Supplementary Online Material For

Supplementary online material for:

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

Pepijn W Kooij*a, Adelina Rogowska-Wrzesinskab, Daniel Hoffmanna, Peter Roepstorffb, Jacobus J Boomsmaa, Morten Schiøtt*a

aCentre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark

bProtein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark.

*Corresponding author: d the above address

Table S1. List of primers used for gene cloning.

Gene / Primer name / Primer function / Primer sequences
AspI / Asp-F1 / 3’ RACE primer / 5’-CGTTCCCTTCGTTGTTGGCCTCC-3’
Asp-R1 / 5’ RACE primer / 5’- GCAAGCTTGTGAAGTCTGAGGGTGTG-3’
AspII / Pro-F4 / Forward degenerate primer / 5’-ACNGGXAGNTCXAAYACNTGG-3’
Pro-R2 / Reverse degenerate primer / 5’-TTRTCNGTIACNGTIGGDAT-3’
Pro-F5 / 3’ RACE primer / 5’-CCGTCGATCTCACGCGAGGAACGCTG-3’
Pro-R5 / 5’ RACE primer / 5’-GCGACGCTGTTGCCGGTCTGCTGAC-3’
MetI / Sp21-F2 / Forward 3’RACE degenerate primer* / 5’-GGTGGTACYTAYGTNCAYGA-3’
Met-R1 / 5’ RACE primer / 5’-AGCGAGTGCCTTGGCGCCAGATTG-3’
Met-R2 / 5’ RACE primer / 5’-CGTAGTCTTGTGTTCCGCCATTTCTGAGG-3’
Met-R3 / 5’ RACE primer / 5’-AGGGCTTGAGAGGCTCGATTGAGTAAG-3’
Met-R4 / 5’ RACE primer / 5’-CCGGAGTTGGTGAAGTTGTACATTGTTGAC-3’
Met-R5 / 5’ RACE primer / 5’-CAAGAGCAACAGCACATCGC-3’
MetII / MetII-full-F3 / Forward primer / 5’-AATGTCAATTCTATCGAATTCAAG-3’
MetII-R1 / Reverse qPCR primer / 5’-CGTGGACGATTGTTCCACCTC-3’
MetII-F1 / Forward qPCR primer / 5’- GGCTATTACTGCAGCGAAGACC-3’
MetII-full-R / Reverse primer / 5’-CAATTCAAGAATTATATGGCAGG-3’
SerI / Sub-F1 / Forward degenerate primer / 5’-GTTGARTTYGARGGXMGNGC-3’
Sub-R1 / Reverse degenerate primer / 5’-TTRTCRTTACCXGCNGCNAC-3’
Sub1-F1 / 3’ RACE primer / 5’-GATCGCCGACAACAAGTATGAAGATGGTG-3’
Sub1-R1 / 5’ RACE primer / 5’-GCACAGTGAGTACCATGACCAACACC-3’
SerII / Sp24-F3 / Forward degenerate primer / 5’-GCTGACCTYCARACNTTYTT-3’
Sp16-R2 / Reverse degenerate primer / 5’-GAGAAACCACCNCCNGARAA-3’
Ser-F1 / 3’ RACE primers / 5’-TAGGCGGTGGCGACTGCCGTACTAATGAC-3’
Ser-R2 / 5’ RACE primer / 5’-GAAACTAGAACCTGCAGCATCGGGGCGG-3’
Ser-R3 / 5’ RACE primer / 5’-CGGAATCTGGGTCAAAAGGAGGGGAACCG-3’
Ser-R4 / 5’ RACE primer / 5’-GTCCATACCGTGCATGACCAGGATCGC-3’

* This primer was used together with the UPM primers from the SMART RACE cDNA kit

Note: X symbolizes inosines

Table S2

The Real Time qPCR primers used in this study.

