Did Convergent Protein Evolution Enable Phytoplasmas to Generate Zombie Plants?

Supplementary Material

Did Convergent Protein Evolution Enable Phytoplasmas to Generate Zombie Plants?

Florian Rümpler1, Lydia Gramzow1, Günter Theißen1 and Rainer Melzer1,2,*

1Department of Genetics, Friedrich Schiller University Jena, Jena, Germany

2School of Biology and Environmental Science, University College Dublin, Dublin, Ireland

Corresponding author: Melzer, R. ()

Figure S1, related to Figure 2

Alignments of the K-domain of MADS-domain TF representatives and the secreted part of SAP54 orthologs performed with (A)ClustalWS[S1], (B)Mafft applying the G-INS-i mode [S2] and (C) Muscle [S3]; visualized in Jalview using the ClustalX coloring scheme (

Table S1, related to Figure 2

Sequence identities between the K-domain of MIKC-type MADS-domain TF representatives and the secreted part of SAP54 orthologs based on the (A)ClustalW, (B)Mafft and (C) Muscle alignments shown in Supplementary Figure 1. Identities were calculated with SIAS (

AGL79 / MAF5 / AGL14 / XAL1 / FUL / AGL6 / AP1 / SEP1 / SEP3 / SOC1
(A) ClustalWalignments
OY-W / 5.74% / 9.19% / 3.44% / 10.34% / 9.19% / 5.74% / 4.59% / 10.34% / 8.04% / 8.04%
A-AY / 6.89% / 10.34% / 4.59% / 11.49% / 10.34% / 6.89% / 5.74% / 11.49% / 8.04% / 9.19%
CA-76 / 6.89% / 10.34% / 4.59% / 11.49% / 10.34% / 6.89% / 5.74% / 11.49% / 8.04% / 9.19%
AVUT / 6.89% / 10.34% / 4.59% / 11.49% / 10.34% / 6.89% / 5.74% / 11.49% / 9.19% / 8.04%
AY2192 / 5.74% / 10.34% / 4.59% / 12.64% / 10.34% / 6.89% / 5.74% / 12.64% / 9.19% / 8.04%
LEO / 5.74% / 9.19% / 3.44% / 10.34% / 9.19% / 5.74% / 4.59% / 10.34% / 9.19% / 6.89%
PYR / 1.72% / 6.89% / 3.44% / 5.17% / 8.62% / 5.17% / 1.72% / 5.17% / 1.72% / 6.89%
SAP54 / 5.74% / 10.34% / 3.44% / 10.34% / 9.19% / 5.74% / 4.59% / 11.49% / 9.19% / 6.89%
CP / 6.89% / 10.34% / 4.59% / 11.49% / 10.34% / 6.89% / 5.74% / 11.49% / 9.19% / 8.04%
