Supporting Information

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Supporting Information

Andrés-Colás et al. 10.1073/pnas.1705815114

SI Materials and Methods

Plant Materials and Growth Conditions.Arabidopsis thaliana ecotype Col-0 and Nicotiana benthamiana were used. Seeds were stratified for 2 d at 4 °C. Growth conditions were a 16-h light/8-h dark photoperiod and white fluorescence light (75μM m^–2·s^–1) at 22 °C.

For growing plants in soil, Jiffy-7 peat pellets were used. For growing seedlings on plates, seeds were surface sterilized with 30%

bleach and 0.02% Triton X-100 and germinated on 1/2 MS medium supplemented with 0.5 g/L Mes, the pH adjusted to 5.7 with KOH, 10 g/L sucrose, and 8 g/L agar.

Plasmid Construction.The complete coding sequences of interest were obtained from A. thaliana cDNA by HiFi PCR using attB- flanked primers (Dataset S2), which introduced the adequate sites for the subsequent cloning by the Gateway system (Invi- trogen) into the pDONR221 vector via BP reaction and were confirmed by sequencing. The At5g44230 (MEF57) coding sequence has a single nucleotide mutation at position 1089, but it does not change any amino acid in the protein sequence.

The verified fragments were cloned under the control of the constitutive CaMV 35S promoter via LR reaction into pK7WGF2 vector for C-terminal fusion with GFP; pB4GWnY and pB4GWnY vectors [kindly provided by Shoji Mano (39)] for C-terminal fusion with the n- or c-split-YFP; pGWB514 or pGWB508 vector [kindly provided by Tsuyoshi Nakagawa (40)] for C-terminal fusion with 3×HA epitope or His-tag. The SLO2 coding sequence was cloned also under the control of its own promoter as described previously (10). The mitochondria-mCherry marker plasmid [mt-rb (41)] was used as mitochondrial marker. The pB4nYFPPEX6 and pB4cYFPAPEM9 plasmids [kindly provided by Tsuyoshi Nakagawa (40)] were used as BiFC controls.

Subcellular Localization and BiFC. Two-dimensional cultures of Agrobacterium tumefaciens harboring the desired constructs and the silencing-suppressor p19 plasmid (42) were pelleted, diluted to an OD600 of 0.1–0.5 in infiltration buffer [0.5% (wt/vol) ^D-glucose, 10 mM Mes, 10 mM MgCl2, 0.1 mM acetosyringone] and coinfiltrated in young N. benthamiana leaves. At 3–6 d after infiltration, small pieces were excised and visualized under confocal imaging.

The MitotrackerOrange CMTMRos dye (Invitrogen) at 250 nM in 1/2 MS liquid medium was infiltrated 4 h before imaging to stain mitochondria. Plasmids carrying the mitochondria-mCherry marker {mt[29 aa ScCOX4]-mCherry-rb (41)} and the mRFP-SKL marker for mitochondria and peroxisomes, respectively, were coinfiltrated together with the constructs of interest.

Leaves infiltrated without GFP/YFP constructs were used as a negative control to reveal the background signal with a high laser gain (Fig. S3A), visualizing chloroplast autofluorescence as well as cellular structure. Samples with sufficiently strong fluorescent signal were assessed under reduced laser gain conditions, thus avoiding chloroplast autofluorescence or signal saturation. Al- though under these conditions detection of weaker signals can be lost, such as the mitochondrial signal, it allows for identifying strong signals in the chloroplasts or clear strong mitochondrial

tested by tagging the proteins with the other half of the split YFP protein.

The subcellular localization experiments were repeated, with similar results, three independent times (agro-infiltrations), except for HSP60.3A-GFP, which was repeated twice.

The protein interaction experiments by BiFC were repeated, with similar results, three independent times (agro-infiltrations), except for combinations with HSP60.3A-/P486-/P487-nYFP and the combinations NUWA-nYFP/HSP60-cYFP, MEF57-cYFP/NUWA-nYFP, cYFP-APEM9/NUWA-nYFP, nYFP-APEM9/NUWA-cYFP, and DYW2-nYFP/DYW2-cYFP, which were repeated twice.

CoIP and Western Blot.Plant material expressing the constructs of interest (transiently transformed N. benthamiana) was grinded in extraction buffer [50 mM Tris·HCl pH 7.5, 150 mM NaCl, 1%

Nonidet P-40 (Igepal CA630), 2× protease inhibitor mix mixture without EDTA (EDTA-free Complete, Roche), 1× phosphatase inhibitor (PhosSTOP, Roche), 50 μM MG132 proteasome inhibitor (C2211, Sigma), 5 mM ATP and 1 mM PMSF]. Nonidet P-40 was diluted to 0.2% by adding extraction buffer without Nonidet P-40 before filtering with 40 μM nylon mesh and centrifuging 1 min at 12,000 rpm (5417R centrifuge, F45-30-11 rotor; Eppendorf, Hauppauge, New York) at 4 °C. A total of 50 μL anti-GFP magnetic Microbeads (Miltenyi Biotec REF 130–

091-125) was added and incubated for 2 h under rotation at 4 °C.

After equilibrating the μ-column (in the magnetic field) with 200μL extraction buffer containing 0.1% Nonidet P-40, the cell lysate was applied. The column was washed four times with 200μL extraction buffer with 0.1% Nonidet P-40 and once with 20 mM Tris·HCl, pH 7.5. The coimmunoprecipitated protein was eluted with 50μL elution buffer preheated at 95 °C [50 mM Tris HCl pH 6.8, 50 mM DTT, 1% SDS, 1 mM EDTA, 0.005%

bromophenol blue, 10% glycerol] after incubating the column with 20μL of preheated elution buffer for 5 min at room tem- perature. The coimmunoprecipitated protein samples were incubated with elution buffer or sample buffer [250 mM Tris·HCl, pH 6.8, 20% glycerol, 4% SDS, 10% β-mercaptoethanol, and 0.025% bromophenol blue] for 5 min at 65 °C. Thirty microli- ters of the coimmunoprecipitated protein samples was run in a 8% SDS/PAGE gel and blotted on a 0.45-μm nitrocellulose membrane (Amersham Protran) at 4 °C, which was probed with 1:100 anti–HA-HRP (clone 3F10 Roche) or 1:100 anti–GFP-HRP (A10260 Invitrogen) in T-TBS buffer (20 mM Tris·HCl, pH 7.5, 0.5 M NaCl, 0.1% Tween-20) and developed by colorimetric assay (0.5 mg/mL DAB in 0.1 M imidazole pH 7 with 0.1μl/mL 30%

