Optobiochemistry: Genetically Encoded Control of Protein Activity by Light.

Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light. Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques.

A New Facet of Vitamin B12: Gene Regulation by Cobalamin-Based Photoreceptors.

Living organisms sense and respond to light, a crucial environmental factor, using photoreceptors, which rely on bound chromophores such as retinal, flavins, or linear tetrapyrroles for light sensing. The discovery of photoreceptors that sense light using 5'-deoxyadenosylcobalamin, a form of vitamin B12 that is best known as an enzyme cofactor, has expanded the number of known photoreceptor families and unveiled a new biological role of this vitamin. The prototype of these B12-dependent photoreceptors, the transcriptional repressor CarH, is widespread in bacteria and mediates light-dependent gene regulation in a photoprotective cellular response. CarH activity as a transcription factor relies on the modulation of its oligomeric state by 5'-deoxyadenosylcobalamin and light. This review surveys current knowledge about these B12-dependent photoreceptors, their distribution and mode of action, and the structural and photochemical basis of how they orchestrate signal transduction and control gene expression.

Light in the Fungal World: From Photoreception to Gene Transcription and Beyond.

Fungi see light of different colors by using photoreceptors such as the White Collar proteins and cryptochromes for blue light, opsins for green light, and phytochromes for red light. Light regulates fungal development, promotes the accumulation of protective pigments and proteins, and regulates tropic growth. The White Collar complex (WCC) is a photoreceptor and a transcription factor that is responsible for regulating transcription after exposure to blue light. In Neurospora crassa, light promotes the interaction of WCCs and their binding to the promoters to activate transcription. In Aspergillus nidulans, the WCC and the phytochrome interact to coordinate gene transcription and other responses, but the contribution of these photoreceptors to fungal photobiology varies across fungal species. Ultimately, the effect of light on fungal biology is the result of the coordinated transcriptional regulation and activation of signal transduction pathways.

Few-fs resolution of a photoactive protein traversing a conical intersection.

The structural dynamics of a molecule are determined by the underlying potential energy landscape. Conical intersections are funnels connecting otherwise separate potential energy surfaces. Posited almost a century ago1, conical intersections remain the subject of intense scientific interest2-5. In biology, they have a pivotal role in vision, photosynthesis and DNA stability6. Accurate theoretical methods for examining conical intersections are at present limited to small molecules. Experimental investigations are challenged by the required time resolution and sensitivity. Current structure-dynamical understanding of conical intersections is thus limited to simple molecules with around ten atoms, on timescales of about 100 fs or longer7. Spectroscopy can achieve better time resolutions8, but provides indirect structural information. Here we present few-femtosecond, atomic-resolution videos of photoactive yellow protein, a 2,000-atom protein, passing through a conical intersection. These videos, extracted from experimental data by machine learning, reveal the dynamical trajectories of de-excitation via a conical intersection, yield the key parameters of the conical intersection controlling the de-excitation process and elucidate the topography of the electronic potential energy surfaces involved.

Darkness inhibits autokinase activity of bacterial bathy phytochromes.

Bathy phytochromes are a subclass of bacterial biliprotein photoreceptors that carry a biliverdin IXα chromophore. In contrast to prototypical phytochromes that adopt a red-light-absorbing Pr ground state, the far-red light-absorbing Pfr-form is the thermally stable ground state of bathy phytochromes. Although the photobiology of bacterial phytochromes has been extensively studied since their discovery in the late 1990s, our understanding of the signal transduction process to the connected transmitter domains, which are often histidine kinases, remains insufficient. Initiated by the analysis of the bathy phytochrome PaBphP from Pseudomonas aeruginosa, we performed a systematic analysis of five different bathy phytochromes with the aim to derive a general statement on the correlation of photostate and autokinase output. While all proteins adopt different Pr/Pfr-fractions in response to red, blue, and far-red light, only darkness leads to a pure or highly enriched Pfr-form, directly correlated with the lowest level of autokinase activity. Using this information, we developed a method to quantitatively correlate the autokinase activity of phytochrome samples with well-defined stationary Pr/Pfr-fractions. We demonstrate that the off-state of the phytochromes is the Pfr-form and that different Pr/Pfr-fractions enable the organisms to fine-tune their kinase output in response to a certain light environment. Furthermore, the output response is regulated by the rate of dark reversion, which differs significantly from 5 s to 50 min half-life. Overall, our study indicates that bathy phytochromes function as sensors of light and darkness, rather than red and far-red light, as originally postulated.

