Supp- Figure 2 Confocal micrograph of N. benthamiana tissues transiently expressing 35S:YFP-PDCB1. PDCB1 was targeted to plasmodesmata (twin punctate

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Supplemental Data. Simpson et al. (009).

The series is illustrated as individual confocal sections at positions, 5 and 0 through a depth of the cell comprising

1 Supplemental Data. Simpson et al. (009). n rabidopsis GPI-anchor plasmodesmal neck protein with callosebinding activity and potential to regulate cell-to-cell trafficking. 5 0 stack Supp- Figure Confocal series through rabidopis leaf epidermal pavement cells expressing 35S:YFP-PDCB. The series is illustrated as individual confocal sections at positions, 5 and 0 through a depth of the cell comprising 6 m. The remaining panel (stack) shows all 0 optical sections combined. The predominant labelling is as a punctate pattern on the wall with little evidence of cytoplasmic labelling. The exception is the single stoma (arrow) present in the centre of the image where the punctate patterning is less evident. Bar = 0 μm.

2 Supp- Figure Confocal micrograph of N. benthamiana tissues transiently expressing 35S:YFP-PDCB. PDCB was targeted to plasmodesmata (twin punctate spots on the cell wall) but with the high levels of transient expression in N. benthamiana, the protein also appeared to accumulate elsewhere on the plasma membrane. Bar = 0 μm.

700 V 800 V 900 V 000 V Supp- Figure 3 Confocal microscopy of unplasmolysed rabidopsis leaf spongy mesophyll cells expressing 35S:YFP-PDCB viewed at increasing power settings.

3 700 V 800 V 900 V 000 V Supp- Figure 3 Confocal microscopy of unplasmolysed rabidopsis leaf spongy mesophyll cells expressing 35S:YFP-PDCB viewed at increasing power settings. Micrographs show the same confocal section viewed with the confocal power settings at 700, 800, 900 and 000 Volts. The image at 000 V is combined with the DIC image to show the cell wall boundaries and the restricted points of contact between adjacent cells. Even at the highest power settings the fluorescence is restricted to Pds. Bars= 0 μm.

In the absence of the inhibitor the apoplastic spaces () have become filled with fluorescent protein leached from the wall; intracellular spaces

4 I I I I Supp- Figure 4 rabidopsis leaf epidermal pavement cells expressing 35S:YFP-PDCB viewed after plasmolysis without pretreatment with the phospholipase C inhibitor. In the absence of the inhibitor the apoplastic spaces () have become filled with fluorescent protein leached from the wall; intracellular spaces (I) remain devoid of fluorescence. In the DIC image (right) the outline of the retracted plasma membrane is indicated with a dotted line. This leaching contrasts with the situation after pretreament with the phospholipase C inhibitor (see Figure D to F). Bar = 0 μm