H2O2and 54μl/mL 0.6% CoCl2). Both antibodies were checked consecutively on the same membrane, with a long washing step (T- TBS buffer for 30 min) in between. The PageRuler prestained protein ladder was used as size marker of 170, 130, 100, 70 (red), 55, 40, 35, 25, and 15 kDa (Thermo Scientific, CN26616). The respective molecular weights were: SLO2-HA, 84.03 kDa; SLO2- GFP, 105.04 kDa; DYW2-GFP, 92.56 kDa; MEF57-GFP, 100.62 kDa; NUWA-HA, 77.23 kDa; NUWA-GFP, 98.24 kDa;

HSP60.2-HA, 67.92 kDa; HSP60.2-GFP, 88.93 kDa; HSP60.3B-

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N. benthamiana leaves infiltrated only with the corresponding HA- tag construct were used.

A positive interaction is shown by a higher signal of the HA constructs in the GFP immunoprecipitate (GFP-IP) with respect to the input signal, when combining the HA and GFP constructs compared with the negative controls for the immunoprecipitation/

binding to the antibody beads (HA construct alone). A negative interaction is indicated by a lower or practically equal signal.

The coimmunoprecipitation experiments were repeated, with similar results, three independent times with a different biological replicate (agro-infiltration) each, for the combinations SLO2- HA/MEF57-GFP and SLO2-HA/DYW2-GFP; for the rest, the experiment was repeated two times with a different biological replicate each, except for the combinations SLO2-GFP/NUWA-HA and HA-APEM9/NUWA-HA, which were performed once with two biological replicates.

T-DNA Insertion, Complemented, and Overexpressing Lines.A. thaliana T-DNA insertion mutant line N585176 from the SALK collec- tion was genotyped from genomic DNA of adult plant leaves by standard PCR with specific primers for the T-DNA and the gene of interest (Dataset S2). The PCR products were visualized on ethidium bromide-stained 2% agarose gel (Fig. S9). The absence of a second T-DNA insertion indicated in the database was confirmed also by standard PCR, after selection for the T-DNA insertion of interest through several generations. The loss of expression of the corresponding gene was checked from RNA by quantitative PCR (qPCR) (Fig. S9).

The homozygous T-DNA insertion mutant line or Col-0 A. thaliana plants were transformed with the 35S::MEF57-GFP and 35S::DYW2-GFP plasmids (in pK7WGF2 backbone), respectively, by the floral-dip method (43). Transformed plants were selected based on Kanamycin resistance. Two independent DYW2-GFP-OE lines were isolated from independent trans- formation experiments. The presence of the insert was confirmed by standard PCR on genomic DNA with specific primers (Dataset S2) and the PCR products were visualized on ethidium bromide-stained 2% agarose gel (Fig. S9). The expression of the corresponding genes was checked from RNA by qPCR (Figs. S7 and S9).

Quantitative PCR.Total RNA was extracted from adult plant leaves, seedlings, or siliques, as indicated, using an RNeasy plant mini kit

(Qiagen). RNA was quantified by UV spectrophotometry (Nano- Photometer, IMPLEN) and its integrity was visually assessed on ethidium bromide-stained 2% agarose gels. Treatment with DNase I Amplification Grade (DNase I, Amp Grade, Invitrogen) was performed.

RNA was reverse transcribed to cDNA by the RNase H+, iScript cDNA synthesis kit (Bio-Rad). Real-time qPCR was performed in a iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad) with the iQ5 Optical System Software Version 2.0 (Bio-Rad).

Specific primers for the genes of interest and three reference genes (Dataset S2) were used. Three biological replicates were taken for each plant line, and three technical repeats were performed for each biological replicate. The data were analyzed with the qbasePLUS2 software (Biogazelle).

Equipment and Settings.The blot images were taken with a Motorola MotoG3 camera at 2340× 4160 pixel resolution and 24 bits per pixel. Images were cropped to the bands of interest using Power- Point. The entire blot images (including the protein size marker) are shown in Fig. S5B. Images of adult plants were taken with the same camera and settings and cropped to the area of interest.

The confocal images were taken from a Nikon ECLIPSE TE2000-S microscope, with fixed spectrum filters, with a Nikon D-ECLIPSE C1 confocal scanhead and the software NIS Element 4.10 from Nikon. The GFP and YFP fluorescences were detected with excitation at 488 nm and emission at 500–530 nm; the red fluorescence from the MitotrackerOrange CMTMRos dye (Invitrogen), the mitochondria-mCherry marker plasmid {mt [29 aa ScCOX4]-mCherry-rb (41)} and the mRFP-SKL marker plasmid (44) were detected with excitation at 552 nm and emission at 593–640 nm. Pictures were taken with a 40× WI objective at 1024× 1024 pixel resolution (12 bit). The LUT is linear and covers the full range of the data. Single fluorochrome images were merged with the Adobe Photoshop program. Im- ages were cropped to a single representative cell with the Pow- erPoint program.

Statistical Analysis.For the IP–MS experiment, a standard two- sided t test with false discovery rate = 0.05 and S0 = 1 was performed with Perseus software (34). Number of biological replicates was n= 3, for each line. No samples were discarded as outliers.