Enzyme / Primer / 5’- sequence -3’ / Annealing temp C / Size
Saccharopepsin (AspI) / Pep-F2 / CCG CCA TTG ACA CTG GTA CC / 53.8 / 289bp
Pep-R2 / GGC GGA GGA AAA CAT CAC CAA / 54.4
Polyporopepsin (AspII) / Pol-F2 / CTC GTT GAC ACC GGG AGT TC / 55.9 / 243bp
Pol-R3 / GAC GGG GCC GAT GCC TAA / 54.9
Peptidyl-Lys metallopeptidase (MetI) / Met-F2 / CGG GAC CGA CTC TAA GGG / 54.9 / 169bp
Met-R6 / TTC ATT GTG AAT ACA TCA TAG ACT TC / 51.7
Peptidyl-Lys metallopeptidase (MetII) / MetII-F1 / GGC TAT TAC TGC AGC GAA GAC C / 56.7 / 307bp
MetII-R1 / CGT GGA CGA TTG TTC CAC CTC / 56.3
Subtilisin (SerI) / Sub-F2 / AGA AAT CTG CTC CAT GGG GC / 53.8 / 300bp
Sub-R1 / CTA CCA TCA GAA CCA AGC ACC / 54.4
Grifolisin (SerII) / Ser-F3P / GCT ACC TTC AAT GGT CTA TAC AAT AAA AC / 56.0 / 310bp
Ser-R4P / TAC CCA GGC CAG TGA CAG GAT / 55.4
Elongation factor 1- (EF1) / EF1-F1 / TTG GAG GAA TCT CCC AAC ATG / 57.9 / 273bp
EF1-R1 / AAC GGA CTT GAC TTC AGT AGT C / 58.4
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) / GAPDH-F4 / TCA ACG GCA AGC TCA CTG GT / 53.8 / 253bp
GAPDH-R4 / ACA AAA TTC CCG TTC AAA GGA ATC / 52.3
Ubiquitin (Ubc) / Ubc-F4 / AAC GAT AAT GGG ACC CGG TG / 53.8 / 239bp
Ubc-R4 / GAT TGG GAT CAG TCA GCA TTG / 52.4

Table S3

Comparison of the sequenced proteases with the MEROPS database showing similarities with known fungal peptidases. For each protease the top ten blast results are shown.

