Article Figures & Data
Figures
- FIG. 1.
NJ phylogenetic tree of deduced amino acid sequences of 149 fungal PKS gene fragments (amplified with KA series primers; marked in red, blue, and green for insect, marine, and lichenized fungi, respectively) and other known NR and reducing fungal PKSs and bacterial PKSs in the NCBI database (in black). Bacterial PKSs were used as the outgroup in the tree. Bootstrap analysis using NJ was conducted with 1,000 replicates, and values of ≥50% are given at nodes.
- FIG. 2.
PCR amplification of PKS genes. Genomic DNA isolated from Pezizomycotina fungi was amplified using the KA series primers KAF1-KAR1 (R1) and KAF1-KAR2 (R2). Duplicate lanes (R1 and R2) are shown for each fungus. Insect-pathogenic fungi: 1, A. pistillaforimis; 2, B. bassiana; 3, C. pseudomilitaris; and 4, P. tenuipes. Marine fungi: 5, Acremonium sp.; 6, Aigialus parvus; 7, H. oceanica; and 8, K. glabrum. Lichenized fungi: 9, Coniothyrium sp.; 10, G. afzelii; 11, Microsphaeropsis sp.; and 12, Lecanora sp. Other fungi: 13, C. gleosporiodes; 14, Phomopsis sp.; and 15, Xylaria sp.
- FIG. 3.
NJ tree of reducing clade III PKSs. The insect-specific clade III group I and the wood/plant-specific group W are shown on the tree. The KA primer-amplified PKS fragments in groups I and W are indicated by triangles and by black ovals, respectively. PKSs from fungal genomes are in parentheses. All PKSs in group I are from the fungi in the order Hypocreales, except for two Aspergillus PKSs from the order Eurotiales (marked with asterisks). The subgroup basal to the insect-specific group is indicated in the shaded area; these PKSs are mostly genomic. Bootstrap values of ≥50% are given at the nodes from the analysis using NJ with 1,000 replicates.
Tables
- TABLE 1.
All 48 fungi used in this studya
Fungus Codeb Taxonomic class Habitat typec Substrate where found Akanthomyces novoguineensis BCC2837 Akn Sordariomycetes Insect Araneae (spider) Akanthomyces pistillariformis BCC1428 Akp Sordariomycetes Insect Lepidoptera (adult moth) Aschersonia tubulata BCC7681 Ast Sordariomycetes Insect Homoptera (nymph) Beauveria bassina BCC1625 Beb Sordariomycetes Insect Insecta (pupa) Cordyceps nipponica BCC1389 Cni Sordariomycetes Insect Neuroptera (nymph) Cordyceps pseudomilitaris BCC1620 Cop Sordariomycetes Insect Lepidoptera (larva) Gibellula sp. BCC14132 Gib Sordariomycetes Insect Araneae (spider) Hirsutella nivea BCC2594 Hin Sordariomycetes Insect Homoptera (hopper) Hymenostilbe sp. BCC1571 Hym Sordariomycetes Insect Isoptera (termite) Hymenostilbe dipterigina MY1105 Hymd Sordariomycetes Insect Diptera (fly) Hypocrella discoidea BCC8239 Hyd Sordariomycetes Insect Homoptera (scale insect) Hypocrella raciborski BCC17141 Hyr Sordariomycetes Insect Homoptera (scale