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order in list

RATIO pSLO2::

SLO2- GFP vs

WT p-value RATIO p35S::

SLO2- GFP vs WT p-value

Protein

s e d i t p e P s

n o i t p i r c s e D n i e t o r P s D I

Unique Peptides

Sequence Coverage

[%]

1 33,289 1.29E-03 3,561 3.03E-03 Q9SIT7;

Q9FFG8

>sp|Q9SIT7|PP151_ARATH

Pentatricopeptide repeat-containing protein At2g13600 (SLO2)

>sp|Q9FFG8|PP417_ARATH

Pentatricopeptide repeat-containing protein At5g44230 (MEF57)

23 23 39.7

2 28,028 2.46E-06 1,699 1.84E-07 Q8L7B5;

F4IVR2;

P21238

>sp|Q8L7B5|CH60B_ARATH Chaperonin CPN60-like 1, mitochondrial At2g33210

(HSP60.2) 30 16 56.6

3 21,128 6.21E-06 1,711 1.71E-07 Q8H1Y0;

P52901;

C0Z306;

Q42066

>sp|Q8H1Y0|ODPA2_ARATH Pyruvate

dehydrogenase E1 component subunit 13 13 35.1

4 20,038 1.45E-03 1,464 4.30E-03 P29197

>sp|P29197|CH60A_ARATH Chaperonin CPN60, mitochondrial At3g23990

(HSP60.3B) 34 20 63.8

5 6,854 1.77E-06 632 5.35E-05 Q9SJ60

>tr|Q9SJ60|Q9SJ60_ARATH Putative

uncharacterized protein At2g35900 6 6 43.2

6 5,328 8.21E-05 295 3.40E-04 CON__P 42212mu t;CON__

P42212 >P42212mut SWISS-PROT:P42212mut|GFP 2 2 8.4 7 5,254 6.39E-06 5,056 1.83E-07 P56805

>sp|P56805|RR15_ARATH 30S ribosomal

8 1 1 c

i t s a l p o r o l h c , 5 1 S n i e t o r p 8 5,237 2.20E-03 2,577 2.90E-03

Q9SR37;

Q42249

>sp|Q9SR37|BGL23_ARATH Beta-glucosidase

2 . 2 4 4 1 9 1 3

2 9 2,508 4.59E-06 3,063 6.35E-08 P59223

>sp|P59223|RS131_ARATH 40S ribosomal

3 4 1 7 n

i e t o r p 10 2,183 2.65E-02 1,845 2.66E-02

Q42044;

Q9LWA0 >tr|Q42044|Q42044_ARATH At2g45180 3 3 17.9

11 2,142 9.18E-06 19 1.17E-01 Q9LEX5

>sp|Q9LEX5|PP290_ARATH

Pentatricopeptide repeat-containing protein

At3g60980 (P486) 9 9 25.5

12 1,896 2.49E-03 217 7.54E-03 Q9ZPW5

;F4JQE9;

O23223

>tr|Q9ZPW5|Q9ZPW5_ARATH AAA-type

ATPase-like protein At2g18330 13 10 24.4

13 1,379 8.56E-05 455 2.19E-06 O04310;

Q56W96;

O04315

>tr|O04310|O04310_ARATH Jacalin-like lectin

7 . 7 3 8 1 9 1 n

i e t o r p g n i n i a t n o c - n i a m o d

14 1,373 4.66E-06 85 1.94E-04 Q9M3A8

>sp|Q9M3A8|PP273_ARATH

At3g49240 (NUWA) 8 8 16.2

15 1,278 3.97E-06 1 1.00E+0

0 Q9LEX6

>sp|Q9LEX6|PP289_ARATH

At3g60960 (P487) 5 5 11.2

16 1,263 2.60E-03 1,094 2.77E-03 O80448;

O80446

>sp|O80448|PDX11_ARATH Pyridoxal

4 . 0 2 4 5 1

. 1 X D P n i e t o r p s i s e h t n y s o i b 17 1,043 9.83E-07 827 4.09E-05

Q8W1E4

;Q9LN82 >tr|Q8W1E4|Q8W1E4_ARATH At1g12650 3 3 9.7

18 1,031 8.68E-03 48 5.36E-02 Q93ZM7

>sp|Q93ZM7|CH60C_ARATH Chaperonin CPN60-like 2, mitochondrial At3g13860

(HSP60.3A) 19 19 42

19 721 7.29E-07 917 2.00E-07 P42732;

B3H631

>sp|P42732|RR13_ARATH 30S ribosomal

2 . 1 1 2 2 c

i t s a l p o r o l h c , 3 1 S n i e t o r p

20 707 1.36E-06 301 7.74E-08 B5TM95;

P93306;

G1C2X4;

G1C2V1;

A7KNI1

>tr|B5TM95|B5TM95_ARATH NADH

9 . 1 1 4 4 7

t i n u b u s e s a n e g o r d y h e d

21 578 2.35E-05 10 1.67E-05 Q9LDZ0;

O49369;

B3H683 >tr|Q9LDZ0|Q9LDZ0_ARATH At5g09590 5 3 7.5 22 492 1.41E-03 281 2.07E-03 Q9C8Y9

>sp|Q9C8Y9|BGL22_ARATH Beta-glucosidase

8 . 4 4 2 1 2 2 2

2

23 387 1.36E-04 77 1.60E-03 Q9ZQE5

>sp|Q9ZQE5|PP153_ARATH

At2g15690 (DYW2) 3 3 6.6

24 365 2.15E-02 278 2.49E-02 Q9LXY9;

Q6NM71

>tr|Q9LXY9|Q9LXY9_ARATH Putative

2 . 8 2 7 7 n

i e t o r p d e z i r e t c a r a h c n u

25 361 1.04E-07 287 1.01E-04 O81293;

F4JHI1;Q

0WLH1 >tr|O81293|O81293_ARATH AT4g02400 1 1 1.5 A

Fig. S1. (Continued)

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>sp|Q9SIT7|PP151_ARATH Pentatricopeptide repeat-containing protein At2g13600 (SLO2)

