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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.

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.