The LOV-domain blue-light receptor LreA of the fungus Alternaria alternata binds predominantly FAD as chromophore and acts as a light and temperature sensor.

Light and temperature sensing are important features of many organisms. Light may provide energy but may also be used by non-photosynthetic organisms for orientation in the environment. Recent evidence suggests that plant and fungal phytochrome and plant phototropin serve dual functions as light and temperature sensors. Here we characterized the fungal LOV-domain blue-light receptor LreA of Alternaria alternata and show that it predominantly contains FAD as chromophore. Blue-light illumination induced ROS production followed by protein agglomeration in vitro. In vivo ROS may control LreA activity. LreA acts as a blue-light photoreceptor but also triggers temperature-shift-induced gene expression. Both responses required the conserved amino acid cysteine 421. We therefore propose that temperature mimics the photoresponse, which could be the ancient function of the chromoprotein. Temperature-dependent gene expression control with LreA was distinct from the response with phytochrome suggesting fine-tuned, photoreceptor-specific gene regulation.

Photoreversible Aggregation of the Biliprotein Containing the First and Second GAF Domains of a Cyanobacteriochrome All2699 in Nostoc sp. PCC7120.

As plant photoreceptors, phytochromes are capable of detecting red light and far-red light, thereby governing plant growth. All2699 is a photoreceptor found in Nostoc sp. PCC7120 that specifically responds to red light and far-red light. All2699g1g2 is a truncated protein carrying the first and second GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains of All2699. In this study, we found that, upon exposure to red light, the protein underwent aggregation, resulting in the formation of protein aggregates. Conversely, under far-red light irradiation, these protein aggregates dissociated. We delved into the factors that impact the aggregation of All2699g1g2, focusing on the protein structure. Our findings showed that the GAF2 domain contains a low-complexity (LC) loop region, which plays a crucial role in mediating protein aggregation. Specifically, phenylalanine at position 239 within the LC loop region was identified as a key site for the aggregation process. Furthermore, our research revealed that various factors, including irradiation time, temperature, concentration, NaCl concentration, and pH value, can impact the aggregation of All2699g1g2. The aggregation led to variations in Pfr concentration depending on temperature, NaCl concentration, and pH value. In contrast, ΔLC did not aggregate and therefore lacked responses to these factors. Consequently, the LC loop region of All2699g1g2 extended and enhanced sensory properties.

A guide to the optogenetic regulation of endogenous molecules.

Genetically encoded tools for the regulation of endogenous molecules (RNA, DNA elements and protein) are needed to study and control biological processes with minimal interference caused by protein overexpression and overactivation of signaling pathways. Here we focus on light-controlled optogenetic tools (OTs) that allow spatiotemporally precise regulation of gene expression and protein function. To control endogenous molecules, OTs combine light-sensing modules from natural photoreceptors with specific protein or nucleic acid binders. We discuss OT designs and group OTs according to the principles of their regulation. We outline characteristics of OT performance, discuss considerations for their use in vivo and review available OTs and their applications in cells and in vivo. Finally, we provide a brief outlook on the development of OTs.

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE.

Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. It is suggested that the absorption change of this subfamily is caused by the bilin C15-Z/C15-E photoisomerization and a subsequent change in the bilin protonation state. However, structural information and direct evidence of the bilin protonation state are lacking. Here, we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a "bucket" consisting of hydrophobic residues, in which the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A- through C-rings coplanar and the D-ring tilted. Three pyrrole nitrogens of the A- through C-rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pKa, whereas they are directly hydrogen bonded in the ß-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the bucket, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the "leaky bucket" structure functions as a proton exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.

Revealing the origin of multiphasic dynamic behaviors in cyanobacteriochrome.