MATKSFLKLAADLSSFTDSSPFAKLLDSCIKSKLSAIYVRYVHASVIKSGFSNEIFIQNRLIDAYSKCGSLEDGRQVFDKMPQRNIYTWNSVVTGLTKLGFLD EADSLFRSMPERDQCTWNSMVSGFAQHDRCEEALCYFAMMHKEGFVLNEYSFASVLSACSGLNDMNKGVQVHSLIAKSPFLSDVYIGSALVDMYSKCG NVNDAQRVFDEMGDRNVVSWNSLITCFEQNGPAVEALDVFQMMLESRVEPDEVTLASVISACASLSAIKVGQEVHGRVVKNDKLRNDIILSNAFVDMYAK CSRIKEARFIFDSMPIRNVIAETSMISGYAMAASTKAARLMFTKMAERNVVSWNALIAGYTQNGENEEALSLFCLLKRESVCPTHYSFANILKACADLAELHL GMQAHVHVLKHGFKFQSGEEDDIFVGNSLIDMYVKCGCVEEGYLVFRKMMERDCVSWNAMIIGFAQNGYGNEALELFREMLESGEKPDHITMIGVLSAC GHAGFVEEGRHYFSSMTRDFGVAPLRDHYTCMVDLLGRAGFLEEAKSMIEEMPMQPDSVIWGSLLAACKVHRNITLGKYVAEKLLEVEPSNSGPYVLLSN MYAELGKWEDVMNVRKSMRKEGVTKQPGCSWIKIQGHDHVFMVKDKSHPRKKQIHSLLDILIAEMRPEQDHTEIGSLSSEEMDYSSNLLWDNAM

>sp|Q9FFG8|PP417_ARATH Pentatricopeptide repeat-containing protein At5g44230 (MEF57)

MTVAHSHRFSTAVNPINISLLSKQLLQLGRTSNNSGTFSEISNQKELLVSSLISKLDDCINLNQIKQIHGHVLRKGLDQSCYILTKLIRTLTKLGVPMDPYARRV IEPVQFRNPFLWTAVIRGYAIEGKFDEAIAMYGCMRKEEITPVSFTFSALLKACGTMKDLNLGRQFHAQTFRLRGFCFVYVGNTMIDMYVKCESIDCARKVF DEMPERDVISWTELIAAYARVGNMECAAELFESLPTKDMVAWTAMVTGFAQNAKPQEALEYFDRMEKSGIRADEVTVAGYISACAQLGASKYADRAVQIA QKSGYSPSDHVVIGSALIDMYSKCGNVEEAVNVFMSMNNKNVFTYSSMILGLATHGRAQEALHLFHYMVTQTEIKPNTVTFVGALMACSHSGLVDQGRQ VFDSMYQTFGVQPTRDHYTCMVDLLGRTGRLQEALELIKTMSVEPHGGVWGALLGACRIHNNPEIAEIAAEHLFELEPDIIGNYILLSNVYASAGDWGGVL RVRKLIKEKGLKKTPAVSWVVDKNGQMHKFFPGNLNHPMSNKIQDKLEELVERLTVLGYQPDLSSVPYDVSDNAKRLILIQHTEKLALAFSLLTTNRDSTITI MKNLRMCLDCHKFMRLASEVTGKVIIMRDNMRFHHFRSGDCSCGDFW

>sp|Q8L7B5|CH60B_ARATH Chaperonin CPN60-like 1, mitochondrial At2g33210 (HSP60.2)

MYRLVSNVASKARIARKCTSQIGSRLNSTRNYAAKDIRFGVEARALMLRGVEDLADAVKVTMGPKGRNVIIEQSWGAPKVTKDGVTVAKSIEFKDRIKNVG ASLVKQVANATNDVAGDGTTCATVLTRAIFTEGCKSVAAGMNAMDLRRGIKLAVDTVVTNLQSRARMISTSEEIAQVGTISANGDREIGELIAKAMETVGKE GVITIQDGKTLFNELEVVEGMKIDRGYISPYFITNPKTQKCELEDPLILIHEKKISNINAMVKVLELALKKQRPLLIVAEDVESDALATLILNKLRANIKVCAVKAP GFGENRKANLHDLAALTGAQVITEELGMNLDNIDLSMFGNCKKVTVSKDDTVVLDGAGDKQAIGERCEQIRSMVEASTSDYDKEKLQERLAKLSGGVAVL KIGGASETEVSEKKDRVTDALNATKAAVEEGIVPGGGVALLYASKELEKLSTANFDQKIGVQIIQNALKTPVYTIASNAGVEGAVVVGKLLEQDNPDLGYDA AKGEYVDMIKAGIIDPLKVIRTALVDAASVSSLLTTTEAVVTEIPTKEVASPGMGGGGMGGMGGMGGMGGMGF

>sp|P29197|CH60A_ARATH Chaperonin CPN60, mitochondrial CPN60 At3g23990 (HSP60.3B)

MYRFASNLASKARIAQNARQVSSRMSWSRNYAAKEIKFGVEARALMLKGVEDLADAVKVTMGPKGRNVVIEQSWGAPKVTKDGVTVAKSIEFKDKIKNV GASLVKQVANATNDVAGDGTTCATVLTRAIFAEGCKSVAAGMNAMDLRRGISMAVDAVVTNLKSKARMISTSEEIAQVGTISANGEREIGELIAKAMEKVG KEGVITIQDGKTLFNELEVVEGMKLDRGYTSPYFITNQKTQKCELDDPLILIHEKKISSINSIVKVLELALKRQRPLLIVSEDVESDALATLILNKLRAGIKVCAIK APGFGENRKANLQDLAALTGGEVITDELGMNLEKVDLSMLGTCKKVTVSKDDTVILDGAGDKKGIEERCEQIRSAIELSTSDYDKEKLQERLAKLSGGVAV LKIGGASEAEVGEKKDRVTDALNATKAAVEEGILPGGGVALLYAARELEKLPTANFDQKIGVQIIQNALKTPVYTIASNAGVEGAVIVGKLLEQDNPDLGYDA AKGEYVDMVKAGIIDPLKVIRTALVDAASVSSLLTTTEAVVVDLPKDESESGAAGAGMGGMGGMDY