insect) Isaria javanica BCC29250 Isj Sordariomycetes Insect Lepidoptera (larva) Metarhizium anisopliae BCC1617 Mea Sordariomycetes Insect Homoptera (hopper nymph) Metarhizium flavoviride BCC7623 Mef Sordariomycetes Insect Homoptera (hopper nymph) Ophiocordyceps irangiensis BCC21423 Opi Sordariomycetes Insect Hymenoptera (ant) Ophiocordyceps myrmecopphila MY163 Opm Sordariomycetes Insect Hymenoptera (ant) Ophiocordyceps sphecocephala BCC21472 Ops Sordariomycetes Insect Hymenoptera (ant) Ophiocordyceps unilaterlais BCC14750 Opu Sordariomycetes Insect Hymenoptera (ant) Ophiocordyceps unilateralis KT3307 Opu2 Sordariomycetes Insect Hymenoptera (ant) Paecillomyces tenuipes BCC1614 Pat Sordariomycetes Insect Lepidoptera (pupa) Acremonium sp. strain BCC9257 Acr Sordariomycetes Marine Wood Aigialus parvus BCC6195 Aip Dothideomycetes Marine Mangrove Corollospora besarispora BCC6732 Cob Sordariomycetes Marine Corollospora maritima BCC12026 Com Sordariomycetes Marine Sand grain Epicoccum sp. strain BCC8702 Epi Dothideomycetes Marine Mangrove Halorosellinia oceanica BCC11430 Hao Sordariomycetes Marine Wood Halosarpheia cf. kandeliae BCC13120 Hak Sordariomycetes Marine Wood Helicascus kanaloanus BCC13107 Hek Dothideomycetes Marine Sonneratia Kallichroma glabrum BCC13115 Kag Sordariomycetes Marine Mangrove Lineolata rhizosphorae BCC11179 Lir Dothideomycetes Marine Driftwood Massarina sp. strain BCC18015 Mas Dothideomycetes Marine Submerged coconut Nais glitra BCC11151 Nag Sordariomycetes Marine Mangrove Torpedospora radiata BCC11269 Tor Ascomycota incertae sedis Marine Driftwood Coniothyrium sp. strain BCC3073 Con Sordariomycetes Lichen Lichen Graphis afzelii BCC12103 Gra Lecanoromycetes Lichen Bark Lecanora sp. strain BCC12113 Lec Lecanoromycetes Lichen Bark Microsphaeropsis sp. strain BCC3050 Mic Anamorphic Ascomycota Lichen Dirinaria applanata lichen Phaeographina sp. strain BCC12100 Pha Lecanoromycetes Lichen Bark Pyrenula sp. strain BCC12115 Pyr Eurotiomycetes Lichen Bark Sarcographa sp. strain BCC12132 Sar Lecanoromycetes Lichen Bark Trypethelium sp. strain BCC9790 Try Eurotiomycetes Lichen Bark Cercospora oryzae Ceo Dothideomycetes Plant Rice Colletotrichum gleosporiodes BCC13327 Cog Sordariomycetes Plant Banana Lasiodiplodia theobromae Lat Dothideomycetes Plant Cocoa Pestalotiopsis psidii Pep Sordariomycetes Plant Guava Phomopsis sp. strain BCC9451 Pho Sordariomycetes Endophyte Lagerstroemia leaves Trichoderma sp. strain BCC7579 Tri Sordariomycetes Soil Soil - TABLE 2.
PKS genes cloned from 48 fungi using PCR amplification with KA series primers
