The fungal root endophyte Serendipita indica modifies extracellular nucleotides to subvert plant immunity

One sentence abstract Immune modulation by metabolites in plant fungus interaction Abstract Extracellular adenosine 5′-triphosphate (eATP) is an essential signaling molecule that mediates different cellular processes through its interaction with membrane-associated receptor proteins in animals and plants. eATP regulates plant growth, development and responses to biotic and abiotic stresses. Its accumulation in the apoplast induces ROS production and cytoplasmic calcium increase mediating a defense response to invading microbes. We demonstrate that perception of eATP is important in plant-fungus interaction and that during colonization by the beneficial root endophyte Serendipita indica accumulation of eATP in the apoplast occurs at early symbiotic stages. We show by liquid chromatography-tandem mass spectrometry, cytological and functional analysis that S. indica subvert eATP host response by secreting SiE5’NT, an enzymatically active ecto-5′nucleotidase capable of hydrolyzing eATP to adenosine. A. thaliana lines producing extracellular SiE5’NT are signi?cantly better colonized and have reduced eATP levels and defense signaling, indicating that SiE5’NT functions as a compatibility factor. Our data show that extracellular bioactive nucleotides play an important role in fungus-root interactions and that fungi can modify plant derived metabolites in the apoplast to modulate host immunity.


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ATP is a coenzyme that serves as an universal energy currency and as building   (Table S1 and Fig. 1A, B). 119 Predictions using ApoplastP (http://apoplastp.csiro.au/) and SecretomeP 120 (http://www.cbs.dtu.dk/services/SecretomeP-1.0/) indicated that 48 of the 102 S. 121 indica proteins are putatively targeted to the plant apoplast (47%) with 9 proteins 122 predicted to be secreted via a non-canonical secretion pathway using a cutoff of 0.6 123 (Table S2). No peptides for GFP were found in any of the apoplastic fluid samples, 124 confirming the GFP western blotting analysis. Twenty proteins were unique at 5 dpi, 125 4 at 10 dpi and 21 at 14 dpi, suggesting differential secretion of S. indica proteins at 126 different stages of colonization (Table S1). 127 Twenty-one of the 102 identified apoplastic proteins were also found in the culture 128 filtrate of S. indica grown in CM, which included a large proportion of enzymes acting 129 on peptide bonds (Fig. 1A, B). The majority of these proteases might conceivably  (Table S1). 137 Remarkably, 19 of the 33 common proteins found in the apoplastic fluid at all 3 time 138 points where not present in the CF samples, implying an induced secretion for these 139 proteins during interaction with the plant roots (Table S1,   were related to purine metabolism, and specifically to ATP metabolic processes 154 (Table S3, Fig. S1B and S2).

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Among the fungal proteins specifically present in the apoplastic fluid of colonized 156 roots, several ATP-scavenging enzymes, including a 5'-nucleotidase (PIIN_01005) 157 and a nucleoside diphosphate kinase (PIIN_00784) were identified (Table S1 and   158 Fig. S1B, C). Twenty one orthologues of the S. indica apoplastic proteins were also 159 found in the apoplastic fluid of rice leaves infected by the rice-blast pathogen 160 Magnaporthe oryzae (Table S4 and  The enrichment for GO terms related to ATP metabolic processes among the fungal-167 derived apoplastic proteins led us to test whether root colonization by S. indica 168 triggers changes in eATP levels at different symbiotic stages and in different plant 169 species. Therefore, a specific luminescent ATP detection assay was used to assess   After confirming the inability of the dorn1-3 mutant to respond to eATP and eADP 184 with cytoplasmic calcium influx (Fig. S3), colonization of this line along with the 185 appropriate Col-0 control (expressing the calcium reporter protein aequorin, Col-0 aq ) 186 was assessed by qPCR at different time points (3, 5, 7, and 10 dpi). We observed 187 that the dorn1-3 mutant supports significant higher fungal colonization in comparison 188 to Col-0 aq control, especially at early symbiotic stages ( Fig. 2C and 4E). Importantly, 189 these observations strongly indicate that eATP accumulates in the apoplast and its 190 detection by the host affects fungal root colonization.

S. indica PIIN_01005 is an ecto-5'-nucleotidase with broad substrate activity
192 against ATP, ADP and AMP 193 Since our data show that detection of eATP plays a role during root-fungus 194 interaction we hypnotized that S. indica uses some intrinsic mechanism to 195 manipulate eATP levels to counteract the ATP-mediated host immune responses.        fungal responsive genes (Fig. 4). In conclusion our work demonstrate that eATP 406 perception plays an important role in plant-fungal interaction in the roots and that S. 407 indica has an intrinsic mechanism to counteract bioactive extracellular nucleotides- under light shaking in 70% ethanol followed by 1 hour in 12% sodium hypochlorite.

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Subsequently, seeds were washed repeatedly for 1 hour with sterile distilled water.

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After germination for three days at 22°C in complete darkness on wet filter paper,  Phusion DNA polymerase (NEB) by respective oligonucleotides (Table S5) EnzChek ® Phosphate Assay Kit (Thermo Fisher Scientific).   The modelling approach was essentially performed as described previously (1). The