PEY / 6.89% / 10.34% / 4.59% / 12.64% / 10.34% / 6.89% / 5.74% / 12.64% / 9.19% / 8.04%
(B) Mafftalignments
OY-W / 16.09% / 12.64% / 19.54% / 17.24% / 13.79% / 17.24% / 17.24% / 16.09% / 11.49% / 14.94%
A-AY / 16.09% / 13.79% / 21.83% / 17.24% / 14.94% / 17.24% / 18.39% / 16.09% / 11.49% / 16.09%
CA-76 / 14.94% / 13.79% / 21.83% / 17.24% / 14.94% / 17.24% / 18.39% / 16.09% / 11.49% / 16.09%
AVUT / 13.79% / 12.64% / 20.68% / 16.09% / 14.94% / 16.09% / 18.39% / 16.09% / 10.34% / 16.09%
AY2192 / 14.94% / 13.79% / 20.68% / 16.09% / 16.09% / 16.09% / 19.54% / 16.09% / 10.34% / 17.24%
LEO / 13.79% / 12.64% / 19.54% / 16.09% / 14.94% / 16.09% / 18.39% / 16.09% / 10.34% / 16.09%
PYR / 8.62% / 6.89% / 20.68% / 8.62% / 13.79% / 13.79% / 18.96% / 13.79% / 10.34% / 15.51%
SAP54 / 14.94% / 13.79% / 19.54% / 14.94% / 16.09% / 17.24% / 19.54% / 16.09% / 10.34% / 17.24%
CP / 13.79% / 12.64% / 20.68% / 16.09% / 14.94% / 16.09% / 18.39% / 16.09% / 10.34% / 16.09%
PEY / 13.79% / 12.64% / 20.68% / 16.09% / 14.94% / 16.09% / 18.39% / 16.09% / 10.34% / 16.09%
(C) Muscle alignments
OY-W / 12.64% / 16.09% / 13.79% / 9.19% / 9.19% / 10.34% / 10.34% / 10.34% / 10.34% / 16.09%
A-AY / 12.64% / 14.94% / 12.64% / 10.34% / 9.19% / 10.34% / 10.34% / 10.34% / 10.34% / 14.94%
CA-76 / 13.79% / 16.09% / 13.79% / 10.34% / 10.34% / 10.34% / 11.49% / 10.34% / 11.49% / 16.09%
AVUT / 12.64% / 14.94% / 13.79% / 9.19% / 9.19% / 10.34% / 10.34% / 10.34% / 11.49% / 14.94%
AY2192 / 12.64% / 16.09% / 12.64% / 9.19% / 9.19% / 9.19% / 10.34% / 10.34% / 11.49% / 13.79%
LEO / 12.64% / 14.94% / 13.79% / 10.34% / 9.19% / 10.34% / 10.34% / 10.34% / 11.49% / 14.94%
PYR / 6.89% / 10.34% / 8.62% / 8.62% / 8.62% / 3.44% / 8.62% / 6.89% / 8.62% / 10.34%
SAP54 / 11.49% / 14.94% / 12.64% / 11.49% / 8.04% / 8.04% / 9.19% / 11.49% / 10.34% / 12.64%
CP / 12.64% / 14.94% / 13.79% / 9.19% / 9.19% / 10.34% / 10.34% / 10.34% / 11.49% / 14.94%
PEY / 12.64% / 14.94% / 13.79% / 10.34% / 9.19% / 10.34% / 10.34% / 10.34% / 11.49% / 14.94%