Cyanobacteriochromes are photoreceptors in cyanobacteria that exhibit a wide spectral coverage and unique photophysical properties from the photoinduced isomerization of a linear tetrapyrrole chromophore. Here, we integrate femtosecond-resolved fluorescence and transient-absorption methods and unambiguously showed the significant solvation dynamics occurring at the active site from a few to hundreds of picoseconds. These motions of local water molecules and polar side chains are continuously convoluted with the isomerization reaction, leading to a nonequilibrium processes with continuous active-site motions. By mutations of critical residues at the active site, the modified local structures become looser, resulting in faster solvation relaxations and isomerization reaction. The observation of solvation dynamics is significant and critical to the correct interpretation of often-observed multiphasic dynamic behaviors, and thus the previously invoked ground-state heterogeneity may not be relevant to the excited-state isomerization reaction.

Formation of an unusual glutamine tautomer in a blue light using flavin photocycle characterizes the light-adapted state.

Blue light using flavin (BLUF) photoreceptor proteins are critical for many light-activated biological processes and are promising candidates for optogenetics because of their modular nature and long-range signaling capabilities. Although the photocycle of the Slr1694 BLUF domain has been characterized experimentally, the identity of the light-adapted state following photoexcitation of the bound flavin remains elusive. Herein hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations of this photocycle provide a nonequilibrium dynamical picture of a possible mechanism for the formation of the light-adapted state. Photoexcitation of the flavin induces a forward proton-coupled electron transfer (PCET) process that leads to the formation of an imidic acid tautomer of Gln50. The calculations herein show that the subsequent rotation of Gln50 allows a reverse PCET process that retains this tautomeric form. In the resulting purported light-adapted state, the glutamine tautomer forms a hydrogen bond with the flavin carbonyl group. Additional ensemble-averaged QM/MM calculations of the dark-adapted and purported light-adapted states demonstrate that the light-adapted state with the imidic acid glutamine tautomer reproduces the experimentally observed spectroscopic signatures. Specifically, the calculations reproduce the red shifts in the flavin electronic absorption and carbonyl stretch infrared spectra in the light-adapted state. Further hydrogen-bonding analyses suggest the formation of hydrogen-bonding interactions between the flavin and Arg65 in the light-adapted state, providing a plausible explanation for the experimental observation of faster photoinduced PCET in this state. These characteristics of the light-adapted state may also be essential for the long-range signaling capabilities of this photoreceptor protein.

The interplay between chromophore and protein determines the extended excited state dynamics in a single-domain phytochrome.

Phytochromes are a diverse family of bilin-binding photoreceptors that regulate a wide range of physiological processes. Their photochemical properties make them attractive for applications in optogenetics and superresolution microscopy. Phytochromes undergo reversible photoconversion triggered by the Z ⇄ E photoisomerization about the double bond in the bilin chromophore. However, it is not fully understood at the molecular level how the protein framework facilitates the complex photoisomerization dynamics. We have studied a single-domain bilin-binding photoreceptor All2699g1 (Nostoc sp. PCC 7120) that exhibits photoconversion between the red light-absorbing (Pr) and far red-absorbing (Pfr) states just like canonical phytochromes. We present the crystal structure and examine the photoisomerization mechanism of the Pr form as well as the formation of the primary photoproduct Lumi-R using time-resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations. We show that the unusually long excited state lifetime (broad lifetime distribution centered at ∼300 picoseconds) is due to the interactions between the isomerizing pyrrole ring D and an adjacent conserved Tyr142. The decay kinetics shows a strongly distributed character which is imposed by the nonexponential protein dynamics. Our findings offer a mechanistic insight into how the quantum efficiency of the bilin photoisomerization is tuned by the protein environment, thereby providing a structural framework for engineering bilin-based optical agents for imaging and optogenetics applications.

Evolution-inspired design of multicolored photoswitches from a single cyanobacteriochrome scaffold.

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1_1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1_1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.

Structural elements regulating the photochromicity in a cyanobacteriochrome.