>sp|Q9LEX5|PP290_ARATH Pentatricopeptide repeat-containing protein At3g60980 (P486)

MSLIGRLNLGRRFCTAVPRRSEDIMSNPDCRPSDLCLRVSYLIRCVGDLDTAAKYARLAVFTSIKSESTTTICQSIIGGMLRDKRLKDAYDLYEFFFNQHNLR PNSHCWNYIIESGFQQGLVNDALHFHHRCINSGQVHDYPSDDSFRILTKGLVHSGRLDQAEAFLRGRTVNRTTYPDHVAYNNLIRGFLDLGNFKKANLVLG EFKRLFLIALSETKDDLHHSNYENRVAFLMATFMEYWFKQGKQVEAMECYNRCVLSNRLLVCAETGNALLKVLLKYGEKKNAWALYHELLDKNGTGKGCL DSDTIKIMVDECFDMGWFSEAMETYKKARPKNDYLSDKYIITRFCENRMLSEAESVFVDSLADDFGYIDVNTYKTMIDAYVKAGRIHDAIKTSNKMIDATLKE VSHLF

>sp|Q9M3A8|PP273_ARATH Pentatricopeptide repeat-containing protein At3g49240 (NUWA)

MSISKAAFLNHLQTLSRSYRHRVLPQPFLAVRYMSFATQEEAAAERRRRKRRLRMEPPVNSFNRSQQQQSQIPRPIQNPNIPKLPESVSALVGKRLDLHN HILKLIRENDLEEAALYTRHSVYSNCRPTIFTVNTVLAAQLRQAKYGALLQLHGFINQAGIAPNIITYNLIFQAYLDVRKPEIALEHYKLFIDNAPLNPSIATFRIL VKGLVSNDNLEKAMEIKEDMAVKGFVVDPVVYSYLMMGCVKNSDADGVLKLYQELKEKLGGFVDDGVVYGQLMKGYFMKEMEKEAMECYEEAVGENS KVRMSAMAYNYVLEALSENGKFDEALKLFDAVKKEHNPPRHLAVNLGTFNVMVNGYCAGGKFEEAMEVFRQMGDFKCSPDTLSFNNLMNQLCDNELLA EAEKLYGEMEEKNVKPDEYTYGLLMDTCFKEGKIDEGAAYYKTMVESNLRPNLAVYNRLQDQLIKAGKLDDAKSFFDMMVSKLKMDDEAYKFIMRALSEA GRLDEMLKIVDEMLDDDTVRVSEELQEFVKEELRKGGREGDLEKLMEEKERLKAEAKAKELADAEEKKKAQSINIAALIPPKAVEEKKETAKLLWENEAGG VEEADVVEMAKGVEAGGSNGQDPPSC

>sp|Q9LEX6|PP289_ARATH Pentatricopeptide repeat-containing protein At3g60960 (P487)

MSLLRRIVRNSTVSKAVPVAKAFMPYPLGRDPSSLPKLDPVSISYIDSRPISLRYRVRAMIEMSNLDEASKLSRLAVLNGFLVDRDTVFICNSVIGAMCSAKR YDDAISLFNYFFNESQTLPNTLSCDLIIKAHCDQGHVDDALELYRHILLDGRVAPGIETYMILAKALVDAKRFDEACVLARSMSCCSFMVYDILIRGFLDIGNF VKASQIFEELKGLDSKLPGREYHKANAIFNVSFMNYWFKQGKDEEAMEILANLEDAQVLNPIVGNRVLQVLVKHGKKTEAWELFGEMIEICDSETVDIMSE YFSEKTVPFERLRKTCYRKMIVSLCEHGKVSDAEKLFAEMFTDVDGGDLLVGPDLLIFRAMINGYVSVGRVDDAIKTLNKMRISNLRKLAIHQAP

>sp|Q93ZM7|CH60C_ARATH Chaperonin CPN60-like 2, mitochondrial At3g13860 (HSP60.3A)

MYRVLSKLSSSIGSSTSRKLVSGRIISSRNYAAKDISFGIGARAAMLQGVSEVAEAVKVTMGPKGRNVIIESSYGGPKITKDGVTVAKSISFQAKAKNIGAEL VKQVASATNKVAGDGTTCATVLTQAILIEGCKSVAAGVNVMDLRVGINMAIAAVVSDLKSRAVMISTPEEITQVATISANGEREIGELIARAMEKVGKEGVIT VADGNTLDNELEVVEGMKLARGYISPYFITDEKTQKCELENPIILIHEKKISDINSLLKVLEAAVKSSRPLLIVAEDVESDALAMLILNKHHGGLKVCAIKAPGF GDNRKASLDDLAVLTGAEVISEERGLSLEKIRPELLGTAKKVTVTRDDTIILHGGGDKKLIEERCEELRSANEKSTSTFDQEKTQERLSKLSGGVAVFKVGG ASESEVGERKDRVTDALNATRAAVEEGIIPGGGVALLYATKALDNLQTENEDQRRGVQIVQNALKAPAFTIAANAGYDGSLVVGKLLEQDDCNFGFDAAK GKYVDMVKAGIIDPVKVIRTALTDAASVSLLLTTTEASVLVKADENTPNHVPDMASMGM

>sp|Q9ZQE5|PP153_ARATH Pentatricopeptide repeat-containing protein At2g15690 (DYW2)