Protease / MEROPS code / MEROPS classification / Protease name / Species / % identity / E-value
AspI / MER122335 / A01.018 / saccharopepsin / Laccaria bicolor / 78.57% / 1.10e-142
MER309581 / A01.018 / saccharopepsin / Serpula lacrymans / 79.56% / 4.40e-141
MER107385 / A01.018 / saccharopepsin / Coprinus cinereus / 78.02% / 1.70e-139
MER309629 / A01.018 / saccharopepsin / Schizophyllum commune / 77.36% / 4.10e-138
MER382986 / A01.018 / saccharopepsin / Stereum hirsutum / 76.88% / 5.20e-138
MER382978 / A01.018 / saccharopepsin / Coniophora puteana / 76.42% / 8.40e-138
MER382983 / A01.018 / saccharopepsin / Punctularia strigosozonata / 77.67% / 1.80e-137
MER382989 / A01.018 / saccharopepsin / Trametes versicolor / 77.12% / 180e-135
MER382982 / A01.018 / saccharopepsin / Dichomitus squalens / 76.42% / 4.80e-135
MER382984 / A01.018 / saccharopepsin / Fomitiporia mediterranea / 74.53% / 1.30e-132
AspII / MER118464 / A01.019 / polyporopepsin / Laccaria bicolor / 71.60% / 1.80e-125
MER383691 / A01.019 / polyporopepsin / Pholiota nameko / 74.53% / 2.70e-122
MER383440 / A01.019 / polyporopepsin / Trametes versicolor / 71.60% / 2.80e-120
MER383572 / A01.019 / polyporopepsin / Dichomitus squalens / 71.74% / 3.20e-190
MER000940 / A01.019 / polyporopepsin / Polyporus tulipiferea / 69.04% / 9.70e-118
MER383537 / A01.019 / polyporopepsin / Stereum hirsutum / 70.15% / 2.00e-117
MER383829 / A01.019 / polyporopepsin / Punctularia strigosozonata / 70.37% / 4.20e-117
MER062431 / A01.019 / polyporopepsin / Phanerochaete chrysosporium / 67.70% / 4.50e-113
MER172923 / A01.019 / polyporopepsin / Laccaria bicolor / 65.43% / 5.30e-110
MER383761 / A01.019 / polyporopepsin / Stereum hirsutum / 64.71% / 1.40e-109
MetI / MER003546 / M35.004 / Peptidyl-Lys metallopeptidase / Pleurotus ostreatus / 56.55% / 1.50e-53
MER005595 / M35.004 / Peptidyl-Lys metallopeptidase / Armillaria mellea / 57.49% / 1.20e-51
MER003545 / M35.004 / Peptidyl-Lys metallopeptidase / Grifola frondosa / 51.50% / 7.90e-50
MER187451 / M35.003 / EcpA peptidase / Corallococcus coralloides / 50.00% / 4.30e-42
MER019534 / M35.003 / EcpA peptidase / Xanthomonas axonopodis / 48.82% / 3.60e-38
MER255520 / M35.003 / EcpA peptidase / Collimonas fungivorans / 45.51% / 5.80e-38
MER089593 / M35.003 / EcpA peptidase / Shewanella loihica / 47.56% / 2.50e-37
MER071568 / M35.003 / EcpA peptidase / Shewanella denitrificans / 48.45% / 4.10e-37
MER075809 / M35.003 / EcpA peptidase / Shewanella amazonensis / 48.50% / 5.20e-37
MER062468 / M35.003 / EcpA peptidase / Xanthomonas campestris / 47.62% / 2.90e-36
MetII / MER003546 / M35.004 / Peptidyl-Lys metallopeptidase / Pleurotus ostreatus / 69.23% / 2.60e-42
MER003546 / M35.004 / Peptidyl-Lys metallopeptidase / Pleurotus ostreatus / 54.90% / 2.60e-42
MER003546 / M35.004 / Peptidyl-Lys metallopeptidase / Pleurotus ostreatus / 47.06% / 2.60e-42
MER003546 / M35.004 / Peptidyl-Lys metallopeptidase / Pleurotus ostreatus / 71.43% / 2.60e-42
MER003546 / M35.004 / Peptidyl-Lys metallopeptidase / Pleurotus ostreatus / 35.48% / 1.60e-21
MER005595 / M35.004 / Peptidyl-Lys metallopeptidase / Armillaria mellea / 69.23% / 6.80e-33
MER005595 / M35.004 / Peptidyl-Lys metallopeptidase / Armillaria mellea / 53.19% / 6.80e-33
MER005595 / M35.004 / Peptidyl-Lys metallopeptidase / Armillaria mellea / 48.48% / 6.80e-33
MER005595 / M35.004 / Peptidyl-Lys metallopeptidase / Armillaria mellea / 54.17% / 4.60e-24
MER255520 / M35.003 / EcpA peptidase / Collimonas fungivorans / 59.18% / 2.60e-32
SerI / MER169844 / S08.UPA / Subfamily S8A unassigned peptidases / Laccaria bicolor / 61.28% / 6.90e-111
MER034994 / S08.UPA / Subfamily S8A unassigned peptidases / Coprinus cinereus / 60.17% / 3.10e-108
MER032520 / S08.UPA / Subfamily S8A unassigned peptidases / Aspergillus oryzae / 52.47% / 3.30e-100
MER032520 / S08.UPA / Subfamily S8A unassigned peptidases / Aspergillus oryzae / 36.11% / 3.30e-100
MER236481 / S08.UPA / Subfamily S8A unassigned peptidases / Cryptococcus gattii / 56.90% / 5.80e-98
MER087598 / S08.UPA / Subfamily S8A unassigned peptidases / Neosartorya fischeri / 61.65% / 9.00e-94
MER087598 / S08.UPA / Subfamily S8A unassigned peptidases / Neosartorya fischeri / 36.11% / 9.00e-94
MER003475 / S08.UPA / Subfamily S8A unassigned peptidases / Aspergillus fumigatus / 61.65% / 1.10e-93
MER003475 / S08.UPA / Subfamily S8A unassigned peptidases / Aspergillus fumigatus / 36.11% / 1.10e-93
MER006088 / S08.UPA / Subfamily S8A unassigned peptidases / Penicillium chrysogenum / 60.43% / 4.90e-93
SerII / MER137045 / S53.010 / grifolisin / Laccaria bicolor / 66.67% / 4.40e-136
MER137535 / S53.010 / grifolisin / Laccaria bicolor / 60.05% / 1.90e-119
MER389943 / S53.010 / grifolisin / Stereum hirsutum / 56.77% / 6.00e-93
MER125099 / S53.010 / grifolisin / Coprinus cinereus / 46.44% / 1.20e-89
MER178229 / S53.010 / grifolisin / Sclerotinia sclerotiorum / 44.78% / 1.30e-85
MER178229 / S53.010 / grifolisin / Sclerotinia sclerotiorum / 40.80% / 1.30e-85
MER405414 / S53.UPW / Family S53 unassigned peptidases / Agaricus bisporus / 66.24% / 1.50e-82
MER078639 / S53.010 / grifolisin / Grifola frondosa / 45.70% / 1.90e-80
MER032166 / S53.010 / grifolisin / Emericella nidulans / 44.53% / 1.10e-79
MER030252 / S53.010 / grifolisin / Aspergillus oryzea / 43.91% / 1.50e-78

Table S4

Results of statistical analyses in R on normalized (relative to the housekeeping genes GAPDH and Ubc) and relative gene expression measurements (gongylidia relative to mycelium)