chimeric functional transcription factor is
reconstituted by the red light-dependent
PhyB/PIF3 interaction (Fig. 12.2a, b), inducing
the expression of a lacZ reporter gene under the
gal1 promoter control (Shimizu-Sato et al.
2002). Similarly, optogenetic systems based on
PhyB/PIF6 interaction have been implemented
in different cell hosts such as yeast (Pathak et al.
2014), cell lines (Mu ¨ller et al. 2013a, b) and
plants (Mu ¨ller et al. 2014). On the other hand,
there are more phytochrome isoforms in A.
thaliana which are potentially useful for building novel optogenetic switches. For instance,
the interaction between phytochrome A
(PhyA) and PIF3 was evaluated in response to
red light (Fig. 12.2a, b), observing high and
comparable levels of lacZ gene expression as
in the PhyB/PIF3 system (Shimizu-Sato et al.
2002). Similarly, PhyA interacts with the far-red
elongated hypocotyl 1 (FHY1) protein in
response to red light (Fig. 12.2a), an interaction
that has been also exploited for implementing
an optogenetic system in yeast (Sorokina et al.
2009). The rest of A. thaliana phytochromes
(PhyC, PhyD, and PhyE) have not been utilized
to generate synthetic light-inducible devices
yet. The red light-responsive phytochromes
can also be found in cyanobacteria, where
the cofactor chromophore corresponds to
phycocyanobilin (PCB). Interestingly, the latter
can replace phytochromobilin in optogenetic
approaches that use plant phytochromes
(Mu ¨ller et al. 2013a, b). However, both chromophores must be supplemented in mammals
because they are absent in these eukaryotic
organisms (Mu ¨ller et al. 2013a, b). Similarly,
an alternative option to implement synthetic
optogenetic systems is the use of bacterial
phytochrome-like photoreceptors. For instance,
CPH1 from Synechocystis sp. is capable of sensing red light, but unlike plant phytochromes, it
forms homodimers in response to the stimulus
(Hughes et al. 1997).
B. Blue Light Photosensitive Modules in
Optogenetic Devices
Besides ChR2, there is a battery of natural flavoproteins that can perceive blue light and use
flavin derivatives as a chromophore. In that
context, the blue light photoreceptor cryptochrome 2 (CRY2) from A. thaliana provides a
versatile optogenetic tool that has been used/
cited in circa 160 articles to date according to
OptoBase, an online database for molecular
optogenetics (Kolar et al. 2018). The CRY2 protein senses blue light by its Photolyase Homology Region (PHR), which specifically binds
flavin adenine dinucleotide (FAD) as a cofactor
(Essen 2006). Upon illumination, CRY2 is capable of interacting with the amino-terminus of
the cryptochrome-interacting basic-helix-loophelix 1 (CIB1) protein (Fig. 12.2a, b) (Liu et al.
2008). Several groups have developed optogenetic approaches based on CRY2/CIB1 interaction to control diverse cellular processes. For
instance, an optogenetic system of this type was
implemented in mammalian cell lines to
manipulate gene expression, subcellular protein localization, and DNA recombination
(Kennedy et al. 2010). First, the authors based
their approach on light-induced reconstitution
of a chimeric GAL4 transcription factor by
genetic fusions, using the lacZ reporter gene
controlled by the gal1 promoter to measure
expression levels in response to blue light.
Additionally, truncated versions of both proteins (CRY2-PHR domain and CIB1 Nterminus) were evaluated, observing higher
expression levels than those using the fulllength proteins. Then, the recruitment of a fluorescent protein to the plasma membrane was
tested by this photoactivated interaction
(Fig. 12.2c). In order to do this, a chimeric
CIB1-GFP was attached to this specific cell location using a CAAX motif. Conversely, a CRY2mCherry fusion was uniformly found in the
cytoplasm before illumination, but upon a
blue light treatment, mcherry fluorescence was
observed at the plasma membrane level in a
dose-dependent manner (Kennedy et al. 2010).
Moreover, the optogenetic heterodimerization
CYR2/CIB1 system successfully reconstituted
the functionality of a split protein such as the
Cre recombinase (Kennedy et al. 2010). The
Cre/loxP recombination system, derived from
bacteriophage P1, can perform diverse changes
at the genome level (i.e., deletion, inversion and
transplacement between strands) depending on
12 Exploiting Fungal Photobiology as a Source of Novel Bio-blocks for Optogenetic Systems
301
reconstituted by the red light-dependent
PhyB/PIF3 interaction (Fig. 12.2a, b), inducing
the expression of a lacZ reporter gene under the
gal1 promoter control (Shimizu-Sato et al.