Table S2, related to Box 2

Hidden-Markov-model search results based on a sequence alignment of SAP54-like sequences against (A) the Protein families (PFAM) database[S4] and (B) the Conserved Domains Database (CDD) [S5] using the HHPred web server [S6]. Entries are ordered according to their probability value, which is a more sensitive estimate of the reliability of the database match than the E-value.

Subject id / Name / Prob / E-value / P-value / Score / Ss / Cols / Query hmm / Template / Hmm
(A) Protein families (PFAM) database
pfam12017 / P element Transposase protein / 43.4 / 25 / 0.0017 / 23.8 / 5.3 / 41 / 46-86 / 9-51 / (236)
pfam15188 / Coiled-coil domain-containing protein 167 / 36.2 / 21 / 0.0014 / 21.6 / 3.5 / 29 / 62-90 / 38-66 / (85)
pfam01806 / Paramyxovirinae P phosphoprotein C-terminal region / 35.4 / 20 / 0.0013 / 25.9 / 3.8 / 35 / 52-86 / 69-110 / (248)
pfam07924 / NuiA Nuclease A inhibitor-like protein / 27.7 / 18 / 0.0012 / 22.9 / 2.3 / 19 / 70-88 / 77-95 / (133)
pfam12824 / MRP-L20 Mitochondrial ribosomal protein subunit L20 / 21.4 / 25 / 0.0017 / 22.8 / 2 / 21 / 43-63 / 92-112 / (164)
pfam11262 / Tho2 Transcription factornuclear export subunit protein 2 / 20.8 / 1.1E+02 / 0.0075 / 20.4 / 5.5 / 48 / 42-89 / 37-88 / (296)
pfam09278 / MerR-DNA-bind MerR, DNA binding / 20.4 / 48 / 0.0032 / 15.4 / 5 / 46 / 43-88 / 18-64 / (65)
pfam15005 / Izumo sperm-egg fusion / 20 / 1.1E+02 / 0.0074 / 19.6 / 5.1 / 39 / 43-81 / 76-115 / (160)
pfam12761 / End3 Actin cytoskeleton-regulatory complex protein END3 / 19.9 / 90 / 0.006 / 21 / 4.8 / 41 / 27-79 / 140-180 / (193)
pfam05766 / NinG Bacteriophage Lambda NinG protein / 19.6 / 1.1E+02 / 0.0073 / 20.3 / 5.1 / 47 / 32-89 / 140-186 / (188)
(B)Conserved Domains Database (CDD)
PF01806 / Paramyxovirinae P phosphoprotein C-terminal region / 32.9 / 1.9E+02 / 0.0037 / 23.5 / 5.9 / 47 / 34-86 / 64-110 / (248)
PF05766 / Bacteriophage Lambda NinG protein / 31.8 / 1.7E+02 / 0.0032 / 22.1 / 5.2 / 58 / 17-89 / 130-187 / (189)
PF01519 / Protein of unknown function DUF16 / 30.5 / 2.1E+02 / 0.0041 / 20.2 / 6.6 / 50 / 33-88 / 43-92 / (102)
PF15188 / Coiled-coil domain-containing protein 167 / 29.1 / 79 / 0.0015 / 21.4 / 2.8 / 29 / 62-90 / 38-66 / (85)
PF12824 / Mitochondrial ribosomal protein subunit L20 / 27.3 / 62 / 0.0012 / 23.5 / 2.2 / 21 / 43-63 / 92-112 / (164)
PF15005 / Izumo sperm-egg fusion / 25.5 / 2.5E+02 / 0.0049 / 20.6 / 5.1 / 39 / 43-81 / 76-115 / (160)
PF11291 / Protein of unknown function DUF3091 / 22.2 / 49 / 0.00095 / 22.9 / 0.9 / 21 / 54-74 / 80-100 / (100)
COG0357 / GidB Predicted S-adenosylmethionine-dependent methyltransferase / 21.1 / 1.0E+02 / 0.002 / 23.2 / 2.4 / 21 / 71-91 / 19-39 / (215)
PF07924 / Nuclease A inhibitor-like protein / 20.5 / 1.2E+02 / 0.0023 / 22.1 / 2.6 / 17 / 72-88 / 79-95 / (133)
cd08780 / Tumor Necrosis Receptor 1-Associated Death Domain protein / 20 / 1.7E+02 / 0.0032 / 19.9 / 3.1 / 47 / 41-88 / 31-77 / (90)

Table S3, related to Box 2

BLAST search results using SAP54 as query, searching the non-redundant protein sequences database. The search set was restricted to the organism group of (A)phytoplasmas and (B)mollicutes excluding phytoplasmas, respectively. No potentially related proteins could be identified that may be considered as true positives (e-value < 0.05), except for SAP54 itself (id 499731772), five previously published orthologs of SAP54 (111610297; 515759348; 156186996; 156186988; 491136134), two, to the best of our knowledge so far undescribed putative orthologs of SAP54 (515760611; 554499954) and a potentially related putative effector protein from peanut witches'-broom phytoplasma (491136155). The names given in brackets correspond to the names in[S7].

