryptochrome (cry)
(Galagan et al. 2003) and an opsin (nop-1)
(Bieszke et al. 1999a, b), whose roles are still
rather obscure. Despite the fact that both pro310
V. Rojas et al.