MSSLMAIRCARTQNIVTIGSLLQLRSSFPRLSSQFHFSGTLNSIPIKHLSTSAAANDYHQNPQSGSPSQHQRPYPPQSFDSQNQTNTNQRVPQSPNQWST QHGGQIPQYGGQNPQHGGQRPPYGGQNPQQGGQMSQYGGHNPQHGGHRPQYGGQRPQYGGPGNNYQNQNVQQSNQSQYYTPQQQQQPQPPR SSNQSPNQMNEVAPPPSVEEVMRLCQRRLYKDAIELLDKGAMPDRECFVLLFESCANLKSLEHSKKVHDHFLQSKFRGDPKLNNMVISMFGECSSITDAK RVFDHMVDKDMDSWHLMMCAYSDNGMGDDALHLFEEMTKHGLKPNEETFLTVFLACATVGGIEEAFLHFDSMKNEHGISPKTEHYLGVLGVLGKCGHL VEAEQYIRDLPFEPTADFWEAMRNYARLHGDIDLEDYMEELMVDVDPSKAVINKIPTPPPKSFKETNMVTSKSRILEFRNLTFYKDEAKEMAAKKGVVYVP DTRFVLHDIDQEAKEQALLYHSERLAIAYGIICTPPRKTLTIIKNLRVCGDCHNFIKIMSKIIGRVLIVRDNKRFHHFKDGKCSCGDYW

B

Fig. S1. Putative SLO2 interacting proteins. (A) Overview of the IP–MS results. The table shows an overview of the first 25 interactors with an indication of the ratio vs. WT and the P value obtained from the three repeats for each genotype used. A P value<0.05 was taken as cutoff value for the level of significance (Perseus statistical analysis). Significant data were ordered based on the pSLO2::SLO2-GFP ratio values and the resulting top 25 candidates are shown. Both IP experiments for pSLO2::SLO2-GFP and p35S::SLO2-GFP are shown with similar results. Red text indicates bait, green text indicates GFP, blue text indicates selected proteins. For row number 1, the actual bait protein is Q9SIT7 (red), but based on the identified peptides, MS analysis cannot distinguish between this protein and Q9FFG8 (blue). (B) Coverage of the detected peptides by IP–MS. The coverage from A is highlighted in gray in the respective protein sequences.

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HSP60.3B-GFP

GFP mitotracker/mt-rb merge ilght

SLO2DYWHSP60P type A

SLO2-GFP

P486-GFP

P487-GFP

HSP60.2-GFP MEF57-GFP

DYW2-GFP

NUWA-GFP

HSP60.3A-GFP

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merge light GFP

DYWP type

DYW2-GFP

NUWA-GFP B

Fig. S2. Subcellular localization of SLO2 and its interacting partners MEF57, DYW2, P486, P487, NUWA, HSP60.2, HSP60.3A, and HSP60.3B. (A) N. benthamiana leaves infiltrated with SLO2-/MEF57-/DYW2-/P486-/P487-/NUWA-/HSP60.2-/HSP60.3A-/HSP60.3B-GFP constructs and analyzed by confocal microscopy 3 d after infiltration. Green and red fluorescences are indicative of the localization of the GFP protein and the mitotracker/mt-rb mitochondria markers, respectively. Representative individual cells of at least two independent experiments are shown, including their merged and light fields. Single cells were cropped from the original image. (Scale bar, 50μm.) White arrows point to a mitochondrial signal dot. The contrast/brightness was adjusted for good visualization.

(B) N. benthamiana leaves infiltrated with DYW2-/NUWA-GFP constructs and analyzed by confocal microscopy 3 d after infiltration, as indicated in A. Yellow arrows point to a chloroplast signal. A second image with lower laser gain is shown for the DYW2-GFP construct.

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YFP mitotracker/mt-rb merge ilght

YFP mRFP-SKL merge ilght

YFP mitotracker/mt-rb merge light

YFP mRFP-SKL merge light

A

cYFP-APEM9 nYFP-PEX6

cYFP-APEM9 nYFP-PEX6 nYFP-APEM9 cYFP-PEX6

nYFP-APEM9 cYFP-PEX6 B

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cYFP-APEM9 HSP60.3B-nYFP

YFP mt-rb merge light

cYFP-APEM9 HSP60.2-nYFP cYFP-APEM9 P487-nYFP cYFP-APEM9 P486-nYFP cYFP-APEM9 SLO2-nYFP

cYFP-APEM9 MEF57-nYFP

cYFP-APEM9 DYW2-nYFP

cYFP-APEM9 NUWA-nYFP

cYFP-APEM9 HSP60.3A-nYFP SLO2DYWP typeHSP60

C

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nYFP-APEM9 HSP60.3B-cYFP

YFP mt-rb merge light

SLO2DYWP typeHSP60

nYFP-APEM9 HSP60.2-cYFP nYFP-APEM9 NUWA-cYFP nYFP-APEM9 SLO2-cYFP

nYFP-APEM9 MEF57-cYFP

nYFP-APEM9 DYW2-cYFP

nYFP-APEM9 HSP60.3A-cYFP D

Fig. S3. BiFC experimental setup. (A) Background of the GFP/YFP signal in the confocal microscopy analysis. N. benthamiana leaves infiltrated without any construct (negative control) and analyzed by confocal microscopy 3–6 d after infiltration. Green and red fluorescences are indicative of the localization of the reconstituted whole YFP protein (protein interaction) and the mitotracker/mt-rb mitochondria markers or mRFP-SKL peroxisome marker, respectively. Rep- resentative individual cells of at least two independent experiments are shown, including their merged and light fields. Single cells were cropped from the original image. (Scale bar, 50μm.) The contrast/brightness was adjusted for good visualization. (B) Positive controls for protein interaction under BiFC experimental conditions. N. benthamiana leaves infiltrated with c/nYFP-APEM9 and n/cYFP-PEX6 constructs and analyzed by confocal microscopy 3 d after infiltration, as indicated in A. Purple arrows point to a peroxisomal signal dot. (C and D) Negative controls for protein interaction under BiFC experimental conditions. N. benthamiana leaves infiltrated with SLO2-/MEF57-/DYW2-/P486-/P487-/NUWA-/HSP60.2-/HSP60.3A-/HSP60.3B-nYFP (C) or SLO2-/MEF57-/DYW2-/

NUWA-/HSP60.2-/HSP60.3A-/HSP60.3B-cYFP (D) together with the respective c/nYFP-APEM9 constructs and analyzed by confocal microscopy 3–6 d after infiltration, as indicated in A.