Habitat type and fungus codea No. of PKS genesb Sizes of PKS genes (kb) Intron sizes (bp)c No. of genes in reducing clade: No. of genes in NR clade I II III IV Noveld Insect Akn 2 0.70, 0.82 no, (70, 60) 1 1 Akp 2 0.78, 0.70 no, no 1 1 Ast 5 0.80, 0.58, 0.73, 0.99, 0.71 no, 59, no, (55, 68, 61), no 3 D2, D5 Beb 3 0.89, 0.71, 0.70 (47, 52, 74), no, no 1 D1, D2 Cni 3 0.82, 0.75, 0.76 50, no, no 1 2 Cop 6 0.72, 0.90, 0.71, 0.77, 0.71, 0.87 no, no, no, no, no, 55 3 1 D1, D4 Gib 1 0.74 30 1 Hin 3 0.75, 0.83, 0.80 58, 40, no 2 1 Hym 4 0.66, 0.75, 0.69, 0.80 no, 40, no, (60, 60) 1 3 Hymd 2 0.52, 0.54 no, no 1 D2 Hyd 2 0.77, 0.69 no, no 1 1 Hyr 1 0.70 no 1 Isj 3 0.70, 0.56, 0.80 no, no, 87 1 1 1 Mea 2 0.81, 0.71 no, no D4, D5 Mef 1 0.71 no 1 Opi 5 0.7, 0.82, 0.85, 0.82, 0.73 (56, 61), 168, no, (85, 60), no 1 1 3 Opm 3 0.83, 0.73, 0.7 (56, 60), no, no 1 2 Ops 3 0.82, 0.70, 0.83 (49, 53, 51), no, 131 1 2 Opu 4 0.95, 0.85, 0.71, 0.76 (48, 50, 68), (53, 58, 61), 65, (47, 53) 1 3 Opu2 4 0.73, 0.79, 0.81, 0.7 (50, 55, 57), no, 83, no 1 1 1 1 Pat 3 0.70, 0.95, 0.76 no, (62, 69), 40 1 1 1 Marine Acr 6 1.02, 0.80, 0.76, 1.02, 0.97, 0.73 104, 62, no, 262, 54, 37 1 1 1 D3, D6(2) Aip 3 0.77, 0.84, 0.79 60, no, 100 1 1 1 Cob 2 0.80, 0.72 60, no 2 Com 2 0.70, 0.87 no, (80, 50) 1 1 Epi 4 0.76, 0.67, 0.95, 0.89 no, no, 120, 51 1 1 1 D1 Hao 4 0.82, 0.76, 0.81, 0.67 62, 48, 53, no 1 1 1 1 Hak 2 0.90, 0.75 no, 40 1 1 Hek 4 0.73, 0.83, 0.94, 0.72 no, 63, (49, 53), no 2 2 Kag 6 0.85, 0.66, 1.01, 0.73, 0.70, 0.66 no, no, 50, no, no, no 1 3 D3, D6 Lir 3 0.81, 0.65, 0.88 83, no, (52, 48) 1 D1, D6 Mas 4 0.79, 0.79, 0.75, 0.77 no, no, no, no 2 1 D6 Nag 4 0.95, 0.84, 0.93, 0.71 (62, 57), (57, 67), 169, no 1 1 1 D1 Tor 1 0.76 31 1 Lichen Con 4 0.95, 0.89, 0.78, 0.81 (62, 57), 141, no, no 2 2 Gra 4 0.92, 1.15, 0.71, 0.96 no, (200, 50, 100), no, no 1 2 D1 Lec 5 1.01, 0.75, 0.88, 0.71, 0.71 (94, 48, 56), no, 49, no, no 4 1 Mic 1 0.88 110 D1 Pha 3 0.79, 0.72, 0.82 no, no, (50, 45) 1 1 1 Pyr 3 0.93, 0.66, 0.93 (50, 54), no, (58, 97, 53) 1 1 D1 Sar 2 0.51, 0.78 no, 46 1 D3 Try 5 1.00, 0.75, 0.69, 0.74, 0.79 100, 70, no, no, no 1 1 2 D3 Plant Ceo 1 0.89 (105, 55) 1 Cog 5 0.89, 0.75, 1.06, 0.80, 0.70 (67, 51, 56, 50), no, (50, 258), no, no 1 2 2 Lat 2 0.81, 0.73 (56, 47, 53), no 1 1 Pep 2 0.81, 0.81 (41, 69), no 1 1 Other Pho 3 0.79, 0.74, 0.9 50, no, 162 2 D2 Tri 2 0.80, 0.87 60, 106 2 ↵ a Fungus codes are given in Table 1.
↵ b PCR products were determined by similarity searches using BLASTX, and the number of PKS genes detected in each fungus is indicated.
↵ c Putative intron sizes correspond to the PKS gene fragments in the adjacent column. “no” indicates that the gene fragment had no introns. Intron sizes in parentheses include the sizes of all the introns found in one PKS gene fragment.
↵ d PKS genes belonging to novel reducing clades D1, D2, D3, D4, D5, and D6. If more than one PKS gene is present in each novel clade, the number of PKS genes is in parentheses.
- TABLE 3.