2002). Similarly, optogenetic systems based on
PhyB/PIF6 interaction have been implemented
in different cell hosts such as yeast (Pathak et al.
2014), cell lines (Mu ¨ller et al. 2013a, b) and
plants (Mu ¨ller et al. 2014). On the other hand,
there are more phytochrome isoforms in A.
thaliana which are potentially useful for building novel optogenetic switches. For instance,
the interaction between phytochrome A
(PhyA) and PIF3 was evaluated in response to
red light (Fig. 12.2a, b), observing high and
comparable levels of lacZ gene expression as
in the PhyB/PIF3 system (Shimizu-Sato et al.
2002). Similarly, PhyA interacts with the far-red
elongated hypocotyl 1 (FHY1) protein in
response to red light (Fig. 12.2a), an interaction
that has been also exploited for implementing
an optogenetic system in yeast (Sorokina et al.
2009). The rest of A. thaliana phytochromes
(PhyC, PhyD, and PhyE) have not been utilized
to generate synthetic light-inducible devices
yet. The red light-responsive phytochromes
can also be found in cyanobacteria, where
the cofactor chromophore corresponds to
phycocyanobilin (PCB). Interestingly, the latter
can replace phytochromobilin in optogenetic
approaches that use plant phytochromes
(Mu ¨ller et al. 2013a, b). However, both chromophores must be supplemented in mammals
because they are absent in these eukaryotic
organisms (Mu ¨ller et al. 2013a, b). Similarly,
an alternative option to implement synthetic
optogenetic systems is the use of bacterial
phytochrome-like photoreceptors. For instance,
CPH1 from Synechocystis sp. is capable of sensing red light, but unlike plant phytochromes, it
forms homodimers in response to the stimulus
(Hughes et al. 1997).
B. Blue Light Photosensitive Modules in
Optogenetic Devices
Besides ChR2, there is a battery of natural flavoproteins that can perceive blue light and use
flavin derivatives as a chromophore. In that
context, the blue light photoreceptor cryptochrome 2 (CRY2) from A. thaliana provides a
versatile optogenetic tool that has been used/
cited in circa 160 articles to date according to
OptoBase, an online database for molecular
optogenetics (Kolar et al. 2018). The CRY2 protein senses blue light by its Photolyase Homology Region (PHR), which specifically binds
flavin adenine dinucleotide (FAD) as a cofactor
(Essen 2006). Upon illumination, CRY2 is capable of interacting with the amino-terminus of
the cryptochrome-interacting basic-helix-loophelix 1 (CIB1) protein (Fig. 12.2a, b) (Liu et al.
2008). Several groups have developed optogenetic approaches based on CRY2/CIB1 interaction to control diverse cellular processes. For
instance, an optogenetic system of this type was
implemented in mammalian cell lines to
manipulate gene expression, subcellular protein localization, and DNA recombination
(Kennedy et al. 2010). First, the authors based
their approach on light-induced reconstitution
of a chimeric GAL4 transcription factor by
genetic fusions, using the lacZ reporter gene
controlled by the gal1 promoter to measure
expression levels in response to blue light.
Additionally, truncated versions of both proteins (CRY2-PHR domain and CIB1 Nterminus) were evaluated, observing higher
expression levels than those using the fulllength proteins. Then, the recruitment of a fluorescent protein to the plasma membrane was
tested by this photoactivated interaction
(Fig. 12.2c). In order to do this, a chimeric
CIB1-GFP was attached to this specific cell location using a CAAX motif. Conversely, a CRY2mCherry fusion was uniformly found in the
cytoplasm before illumination, but upon a
blue light treatment, mcherry fluorescence was
observed at the plasma membrane level in a
dose-dependent manner (Kennedy et al. 2010).
Moreover, the optogenetic heterodimerization
CYR2/CIB1 system successfully reconstituted
the functionality of a split protein such as the
Cre recombinase (Kennedy et al. 2010). The
Cre/loxP recombination system, derived from
bacteriophage P1, can perform diverse changes
at the genome level (i.e., deletion, inversion and
transplacement between strands) depending on
12 Exploiting Fungal Photobiology as a Source of Novel Bio-blocks for Optogenetic Systems
301