The three-dimensional (3D) crystal structures of the GAF3 domain of cyanobacteriochrome Slr1393 (Synechocystis PCC6803) carrying a phycocyanobilin chromophore could be solved in both 15-Z dark-adapted state, Pr, λmax = 649 nm, and 15-E photoproduct, Pg, λmax = 536 nm (resolution, 1.6 and 1.86 Å, respectively). The structural data allowed identifying the large spectral shift of the Pr-to-Pg conversion as resulting from an out-of-plane rotation of the chromophore's peripheral rings and an outward movement of a short helix formed from a formerly unstructured loop. In addition, a third structure (2.1-Å resolution) starting from the photoproduct crystals allowed identification of elements that regulate the absorption maxima. In this peculiar form, generated during X-ray exposition, protein and chromophore conformation still resemble the photoproduct state, except for the D-ring already in 15-Z configuration and tilted out of plane akin the dark state. Due to its formation from the photoproduct, it might be considered an early conformational change initiating the parental state-recovering photocycle. The high quality and the distinct features of the three forms allowed for applying quantum-chemical calculations in the framework of multiscale modeling to rationalize the absorption maxima changes. A systematic analysis of the PCB chromophore in the presence and absence of the protein environment showed that the direct electrostatic effect is negligible on the spectral tuning. However, the protein forces the outer pyrrole rings of the chromophore to deviate from coplanarity, which is identified as the dominating factor for the color regulation.

Photoactive Yellow Protein Represents a Distinct, Evolutionarily Novel Family of PAS Domains.

Photoactive yellow protein (PYP) is a model photoreceptor. It binds a p-coumaric acid as a chromophore, thus enabling blue light sensing. The first discovered single-domain PYP from Halorhodospira halophila has been studied thoroughly in terms of its structural dynamics and photochemical properties. However, the evolutionary origins and biological role of PYP homologs are not well understood. Here, we show that PYP is an evolutionarily novel domain family of the ubiquitous PAS (Per-Arnt-Sim) superfamily. It likely originated from the phylum Myxococcota and was then horizontally transferred to representatives of a few other bacterial phyla. We show that PYP is associated with signal transduction either by domain fusion or by genome context. Key cellular functions modulated by PYP-initiated signal transduction pathways likely involve gene expression, motility, and biofilm formation. We identified three clades of the PYP family, one of which is poorly understood and potentially has novel functional properties. The Tyr42, Glu46, and Cys69 residues that are involved in p-coumaric acid binding in the model PYP from H. halophila are well conserved in the PYP family. However, we also identified cases where substitutions in these residues might have led to neofunctionalization, such as the proposed transition from light to redox sensing. Overall, this study provides definition, a newly built hidden Markov model, and the current genomic landscape of the PYP family and sets the stage for the future exploration of its signaling mechanisms and functional diversity. IMPORTANCE Photoactive yellow protein is a model bacterial photoreceptor. For many years, it was considered a prototypical model of the ubiquitous PAS domain superfamily. Here, we show that, in fact, the PYP family is evolutionarily novel, restricted to a few bacterial phyla and distinct from other PAS domains. We also reveal the diversity of PYP-containing signal transduction proteins and their potential mechanisms.

Photosensing and quorum sensing are integrated to control Pseudomonas aeruginosa collective behaviors.

Bacteria convert changes in sensory inputs into alterations in gene expression, behavior, and lifestyles. A common lifestyle choice that bacteria make is whether to exhibit individual behavior and exist in the free-living planktonic state or to engage in collective behavior and form sessile communities called biofilms. Transitions between individual and collective behaviors are controlled by the chemical cell-to-cell communication process called quorum sensing. Here, we show that quorum sensing represses Pseudomonas aeruginosa biofilm formation and virulence by activating expression of genes encoding the KinB-AlgB two-component system (TCS). Phospho-AlgB represses biofilm and virulence genes, while KinB dephosphorylates and thereby inactivates AlgB. We discover that the photoreceptor BphP is the kinase that, in response to light, phosphorylates and activates AlgB. Indeed, exposing P. aeruginosa to light represses biofilm formation and virulence gene expression. To our knowledge, P. aeruginosa was not previously known to detect and respond to light. The KinB-AlgB-BphP module is present in all pseudomonads, and we demonstrate that AlgB is the partner response regulator for BphP in diverse bacterial phyla. We propose that in the KinB-AlgB-BphP system, AlgB functions as the node at which varied sensory information is integrated. This network architecture provides a mechanism enabling bacteria to integrate at least two different sensory inputs, quorum sensing (via RhlR-driven activation of algB) and light (via BphP-AlgB), into the control of collective behaviors. This study sets the stage for light-mediated control of P. aeruginosa infectivity.

Light-induced production of isobutanol and 3-methyl-1-butanol by metabolically engineered cyanobacteria.

