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SummaryThe intracellular colonization of plant roots by the beneficial fungal endophyteSerendipita indicafollows a biphasic strategy. After an early biotrophic phase, the interaction transitions to a host cell death phase restricted to the epidermal and cortex layers of the root. Host cell death contributes to the successful accommodation of the fungus during the beneficial interaction inArabidopsis thaliana. How host cell death is initiated and controlled is largely unknown. Here we show that two fungal enzymes, the ecto-5’-nucleotidaseSiE5NT and the nucleaseSiNucA, act synergistically in the plant apoplast at the onset of cell death to produce deoxyadenosine (dAdo), a potent cell death inducer in animal systems. The uptake of extracellular dAdo, but not the structurally related adenosine (Ado), activates a previously undescribed cell death mechanism inA. thaliana. Mutation of the equilibrative nucleoside transporter ENT3 inA. thalianaresults in resistance to cell death triggered by extracellular dAdo and reduced fungal-mediated cell death during root colonization. A library screen ofA. thalianaT-DNA insertion lines identified a toll/interleukin-1 receptor nucleotide-binding leucine-rich repeat (TIR-NLR) protein as an additional intracellular component in dAdo-triggered cell death. Mutation of this previously uncharacterised TIR-NLR, which we have named ISI (induced byS.indica), affects host cell death, fungal colonization and growth promotion, suggesting a key role in the regulation of root cell death and plant-microbe interaction. Our data show that the combined activity of two fungal apoplastic enzymes leads to the production of a metabolite that, upon uptake, triggers TIR-NLR-modulated plant cell death, providing a link to immunometabolism in plants.Short summaryEfficient intraradical colonization by the beneficial fungal endophyteSerendipita indicarequires restricted host cell death. How this symbiotic host cell death is initiated and controlled is largely unknown. Here we show that two fungal enzymes, the ecto-5’-nucleotidaseSiE5NT and the nucleaseSiNucA, act synergistically in the apoplast at the onset of cell death to produce deoxyadenosine (dAdo), a potent cell death inducer in animal systems. Uptake of extracellular dAdo activates a previously undescribed cell death mechanism in plants. Mutation of theA. thalianaequilibrative nucleoside transporterENT3leads to resistance to cell death triggered by uptake of extracellular dAdo and to reduced fungal-mediated cell death during colonization. A library screen ofA. thalianaT-DNA insertion lines identified a TIR-NLR protein as an additional intracellular component in dAdo-triggered cell death, providing a link to immunometabolism in plants.In a nutshellRegulated host cell death is part of the plant defense strategy against pathogens, but it is also involved in the accommodation of certain beneficial microbes in the roots. We have identified extracellular metabolites and intracellular metabolic signals that contribute to colonization by beneficial root fungal endophytes and uncovered a conserved cell death mechanism likely co-opted for establishing plant-endophyte symbiosis.

Summary In contrast to the current paradigm of using microbial mono‐cultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed‐species consortia to perform functions that are difficult to programme into individual strains. In this work, we developed a synthetic microbial consortium composed of two genetically engineered microbes, a cyanobacterium (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). These microbial species specialize in the co‐culture: cyanobacteria fix CO2 through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P. putida, which has been engineered to consume sucrose and to degrade the environmental pollutant 2,4‐dinitrotoluene (2,4‐DNT). By encapsulating S. elongatus within a barium–alginate hydrogel, cyanobacterial cells were protected from the toxic effects of 2,4‐DNT, enhancing the performance of the co‐culture. The synthetic consortium was able to convert 2,4‐DNT with light and CO2 as key inputs, and its catalytic performance was stable over time. Furthermore, cycling this synthetic consortium through low nitrogen medium promoted the sucrose‐dependent accumulation of polyhydroxyalkanoate, an added‐value biopolymer, in the engineered P. putida strain. Altogether, the synthetic consortium displayed the capacity to remediate the industrial pollutant 2,4‐DNT while simultaneously synthesizing biopolymers using light and CO2 as the primary inputs., This work describes the development of a synthetic microbial consortium composed of a cyanobacterium engineered to use light to fix inorganic carbon, and a heterotrophic bacterium engineered to degrade the environmental pollutant, 2,4‐dinitrotoluene. Together, the synthetic consortium displayed the capacity to remediate the industrial compound 2,4‐DNT while simultaneously synthesizing biopolymers using light and carbon dioxide as the primary inputs.

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner insoluble cell wall layer and an outer soluble extracellular polysaccharide (EPS) matrix. Here, we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, and hence likely have different biological functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose branches and can scavenge reactive oxygen species. Exogenous application of β-GD leads to enhanced fungal colonization in barley, confirming its role in the fungal counter-defensive strategy to subvert host immunity. Our data highlight the hitherto undescribed capacity of this often-overlooked EPS matrix from plant-associated fungi to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant–microbe interface.

Design and implementation of synthetic biological circuits highly depends on well-characterized, robust promoters with predictable input-output responses. While great progress has been made with heterotrophic model organisms such as Escherichia coli, the available variety of tunable promoter parts for phototrophic cyanobacteria is still limited. Commonly used synthetic and semisynthetic promoters show weak dynamic ranges or no regulation at all in cyanobacterial models. Well-controlled alternatives such as native metal-responsive promoters, however, pose the problems of inducer toxicity and lacking orthogonality. Here, we present the comparative assessment of dose-response functions of four different inducible promoter systems in the model cyanobacterium Synechocystis sp. PCC 6803. Using the novel bimodular reporter plasmid pSHDY, dose-response dynamics of the re-established vanillate-inducible promoter PvanCC was compared to the previously described rhamnose-inducible Prha, the anhydrotetracycline-inducible PL03, and the Co2+-inducible PcoaT. We estimate individual advantages and disadvantages regarding dynamic range and strength of each promoter, also in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in cyanobacteria, facilitating the scalability and robustness of synthetic regulatory network designs and of complex metabolic pathway engineering strategies.

Research in the field of synthetic biology highly depends on efficient, well-characterized promoters. While great progress has been made with other model organisms such asEscherichia coli, photosynthetic cyanobacteria still lag behind. Commonly used promoters that have been tested in cyanobacteria show weaker dynamic range or no regulation at all. Alternatives such as native metal-inducible promoters pose the problem of inducer toxicity.Here, we evaluate four different inducible promoters, both previously published and new, using the modular plasmid pSHDY, in the model cyanobacteriumSynechocystis sp. PCC 6803 - namely the vanillate-inducible promoter PvanCC, the rhamnose-inducible Prha, and the aTc-inducible PL03, and the Co2+-inducible PcoaT. We estimate individual advantages and disadvantages, as well as dynamic range and strength of each promoter in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in order to facilitate the construction of more complex regulatory synthetic networks, as well as more complicated biotechnological pathways for cyanobacteria.

Inducible promoters are an important tool for synthetic biology. They enable temporal control of gene expression, as well as controlled expression of toxic genes. However, prior to working with an inducible promoter system, it is important to assess whether the inducer compount might positively or negatively affect growth or viability of the cell. This protocol describes the methodology for toxicity assays in the unicellular cyanobacterium Synechocystis sp. PCC 6803 to quantify its growth in the presence of different concentrations of inducer.

Inducible promoters are an important tool for synthetic biology. They enable temporal control of gene expression, as well as controlled expression of toxic genes. This protocol describes the methodology for fluorescence-based reporter assays in the unicellular cyanobacterium Synechocystis sp. PCC 6803 to quantify the dose-dependent response of an inducible promoter to its inducer.