using white light, which covers a wide spectrum
of wavelengths. Furthermore, a second challenge in optogenetics is to expand the repertory of available photoreceptors, describing
novel bio-blocks for these synthetic systems,
and where fungal photobiology may play an
important role as a source of new opto-modules.
III. Exploiting Fungal Photobiology as
a Source of Novel Bio-blocks for
Optogenetic Systems
In the former section, we have focused our
attention on optogenetic systems that use
light-responsive domains belonging to bacteria,
archaea, plants, and animals. However, what
about the fungal kingdom? As mentioned
earlier, light is a strong signal that provides
important environmental information affecting several cellular processes. In fungi, these
processes include developmental decisions, utilization of complex substrates, pigmentation,
phototropism, and virulence (Schumacher
2017; Fuller et al. 2018; Schmoll 2018; Corrochano 2019). Specifically, the majority of the
studies involving light responses in fungi have
been performed in filamentous species, with
some of them having a plethora of photoreceptors that contain light-sensing modules, including opsins, phytochromes, cryptochromes,
and LOV-proteins (Corrochano 2007, 2019;
Idnurm et al. 2010). Interestingly, however,
genome analyses indicate a complete lack of
photoreceptor-encoding genes in the unicellular yeast S. cerevisiae, which has therefore
already been used as a platform to test multiple
optogenetic systems, since light can be considered an orthogonal stimulus in this organism
(Goffeau et al. 1996).
A. Utilizing Light-Responsive Domains from
Fungal Species
There are only few examples of light-inducible
genetic devices using fungal photoreceptors,
which could be due, in part, to the fact that
most of them have not been fully characterized
yet. In that context, studies carried out in the
filamentous fungus Neurospora crassa have
established clear examples of light-controlled
mechanisms in fungi. This has led to the translation of information about N. crassa photobiology into applied optogenetic systems. For
instance, Wang et al. (2012) reported an optogenetic system using the VIVID protein (VVD)
from N. crassa. This protein possesses only 186
amino acids, being the smallest LOV-domain
containing photoreceptor (Schwerdtfeger and
Linden 2003). The authors took advantage of
VVD homodimerization in response to blue
light to develop a synthetic gene expression
system in mammalian cells (Fig. 12.2a). They
fused VVD to the p65-AD and the GAL4 DBD,
the latter without its dimerization sequence,
generating a chimeric transcription factor
called GAVP. Thus, the blue light-dependent
interaction VVD/VVD by their LOV domains
was the unique possibility to form the functional dimer with the ability to turn on the
reporter gene expression (Wang et al. 2012).
To probe this synthetic design, the authors
transfected GAVP into mammalian cell lines
containing the luciferase reporter gene (luc)
under the control of the gal1 promoter.
Although luc expression was high in blue light
conditions, the background activity in darkness
had to be reduced by point mutations in VVD.
This optimized version was named GAVPO,
and it was used to implement the onecomponent “LightOn” optogenetic system
(Wang et al. 2012). The latter showed fast
dose-dependent responses depending on the
number of light pulses, length of the pulse,
and light irradiance. Furthermore, the spatial
resolution of the “LightOn” system was validated in mice, where liver and kidney were
specifically irradiated by blue light and expression of mcherry and lacZ reporters confirmed
the feasibility to control biological processes
with accuracy in a complex free-moving animal
(Wang et al. 2012). Importantly, decreased
blood glucose levels are observed in diabetic
mice by light-induced production of insulin
(Wang et al. 2012). Subsequently, various parameters of the “LightOn” system were modified
to optimize the light/dark fold-induction. Over306
V. Rojas et al.
of wavelengths. Furthermore, a second challenge in optogenetics is to expand the repertory of available photoreceptors, describing
novel bio-blocks for these synthetic systems,
and where fungal photobiology may play an
important role as a source of new opto-modules.
III. Exploiting Fungal Photobiology as
a Source of Novel Bio-blocks for
Optogenetic Systems
In the former section, we have focused our
attention on optogenetic systems that use
light-responsive domains belonging to bacteria,
archaea, plants, and animals. However, what
about the fungal kingdom? As mentioned
earlier, light is a strong signal that provides
important environmental information affecting several cellular processes. In fungi, these
processes include developmental decisions, utilization of complex substrates, pigmentation,
phototropism, and virulence (Schumacher
2017; Fuller et al. 2018; Schmoll 2018; Corrochano 2019). Specifically, the majority of the
studies involving light responses in fungi have
been performed in filamentous species, with
some of them having a plethora of photoreceptors that contain light-sensing modules, including opsins, phytochromes, cryptochromes,
and LOV-proteins (Corrochano 2007, 2019;
Idnurm et al. 2010). Interestingly, however,
genome analyses indicate a complete lack of
photoreceptor-encoding genes in the unicellular yeast S. cerevisiae, which has therefore
already been used as a platform to test multiple
optogenetic systems, since light can be considered an orthogonal stimulus in this organism
(Goffeau et al. 1996).
A. Utilizing Light-Responsive Domains from
Fungal Species
There are only few examples of light-inducible
genetic devices using fungal photoreceptors,
which could be due, in part, to the fact that
most of them have not been fully characterized
yet. In that context, studies carried out in the
filamentous fungus Neurospora crassa have
established clear examples of light-controlled
mechanisms in fungi. This has led to the translation of information about N. crassa photobiology into applied optogenetic systems. For
instance, Wang et al. (2012) reported an optogenetic system using the VIVID protein (VVD)
from N. crassa. This protein possesses only 186
amino acids, being the smallest LOV-domain
containing photoreceptor (Schwerdtfeger and
Linden 2003). The authors took advantage of
VVD homodimerization in response to blue
light to develop a synthetic gene expression
system in mammalian cells (Fig. 12.2a). They
fused VVD to the p65-AD and the GAL4 DBD,
the latter without its dimerization sequence,
generating a chimeric transcription factor
called GAVP. Thus, the blue light-dependent
interaction VVD/VVD by their LOV domains
was the unique possibility to form the functional dimer with the ability to turn on the
reporter gene expression (Wang et al. 2012).
To probe this synthetic design, the authors
transfected GAVP into mammalian cell lines
containing the luciferase reporter gene (luc)
under the control of the gal1 promoter.
Although luc expression was high in blue light
conditions, the background activity in darkness
had to be reduced by point mutations in VVD.
This optimized version was named GAVPO,
and it was used to implement the onecomponent “LightOn” optogenetic system
(Wang et al. 2012). The latter showed fast
dose-dependent responses depending on the
number of light pulses, length of the pulse,
and light irradiance. Furthermore, the spatial
resolution of the “LightOn” system was validated in mice, where liver and kidney were
specifically irradiated by blue light and expression of mcherry and lacZ reporters confirmed
the feasibility to control biological processes
with accuracy in a complex free-moving animal
(Wang et al. 2012). Importantly, decreased
blood glucose levels are observed in diabetic
mice by light-induced production of insulin
(Wang et al. 2012). Subsequently, various parameters of the “LightOn” system were modified
to optimize the light/dark fold-induction. Over306
V. Rojas et al.