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DYW2-cYFP HSP60.3B-nYFP MEF57-cYFP HSP60.3B-nYFP SLO2-cYFP HSP60.3A-nYFP

HSP60

SLO2-cYFP HSP60.2-nYFP

SLO2-cYFP HSP60.3B-nYFP SLO2

HSP60 DYW

MEF57-cYFP HSP60.2-nYFP

MEF57-cYFP HSP60.3A-nYFP

MEF57-nYFP HSP60.3B-cYFP MEF57-nYFP HSP60.2-cYFP

DYW2-cYFP HSP60.2-nYFP

DYW2-cYFP HSP60.3A-nYFP

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NUWA-nYFP HSP60.3B-cYFP P487-nYFP HSP60.3B-cYFP P486-nYFP HSP60.3B-cYFP

HSP60 P type

P487-nYFP HSP60.2-cYFP

P487-nYFP HSP60.3A-cYFP P486-nYFP HSP60.2-cYFP

P486-nYFP HSP60.3A-cYFP

NUWA-nYFP HSP60.2-cYFP

NUWA-nYFP HSP60.3A-cYFP

Fig. S4. Interaction of SLO2 and its PPR partners MEF57, DYW2, P486, P487, and NUWA with mtHSP60 import factors in mitochondria. N. benthamiana leaves infiltrated with SLO2-/MEF57-/DYW2-cYFP or MEF57-/P486-/P487-/NUWA-nYFP together with HSP60.2-/HSP60.3A-/HSP60.3B-nYFP or HSP60.2-/HSP60.3A-/

HSP60.3B-cYFP constructs, respectively, and analyzed by confocal microscopy 3 d after infiltration, as indicated in Fig. S3A. White arrows point to a mitochondrial signal dot.

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NUWA-HA DYW2-GFP

NUWA-HA

v

HA-APEM9 DYW2-GFP HA-APEM9 HA-APEM9 NUWA-GFP

SLO2-HA GFP-APEM9

SLO2-HA

SLO2-HA MEF57-GFP

SLO2-HA

SLO2-HA MEF57-GFP

SLO2-HA

GFP-IP INPUT

SLO2-HA DYW2-GFP

SLO2-HA SLO2-HA DYW2-GFP

SLO2-HA

GFP-IP INPUT

GFP-Ab

HA-Ab (+GFP-Ab)

GFP-Ab

NUWA-HA SLO2-GFP

NUWA-HA

HA-Ab (+GFP-Ab)

GFP-Ab

HA-Ab (+GFP-Ab)

GFP-Ab GFP-IP

INPUT

SLO2-HA HSP60.3B-GFP

SLO2-HA

HSP60.3B-HA DYW

2-GFP

HSP60.3B-HA NUW

A-GFP

HSP60.3B-HA HSP60.2-HA DYW2-GFP HSP60.2-HA NUWA-GFP

HSP60.2-HA

SLO2-HA HSP60.2-GFP

SLO2-HA

HA-Ab (+GFP-Ab)

GFP-Ab

HA-Ab (+GFP-Ab) GFP-IP

INPUT B

HA-Ab (+GFP-Ab)

GFP-Ab

HA-Ab (+GFP-Ab)

GFP-Ab GFP-IP

INPUT

SLO2-HA DYW2-GFP

SLO2-HA +NUWA-His SLO2-HA DYW2-GFP +NUWA-His

HA-APEM9 HA-APEM9 NUWA-GFP

SLO2-HA GFP-APEM9

SLO2-HA

HA-Ab GFP-Ab HA-Ab GFP-Ab GFP-IP

INPUT

SLO2 DYW2 NUWA

HA-APEM9 DYW2-GFP

A

Fig. S5. CoIP experiment setup. (A) Negative controls for protein interaction under CoIP experimental conditions. Total protein extracts (input) and GFP-immunoprecipitated proteins (GFP-IP) from N. benthamiana leaves infiltrated with SLO2-HA or HA-APEM9 together with GFP-APEM9 or DYW2-/NUWA-GFP constructs, 3–6 d after infiltration, analyzed by Western blot with anti-HA and anti-GFP antibodies (HA-Ab and GFP-Ab). N. benthamiana leaves infiltrated with the corresponding HA-tag constructs alone were taken as negative controls of the immunoprecipitation. Representative blots of at least two independent replicates for each combination are shown. Blots were cropped to the bands of interest (full-length blots in B). The contrast/brightness was adjusted for good visualization. (B) Full-length blots of the CoIP experiments. Both antibodies were checked consecutively on the same membrane (first anti-GFP, second anti-HA) and pictures after each antibody checking are shown. Black arrows point at the bands of interest for each antibody.

White squares hide nonrelevant runs/samples for visual and interpretation clarity. The contrast/brightness was adjusted for good visualization. Size markers are as follows: 170, 130, 100, 70 (red), 55, 40, 35, 25, and 15 kDa. The respective molecular weights are as follows: SLO2-HA, 84.03 kDa; SLO2-GFP, 105.04 kDa; DYW2-GFP, 92.56 kDa; MEF57-GFP, 100.62 kDa;

NUWA-HA, 77.23 kDa; NUWA-GFP, 98.24 kDa; HSP60.2-HA, 67.92 kDa; HSP60.2-GFP, 88.93 kDa; HSP60.3B-HA, 66.41 kDa; HSP60.3B-GFP, 87.42 kDa; HA-APEM9, 43.41 kDa; and GFP- APEM9, 64.42 kDa.

