Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

Abstract

The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5-7, but the aspartic proteases were inactive across a pH range of 3-10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi.

Figure 1

Enzyme activity in milliunits of proteases (mU) in fecal fluid of workers of the leaf-cutting ant A. echinatior kept on natural fungus-garden-diet and on a diet of only sugar water and possibly plant sap as control (t₁₇=14.8972, P<0.0001). Whiskers are s.e. of the mean.

Figure 2

Activity of metalloendoproteases, serine proteases, aspartic proteases and cysteine proteases in milliunits (mU). Both metalloendoproteases (z=10.824, P<0.0001) and serine proteases (z=2.004, P<0.05) showed significantly enhanced activity at pH 6 relative to the zero baseline. Serine proteases had their peak activity at pH 7 (z=4.654, P<0.0001) and activities of metalloendoproteases at pH levels >7 remained equally significant as those at pH 6. Whiskers are s.e. of the mean.

Figure 3

Average relative gene expression (fold change) across gongylidia collected from fungus gardens of five colonies of A. echinatior: dark grey=metalloendoproteases, grey=aspartic proteases, light grey=serine proteases. (a) Relative expression fold changes (mean±s.e.) in gongylidia compared with mycelium, with the dashed horizontal line representing no change in expression. Target genes were significantly upregulated in the gongylidia (P=0.00004, 0.00001, 0.00004, 0.99492, 0.00001 and 0.00001 for MetI, MetII, AspI, AspII, SerI and SerII, respectively, marked with asterisk (*)), except for polyporopepsin (AspII). (b) Relative expression fold-change plotted against gene expression normalized for the housekeeping genes GAPDH and Ubc (means±s.e.). The three upregulated serine (SerI and SerII) and aspartic (AspI) proteases had fold-changes proportional to normalized gene expression (being very close to the dashed line diagonal), but peptidyl-Lys metallopeptidase I (MetI) combined a high normalized gene expression with a relatively low gene fold-change, and peptidyl-Lys metallopeptidase II (MetII) combined a low normalized gene expression with a relatively high gene fold-change.