However, the Acromyrmex strains share biosynthesis pathways for riboflavin vitamin, queuosine and a wide range of antioxidant enzymes likely to be beneficial for the ant fungus-farming symbiosis. Comparison with 54 Rhizobiales genomes showed that all insect-associated Rhizobiales lost the ability to fix nitrogen and that the Acromyrmex symbionts had exceptionally also lost the urease genes. We obtained genomes of three strains associated with Acromyrmex leaf-cutting ants and show that, in spite of being extracellular gut symbionts, they lost all pathways for essential amino acid biosynthesis, making them fully dependent on their hosts. Rhizobiales are well-known plant-root nitrogen-fixing symbionts, but the functions of insect-associated Rhizobiales are poorly understood. echinatior: Acromyrmex echinatior ANOVA: Analysis of Variance AZCL: Azurine-Crosslinked BLAST: Basic Local Alignment Search Tool cDNA: Complementary Deoxyribonucleic Acid DNA: Deoxyribonucleic Acid ELISA micro plate reader: Enzyme-linked Immunosorbent Assay micro plate reader GAL1: Galactokinase GAPDH: Glyceraldehyde 3-Phosphate Dehydrogenase GH12: Glycoside Hydrolase Family 12 glht: General Linear Hypotheses INVSc1: Saccharomyces cerevisiae Yeast strain used in this study LiOAc: Lithium Acetate MS/MS: Tandem Mass Spectrometry PCR: Polymerase Chain Reaction PEG: Polyethylene Glycol pYES2: Yeast expression vector used in this study qPCR: Quantitative Polymerase Chain Reaction RNA: Ribonucleic Acid RT-qPCR: Real Time Quantitative Polymerase Chain Reaction SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SE: Standard Error SMART RACE: Switching Mechanism At the 5'-end of RNA Transcript Rapid Amplification of cDNA Ends Tris: Tris(hydroxymethyl)aminomethane Ubc: Ubiquitin UPM: Universal Primer Mix URA3: Orotidine 5'-phosphate decarboxylase Xeg1: Xyloglucanase described in this study. Our results support the hypothesis that the ant-fungus symbiosis prioritizes access to the protein-rich contents of live plant cells and that carbohydrates are not a limiting resource.Ībbreviations A. Overall high xyloglucanase activity in old mycelium that is (about to be) discarded is striking and quite possibly serves defensive purposes by precluding that competing microorganisms can grow.
Xeg1 does not play a major decomposition role in the middle layer of fungus gardens where it is produced by the gongylidia. Our results are consistent with Xeg1 playing a role in the initial breakdown of plant cell wall hemicellulose to provide sugars for aggressive hyphal growth before intracellular proteins become available. We confirm substrate specificity of fungal Xeg1 towards xyloglucan by heterologous expression in yeast and show that xyloglucanase activity is higher in the oldest, bottom layers of fungus gardens and in discarded debris material than in the upper and middle layers of fungus gardens. Xeg1 is 4-5 times more active in fecal fluid when ants ingest their normal fungal food, compared to a sucrose control diet, as expected when they cannot produce Xeg1 themselves. Similar to other enzymes ingested this way, also Xeg1 is not digested but vectored to the fresh leaf-fragment pulp at the top of fungus gardens via ant fecal fluid. The fungal symbiont provides a single xyloglucanase (Xeg1) to its ant farmers by upregulating the expression of this protein in the inflated hyphal tips (gongylidia) that the ants ingest. However, many of these ants discard much of their harvested plant material after partial degradation, which has led to the hypothesis that the fungal symbionts are primarily producing cell wall degrading enzymes to gain access to intracellular nutrients rather than for obtaining sugars from recalcitrant cell wall polymers, such as (hemi-)cellulose. Leaf-cutting ants are major insect herbivores in the Neo-Tropics that rely on fungus-garden enzymes for degrading plant cell walls. Xyloglucan is an important component in plant cell walls that herbivores cannot digest without microbial symbionts.
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