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SLO2-nYFP MEF57-cYFP

SLO2-nYFP DYW2-cYFP SLO2-nYFP MEF57-cYFP +HSP60.2-HA or +HSP60.3A-HA or +HSP60.3B-HA or +P486-HA or +P487-HA

SLO2-nYFP MEF57-cYFP +NUWA-HA

SLO2-nYFP DYW2-cYFP +HSP60.2-HA or +HSP60.3A-HA or +HSP60.3B-HA or +P486-HA or +P487-HA

SLO2-nYFP DYW2-cYFP +NUWA-HA DYW

P type SLO2

SLO2

SLO2-cYFP P486-nYFP

SLO2-cYFP P487-nYFP

SLO2-cYFP NUWA-nYFP A

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P type DYW

MEF57-cYFP NUWA-nYFP MEF57-cYFP P486-nYFP

MEF57-cYFP P487-nYFP

DYW2-cYFP NUWA-nYFP DYW2-cYFP P486-nYFP

DYW2-cYFP P487-nYFP B

Fig. S6. Interaction between SLO2 and its PPR interacting partners. (A) Interaction of SLO2 with MEF57, DYW2, P486, P487, and NUWA in mitochondria.

N. benthamiana leaves infiltrated with SLO2-nYFP together with MEF57-/DYW2-cYFP in the absence or presence of HSP60.2-/HSP60.3A-/HSP60.3B-/P486-/P487- HA or NUWA-HA constructs, or with SLO2-cYFP together with P486-/P487-/NUWA-nYFP constructs, analyzed by confocal microscopy 3–6 d after infiltration, as indicated in Fig. S3A. White arrows point to a mitochondrial signal dot. Images for combinations without NUWA-HA or NUWA-nYFP, with really low fluorescent signal, were taken at a higher laser gain condition. For a proper comparison between proteins, images for both DYW-type PPR proteins (MEF57 and DYW2) were taken under the same conditions. For the combinations in the presence of HSP60.2-/HSP60.3A-/HSP60.3B-/P486-/P487-HA constructs, the SLO2- nYFP/MEF57-cYFP+ HSP60.3A-HA and the SLO2-nYFP/DYW2-cYFP + P486-HA images are shown as representative of all of the listed combinations.

(B) Interaction of the SLO2 binding partners MEF57 and DYW2, with P486, P487, and NUWA in mitochondria. N. benthamiana leaves were infiltrated with MEF57-/DYW2-cYFP together with P486-/P487-/NUWA-nYFP constructs, analyzed by confocal microscopy 3 d after infiltration, as indicated in Fig. S3B. White arrows point to a mitochondrial signal dot and yellow arrows to a chloroplast signal. Images for combinations without NUWA-nYFP, with really low fluorescent signal, were taken at a higher laser gain condition. For a proper comparison between proteins, images for both DYW-type PPR proteins (MEF57 and DYW2) were taken under the same conditions.

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A

B 0 2 4 6 8 10 12

DYW2relativeexpressionlevel

WT DYW2-GFP-OE1 DYW2-GFP-OE2

0 1 2 3 4 5 6

NUWArelativeexpressionlevel

WT DYW2-GFP-OE1 DYW2-GFP-OE2

Fig. S7. DYW2 and NUWA expression levels in DYW2-GFP-OE lines. Total RNA from rosette leaves of WT and two independent DYW2-GFP-OE lines analyzed by RT-qPCR with specific primers for DYW2 (A) and NUWA (B). The relative expression levels with respect to three reference genes (Actin 2, Elongation factor 1 ALPHA, and RGS1-HXK1 INTERACTING PROTEIN 1) are shown. The columns and error bars represent the mean and SD values, respectively, of three biological replicates (represented by dots).

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nad1-743 (L248P) A

nad9-92 (F31S) B

slo2-3 Complemented

slo2-3

WT slo2-2 Complemented

slo2-2

Complemented mef57

WT mef57

AT T T G AT C T G AT T T G

Fig. S8. RNA editing analysis of slo2 and mef57 mutants. (A) Editing analysis of seedlings from WT, slo2-2, slo2-3, complemented slo2-2 and complemented slo2-3 lines, for the nad1-743 site. The sequencing chromatograms are shown. The editing site is marked with a square. The amino acid change is indicated on the left. The WT line was analyzed twice and each independent mutant line once. (B) Editing analysis of WT, mef57, and complemented mef57 lines, for the nad9-92 site, as indicated in A. Seedlings, leaves, and siliques of each line were analyzed once, with similar results.

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0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

MEF57 relativeecpressionlevel

MEF57

F R

1,534 bp LB

N585176

1,974 bp / 657 aa

DYW

L1 P S S P L1 S P L2 S E E+

KO WT KO WT AT3G43442 (LB/R) AT3G43442 (F/R)

GFP (F/R) KO COMP A

B

C

MEF57 (LB/F) COMP1 2 3 4

MEF57 (F/R) COMP1 2 3 4 MEF57 (LB/F)

KO WT

MEF57 (F/R) KO WT

WT mef57 Complemented mef57 KO WT AT3G43442 (LB/F)

qF qR

Fig. S9. Isolation of a homozygous T-DNA insertion mef57 line and the respective complemented line. (A) Scheme of the primary structure of MEF57 and T-DNA insertion site. The ID code is indicated on the Left and the sizes in base pairs (bp) and amino acids (aa) on the Right. The different PPR protein motifs are indicated with colors and capital letters (L1, brown; L2, red; S, yellow; P, orange; E, light green; E⁺, dark green; DYW, blue). The T-DNA insertion site is indicated with a big white triangle, with the name of the corresponding mutant line inside and the distance to the start codon in base pairs outside. The position and name of the primers used for genotyping and expression analyses are indicated with continuous and dashed arrows, respectively. (B) Genotyping of the mef57 and the complemented mef57 lines. gDNA from leaves analyzed by standard PCR with specific primers, as indicated. The contrast/brightness was adjusted for good visualization. (C) MEF57 expression levels in mef57 and the complemented mef57 lines. Total RNA from siliques of WT, mef57, and complemented mef57 lines were analyzed by RT-qPCR with MEF57 specific primers. The relative expression levels with respect to two reference genes (Elongation factor 1 ALPHA and RGS1-HXK1 INTERACTING PROTEIN 1) are shown. The columns and error bars represent the mean and SD values, respectively, of three biological replicates (represented by dots).

Dataset S1. RNA editing analysis of DYW2-GFP-OE lines

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Dataset S2. Oligonucleotides used for cloning (A), genotyping (B), and qPCR analyses (C) Dataset S2