Subject id / Name / Organism / Identity (%) / Positives (%) / Alignment length / Mis-matches / Gap opens / E-value / Bit score
(A)Phytoplasmas
499731772 / hypothetical protein (SAP54) / Aster yellows witches'-broom phytoplasma / 100 / 100 / 91 / 0 / 0 / 2E-55 / 169
111610297 / hypothetical protein (AY1) / Maryland aster yellows phytoplasma / 87.91 / 94.51 / 91 / 11 / 0 / 2E-47 / 148
515759348 / hypothetical protein (JR1) / Poinsettia branch-inducing phytoplasma / 89.01 / 94.51 / 91 / 10 / 0 / 3E-47 / 148
156186996 / hypothetical protein (SP1) / Spiraea stunt phytoplasma / 86.81 / 93.41 / 91 / 12 / 0 / 1E-46 / 147
156186988 / hypothetical protein (TY) / Tomato yellows phytoplasma / 86.81 / 92.31 / 91 / 12 / 0 / 7E-46 / 144
515760611 / hypothetical protein / Italian clover phyllodyphytoplasma / 86.3 / 93.15 / 73 / 10 / 0 / 7E-36 / 118
491136134 / AYWB SAP54-like protein / Peanut witches'-broom phytoplasma / 52.75 / 65.93 / 91 / 42 / 1 / 1E-17 / 72
554499954 / hypothetical protein / CandidatusPhytoplasmasolani / 51.14 / 64.77 / 88 / 39 / 2 / 3E-15 / 65.1
491136155 / putative effector / Peanut witches'-broom phytoplasma / 37.1 / 56.45 / 62 / 39 / 0 / 0.006 / 32
213389875 / sugar-binding periplasmic protein / Palm lethal yellowing phytoplasma / 40 / 72 / 25 / 15 / 0 / 0.42 / 26.9
515759765 / hypothetical protein / Milkweed yellows phytoplasma / 42.86 / 65.71 / 35 / 20 / 0 / 1 / 25.8
515758879 / DNA primase / Poinsettia branch-inducing phytoplasma / 35.87 / 52.17 / 92 / 50 / 5 / 4.5 / 23.9
499474371 / ATPase AAA / Onion yellows phytoplasma / 24.36 / 46.15 / 78 / 49 / 1 / 4.7 / 23.9
503948597 / RNA replicase / 'Brassica napus' phytoplasma / 24.36 / 46.15 / 78 / 49 / 1 / 5.4 / 23.9
501496294 / hypothetical protein / CandidatusPhytoplasma mali / 29.41 / 49.02 / 51 / 34 / 1 / 6.7 / 23.5
554499772 / ATP-binding protein / CandidatusPhytoplasmasolani / 25.33 / 57.33 / 75 / 46 / 3 / 6.9 / 23.5
515758830 / translation initiation factor IF-2 / Poinsettia branch-inducing phytoplasma / 40 / 60 / 25 / 15 / 0 / 8.6 / 23.1
515760354 / translation initiation factor IF-2 / Italian clover phyllodyphytoplasma / 40 / 60 / 25 / 15 / 0 / 8.8 / 23.1
515760223 / DNA primase / Italian clover phyllodyphytoplasma / 34.78 / 52.17 / 92 / 51 / 5 / 8.9 / 23.1
515760768 / translation initiation factor IF-2 / Vaccinium witches'-broom phytoplasma / 40 / 60 / 25 / 15 / 0 / 9.1 / 23.1
(B)Mollicutes excluding phytoplasmas
667884065 / excinuclease ABC subunit C / Mycoplasma iowae / 52 / 76 / 25 / 12 / 0 / 1.4 / 28.9
525041233 / cobalt ABC transporter ATP-binding subunit / Spiroplasmataiwanense / 36.59 / 60.98 / 41 / 26 / 0 / 5.8 / 26.9
502618446 / glutamyl-tRNA(gln)amidotransferase subunit C / Mycoplasma hominis / 36.36 / 59.09 / 44 / 24 / 1 / 5.9 / 26.6
516294572 / hypothetical protein / Mycoplasma mycoides / 30.88 / 57.35 / 68 / 40 / 1 / 8.2 / 26.6
652845909 / hypothetical protein / Mycoplasma molare / 32.89 / 44.74 / 76 / 40 / 3 / 8.9 / 26.6
490126330 / hypothetical protein / Ureaplasmaurealyticum / 30.16 / 55.56 / 63 / 42 / 1 / 9.3 / 26.6

Supplementary References

S1Larkin, M.A., et al. (2007) Clustal W and clustal X version 2.0.Bioinformatics 23, 2947-2948.

S2Katoh, K. and D.M. Standley (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution 30, 772-780.

S3Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput.Nucleic Acids Research 32, 1792-1797.

S4Finn, R.D., et al. (2014) Pfam: the protein families database. Nucleic Acids Research 42, D222-D230.

S5Sayers, E.W., et al. (2012) Database resources of the National Center for Biotechnology Information. Nucleic Acids Research 40, D13-D25.

S6Soding, J., A. Biegert, and A.N. Lupas (2005) The HHpred interactive server for protein homology detection and structure prediction.Nucleic Acids Research 33, W244-W248.

S7Maejima, K., et al. (2014) Recognition of floral homeotic MADS domain transcription factors by a phytoplasmal effector, phyllogen, induces phyllody. Plant Journal 78, 541-554.