BACKGROUND: Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803). RESULTS: To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO2) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L-1 and 75 mg L-1, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers. CONCLUSION: A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO2 is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses.

Unusual ring D fixation by three crucial residues promotes phycoviolobilin formation in the DXCF-type cyanobacteriochrome without the second Cys.

Cyanobacteriochromes are linear tetrapyrrole-binding photoreceptors produced by cyanobacteria. Their chromophore-binding GAF domains are categorized into many lineages. Among them, dual Cys-type cyanobacteriochrome GAF domains possessing not only a highly conserved 'first Cys' but also a 'second Cys' are found from multiple lineages. The first Cys stably attaches to C31 of the A-ring, while the second Cys mostly shows reversible ligation to the C10 of the chromophore. Notably, the position of the second Cys in the primary sequence is diversified, and the most abundant dual Cys-type GAF domains have a 'second Cys' within the DXCF motif, which are called DXCF GAF domains. It has been long known that the second Cys in the DXCF GAF domains not only shows the reversible ligation but also is involved in isomerization activity (reduction in C4=C5 double bond) from the initially incorporated phycocyanobilin to phycoviolobilin. However, comprehensive site-directed mutagenesis on the DXCF GAF domains, AM1_6305g1 and AM1_1499g1, revealed that the second Cys is dispensable for isomerization activity, in which three residues participate by fixing the C- and D-rings. Fixation of the chromophore on both sides of the C5 bridge is necessary, even though one side of the fixation site is far from this bridge, with the other side at C31 fixed by the first Cys.

Rational conversion of chromophore selectivity of cyanobacteriochromes to accept mammalian intrinsic biliverdin.

Because cyanobacteriochrome photoreceptors need only a single compact domain for chromophore incorporation and for absorption of visible spectra including the long-wavelength far-red region, these molecules have been paid much attention for application to bioimaging and optogenetics. Most cyanobacteriochromes, however, have a drawback to incorporate phycocyanobilin that is not available in the mammalian cells. In this study, we focused on biliverdin (BV) that is a mammalian intrinsic chromophore and absorbs the far-red region and revealed that replacement of only four residues was enough for conversion from BV-rejective cyanobacteriochromes into BV-acceptable molecules. We succeeded in determining the crystal structure of one of such engineered molecules, AnPixJg2_BV4, at 1.6 Å resolution. This structure identified unusual covalent bond linkage, which resulted in deep BV insertion into the protein pocket. The four mutated residues contributed to reducing steric hindrances derived from the deeper insertion. We introduced these residues into other domains, and one of them, NpF2164g5_BV4, produced bright near-infrared fluorescence from mammalian liver in vivo. Collectively, this study provides not only molecular basis to incorporate BV by the cyanobacteriochromes but also rational strategy to open the door for application of cyanobacteriochromes to visualization and regulation of deep mammalian tissues.

A light life together: photosensing in the plant microbiota.

Bacteria and fungi of the plant microbiota can be phytopathogens, parasites or symbionts that establish mutually advantageous relationships with plants. They are often rich in photoreceptors for UVA-Visible light, and in many cases, they exhibit light regulation of growth patterns, infectivity or virulence, reproductive traits, and production of pigments and of metabolites. In addition to the light-driven effects, often demonstrated via the generation of photoreceptor gene knock-outs, microbial photoreceptors can exert effects also in the dark. Interestingly, some fungi switch their attitude towards plants in dependence of illumination or dark conditions in as much as they may be symbiotic or pathogenic. This review summarizes the current knowledge about the roles of light and photoreceptors in plant-associated bacteria and fungi aiming at the identification of common traits and general working ideas. Still, reports on light-driven infection of plants are often restricted to the description of macroscopically observable phenomena, whereas detailed information on the molecular level, e.g., protein-protein interaction during signal transduction or induction mechanisms of infectivity/virulence initiation remains sparse. As it becomes apparent from still only few molecular studies, photoreceptors, often from the red- and the blue light sensitive groups interact and mutually modulate their individual effects. The topic is of great relevance, even in economic terms, referring to plant-pathogen or plant-symbionts interactions, considering the increasing usage of artificial illumination in greenhouses, the possible light-regulation of the synthesis of plant-growth stimulating substances or herbicides by certain symbionts, and the biocontrol of pests by selected fungi and bacteria in a sustainable agriculture.