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Figure S1. Stability of TENGU-GST protein.
TENGU-GST recombinant protein was incubated with Tris buffer at room temperature and analyzed by SDS-PAGE.
Figure S2. Characterization of TENGU-GSTprocessing product by Edman sequencing.
The “control” panel on the top indicates the retention time of each amino-acid.
Panels below show the results of cycle1 to cycle 5. Arrowheads indicate the detected peak in each cycle.
Figure S3. Conservation of tengu homologs among phytoplasmas.
A, Phyllogenetic tree of Phytoplasmas based on 16S ribosomal DNA sequences. Phytoplasma strains that possess tengu or tengu homolog are shown in Bold. Acholeplasma laidlawii was used as the outgroup and numbers on branches are the bootstrap values (only values>80% are shown). Four phytoplasma strains of which complete genomic sequences have been determined were underlined.
Abbreviations: AlloY=Allocasuarina yellows; AlmWB=Almond witches’ broom; APPTW=American potato purple top wilt; AshY=Ash yellows; BGWL=Bermuda grass white leaf; BVGY=Buckland valley grapevine yellows; BWB=Buchthorn witches’-broom;CbY=Chinaberry yellows; CnWB=Chestnut witches’ broom; CP=Cloverproliferation; ESFY=European stone fruit yellows; EY, Elm yellows; FD, Flavescence dorée of grapevine; HibWB=Hibiscus witches’ broom; JHP=Japanese Hydrangea phyllody; JWB=Jujube witches’ broom; LD, LDN, LDT, LY=Coconut lethal yellowing; LfWB=Loofah witches’ broom; OAY=Oenothera aster yellows; PAY= Papaya; PD=Pear decline;PinP=Pine; PPWB=Pigeon pea witches’ broom; RYD=Rice yellow dwarf; SCYLP=Sugarcane yellow leaf syndrome; SCWL=Sugarcane white leaf; SpaWB=Spartinum witches’-broom; SPLL=Sweet potato little leaf; StrawY=Strawberry yellows; StLL=Stylosanthes little leaf; STOL=Stolbur of Capsicum annuum; ViLL=Vigna little leaf; WBDL=Witches’-broom disease of lime; WX=Western X disease.
Figure S4. Conservation ofthe11-amino acidfunctional region among TENGU homologs.
Amino acid alignment of the secreted region of TENGU and its homologs. Residues identical to TENGU (from OY-M strain) are shown as “.”. Sequences from PPT and KV strains are identical. The site of TENGU processing is indicated with an arrowhead and the resulting N-terminal region that possesses symptom inducing activity is shaded.
Onion yellows phytoplasma line M / OY-M / AP006628, PAM_r001
Onion yellows phytoplasma line W / OY-W / D12569
Paulownia witches’ broom phytoplasma / PaWB / AF279271
Sumac witches’ broom phytoplasma / SWB / AB693125
Bamboo witches’ broom phytoplasma / BamWB / AY792328
Garlic yellows phytoplasma / GY / AB750363
Water dropwort witches’ broom phytoplasma / WDWB / AB078436
Mulberry dwarf phytoplasma / MD / FJ844442
Porcelain vine witches’ broom phytoplasma / PvWB / AB693126
Clover phyllody phytoplasma / KV / X83870
Potato purple top phytoplasma / PPT / AF217247
Apricot chlorotic leafroll phytoplasma / ACLR / X68338
Aster yellows phytoplasma strainwitches’ broom / AY-WB / CP000061, AYWB_r01
Australian grape-vine yellows phytoplasma / AUSGY / AM422018
Apple proliferation phytoplasma / AP / CU469464
Table S1. Phytoplasma strains used in this study.
Phytoplasma strain names, abbreviations, and GenBank accession numbers of the 16SrDNA sequence are listed.
Primer name / Sequence (5'-3')N19-R / TGA CCC GGG TTA TTG ATT CTC TTT AGT TTC AAT TAG AGT TAT CAC GTT TTC AA
C19-F / ACG CGT CGA CAT GAC AGA ACA AAT AAA AAT ACA ATG TCA AGA TTT ATT GCA AAA GGG
N10-F / TCG CGT CGA CAT GGA CCA AGA TGA TGA TAT TGA AAA CGT
N10-R / TGA CCC GGG TTA TAT CAC GTT TTC AAT ATC ATC ATC TTG G
N8-F / ACG CGT CGA CAT GGA CCA AGA TGA TGA TAT TG
N8-R / TGA CCC GGG TTA GTT TTC AAT ATC ATC ATC TTG G
N1211-F / ACG CGT CGA CAT GGA CCA AGA TGA TGA TAT TGA AAA CGT GA
N12-R / TGA CCC GGG TTA TAG AGT TAT CAC GTT TTC AAT ATC ATC A
N11-R / TGA CCC GGG TTA TCA ATA GTG CAA AAG TTA TAG TAG TAG A
1213A-F / GGC ATA TGG ACC AAG ATG ATG ATA TTG AAA ACG TGA TAA CTG CTG CTG AAA C
1213E-F / GGC ATA TGG ACC AAG ATG ATG ATA TTG AAA ACG TGA TAA CTG AAG AAGAAA C
pETart-F / AGA AGG AGA TAT ACA TAT GGA CCA AGA TGA TGA TAT T
pETart-R / TGT CGA CGG AGC TCG CTA ATC CGA TTT TGG AGG ATG G
tenguGST-F / AAG GGC GAG AAG ATG CCA TGT CCC CTA TAC TAG GTT AT
TENGUgst-R / ATA ACC TAG TAT AGG GGA CAT GGC ATC TTC TCG CCC TT
GSTecI-R / CGG AAT TCC TAA TCC GAT TTT GGA GGA TGG
PAM765-F / ACG CGT CGA CAT GGA CCA AGA TGA TGA TAT TGA AAA CGT GAT AAC TC
PAM765-R / TGA CCC GGG TTA GGC ATC TTT CTC GCC CTT TTG CAA TAA ATC TTG ACA
pGEXTENGU-F / CGG GAT CCG ACC AAG ATG ATG ATA TTG AAA ACG
pETTENGU-R / TGAGCTCGAGGGCATCTTTCTCGCCCTTTTGCAATAAATCTTGACA
GST-F / ACG CGT CGA CAT GTC CCC TAT ACT AGG TTA TTG GAA
GST-R / GGA ATT CTT AGG ATC CAC GCG GAA CCA GA
pGEXTENGU-R / GGA ATT CTT AGG CAT CTT TCT CGC CCT TTT GCA ATA AAT CTT GAC A
PAM485F / GAA CCG GAA CCA CCT GAA GGA G
PAM486R / CAG CAT TTA TTC CAA GTG CTT TTG C
Table S2. Primers used in this study.
In all primers, restriction enzyme sites are indicated in bold. In the primers 1213A-F and 1213E-F, mutated codons are indicated in italic. In the primers pETart-F, pETart-R, tenguGST-F, and TENGUgst-R the sequences matching the tengu gene are underlined.