PKS genes in 13 fungal genomes
Fungus No. of PKS genesa Total NR clades Reducing clades Bacterial PKSd Subtotal I II III IVb Subtotal I II III IV D1 D2 D3 D4 Otherc Aspergillus clavatus 21 4 1 2 1 17 4 7 2 2 1 M Aspergillus fumigatus 15 6 1 1 2 2 9 1 3 3 1 1 Aspergillus nidulans 27 13 3 1 5 4 14 3 2 4 1 2 1 D6 Aspergillus niger 30 2 1 1 28 10 6 2 8 2 Aspergillus oryzae 22 6 3 1 2 16 2 4 1 5 1 1 1 1 Aspergillus terreus 30 10 2 2 4 2 20 3 2 1 5 1 2 3 1 D5, M Chaetomium globosum 26 7 1 5 1 18 1 6 2 3 3 2 D5 1 Magnaporthe grisea 21 4 1 1 2 16 7 4 2 2 1 1 Neosartorya fischeri 17 7 1 2 2 2 10 1 2 4 2 1 Phaeosphaeria nodorum 23 7 2 1 2 2 16 2 2 1 7 1 1 D5, M Podospora anserina 20 6 1 1 3 1 14 1 4 1 4 1 1 D5(2) Pyrenophora tritici-repentis 15 4 2 1 1 11 5 3 1 D6, M Sclerotinia sclerotiorum 16 3 1 2 13 4 2 1 2 1 D6(3) ↵ a PKS genes were determined using annotated and BLAST searches (NCBI) against each fungal genome.
↵ b The NR clade basal to NR clades I and II in the phylogenetic tree (Fig. 1).
↵ c PKS genes belonging to other reducing clades: D5, D6, and MSAS group (M). If more than one PKS gene is present in each of these clades, the number of PKS genes is in parentheses.
↵ d The PKS gene is clustered with the bacterial PKS group.
- TABLE 4.
Conserved amino acid residues of reducing clade III PKSs specific for different groups of fungi
Domain Relative positionsa Conserved domain in the alignmentb Conserved residues All insect-pathogenic fungi Insect biocontrol fungic Ophiocordyceps genus Wood-inhabiting fungi KS 65/65 a E(Q)KA KH(T)P 74/74 b LDE(D) FKE(A) 116/116 c LS FT Interdomain 132/129/132 d V(I)VPR -HK(R) PTRK 139/139 e SGNDEGAAKRNATAI TANDKASLEAVLKNL 159/159 f H R 167/167 g RIIR ALMA(S) 178/178 h ERRT(S)H QRRSL 183/179/183 i HS(N)Y L(M)PW LQW 199/199 j STLNSEAM EAINGQR(K)L(P) ↵ a Relative position using the residue number of the first member of group in the multiple alignment: IsjF1R1C4 (all insect-pathogenic fungi and insect biocontrol fungi), Opu2F1R2_D9 (Ophiocordyceps genus), and PyrF1R2B1 (wood-inhabiting fungi).
↵ b Conserved domains are shown in the multiple-sequence alignment in Fig. S2 in the supplemental material.
↵ c The insect biocontrol fungal group includes B. bassina, C. pseudomilitaris, I. javanica, M. flavoviride, and P. tenuipes.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Neighbor-joining tree of deduced amino acid sequences of newly cloned PKS gene fragments, reference NR and reducing fungal
PKSs and bacterial PKSs in the NCBI database, and genomic PKSs (Fig. S1).
MS PowerPoint file, 337K. - Supplemental file 2
-
Multiple alignment of amino acid sequences of the reducing clade III PKS gene fragments amplified by the KA-series primers
and reference clade III PKSs (Fig. S2).
MS PowerPoint file, 245K. - Supplemental file 3
-
Legends to Fig. S1 and S2.
MS Word document, 28K. - Supplemental file 4
-
Polyketide synthases cloned in this study and their GenBank accession numbers (Table S1).
MS Word document, 128K. - Supplemental file 5
-
Reference PKSs and PKSs from Xylaria sp. strain BCC1067 with their GenBank accession numbers (Table S2).
MS Word document, 139K. - Supplemental file 6
-
Genomic PKSs from 13 fungal genomes and their GenBank accession numbers (Table S3).
MS Word document, 300K.
- Supplemental file 1
-
Neighbor-joining tree of deduced amino acid sequences of newly cloned PKS gene fragments, reference NR and reducing fungal
PKSs and bacterial PKSs in the NCBI database, and genomic PKSs (Fig. S1).