5 CLUSTL W(.60) multiple sequence alignment of X8 domains atg43670 atg7850 atg43660 at3g850 at4g665 atg66870 atg66855 at5g6340 at5g6350 at4g09090 at4g09465 at5g53600 at5g5360 at5g6330 atg03505 atg3830 atg6995 atg6450 at5g630 at5g08000 atg8650 at4g3600 atg9380 Ole-E0 atg30933 at5g35740 atg0490 atg79480 atg09460 at3g5800 atg4930 atg43670 atg7850 atg43660 at3g850 at4g665 atg66870 atg66855 at5g6340 at5g6350 at4g09090 at4g09465 at5g53600 at5g5360 at5g6330 atg03505 atg3830 atg6995 atg6450 at5g630 at5g08000 atg8650 at4g3600 atg9380 Ole-E0 atg30933 at5g35740 atg0490 atg79480 atg09460 at3g5800 atg4930 WCVKPSTDNERLQENINFCSIDCQIISEGGCYLPDSIISRSVMNLYYQQGRHFW WCVKPSSDQVLQDNINFCSVDCRVLLSGCPCYSPSNLINHSIMNLYYQNGRNYW WCVKPGTPIKQLVKNLNNVCSVHCEVVSEGGCYDPINLYNSSVVMNLYYQNQGRQYS WCVKPGTLTEQLINNLNYCSVDCQIISCYSPDNIYNMSVVMNLYYQEGRNFW WCVNPSSTQLQNIDWLCS-GCVLIGPGGSCFEPNNVINHSFVMNDYYQLQGSTEE WCVNVSSTQLQNIDWCS-DCTINPGGSCFDPDTLVSHSFVMNDFYQNHGSTEE WCVSTDTQLQNIDWCN-DCVKINPGGVCYEPNTLTSHSFVMNDYYRNHGSIEE WCIGDKTDKQLQNIDWVCSRDCGLNSGGPCFEPNTVRDHSFMNLYYQNLGTKE WCVNKKTDEQLQNIDWCCSRDCTQINPGGVCYEPNTLRDHSYVMNLYYQNLGRTKD WCVKMNTNQLQGNINFGCSVDCGPIQPGGSCYIPNSLVNHSFVMNYYQSHGRTKK WCITLITNQLQNINFCSVDCRPIRPGGSCFIPNNLNHSFVMNSYYQTHGRTNK WCTMPTSTTEQLQSNINFCNVDCPIQPGGFCYYPNTLLDHFMTRYYRSQGHTY WCSMPSSTPEQLQNIQLCSVDCTPIQPGGFCYYPNTLLDHSFVMNSYYKSQGRTY WCMMPNTGEQLQNIDYCSVDCTPIQPGGTCYEPNTLLDHSFMNYYQSHGRIED YCLCRDGVGEKDLQTSIDYCGKDCNPIHEKGPCYQPNTIKSHCDWVNTYFQRFGQISG YCLCKDGIGDTELQTSIDYCGDCNPIHDKGTCYQPDTIKSHCDWVNSYFQNQVPG YCLCKEGN-EQVLQKIDYCGDCTQIQPTGCYQPNTVKNHCDVVNSYYQKKSSG VCVCKDN-ELDLQKVIDFCGDCQIQTTGCYQPNTLKNHCDVVNSYYQKKSTG WCVCKTGLSDTVLQTLDYCGDCNPTKPKQSCFNPDNVRSHCNYVNSFFQKKGQSPG WCVCKTGLSDSVLQKTLDYCGDCNPTHPKGSCFNPDNVRHCNYVNSFFQKKGQSE WCVCKEGLSEMLQKTLDYCGDCGPIHQTGPCFNPNTVKSHCSYVNSFFQKKGQSLG WCVRFDVTSQLQLDYCDCPIQPNGLCFLPNTVQHSYFNSYFQRMPG WCIKNSPTSLQVLDYCGDCGQIQQGCYEPNTIRDHSFFNSYYQKHPGSD- WCVPKETDQLQSNIDYVCSMDCGPIQNGCFNPNTVRHSYMNSWYQSKGRNDF WCVRENVKMLQLDYCGDCSEIQEGGNCYNPNSLRHSFFNSYYQKNPIPS- WCIDEQTPDDELQLDWCGDCSKMQQNQPCFLPNTIRDHSFFNSYYQTYKNKGG WCVDGQIPDNVIQVDWCQDCSTIQPNQPCFLPNTVKDHSVVFNNYYQRYKRNGG WCVKPSVPDPIIQEMNFCGDCHSIQPNGPCFKPNTLWHSFYNSYWQRTKSTGG WCVKPGSQVSLQQLDYCGDCSQLQQGGNCYSPISLQSHSFFNSYYQKN-PSPQ WCVKNNEDSSLQTIEWCGDCGPIQQGGPCNDPTDVQKMSFVFNNYYLKNGEEDE WCMENPYYFRRVISSLKWCKDCSPLEKGGRCQDLDNYRSQSYFNDYYQKN-PIPR NCNFEGSGLIGITDPSCIY NCNFKNSGLITITNPS--F KCDFEGSGIISVTDPSCIY NCNFGDSGLVITDPSLLF CNFSGSGRIIDTNPSCVY CNFTGTGQVVTDPSCVY CEFNHTGQIISGDPSCRY QCNFHNTGIEVSTDPSCIF QCTFNGSGSEVTKDPSCIF CSFKNTGTFVTDLS--- CSFKNTGTFTDPSCVY CSFGNTGYIISSDPSCIF CSFGNTGYLIYSDPSCEF CRFGRTGCFVFIDPSCIY SCNFSGTTTSQNLPSCLY SCNFSGTTTNPNPPSCIY TCDFNGSPSTTPPSCLT TCDFNGVISTSPPSCLS SCNFDGTTPTNSDPSCF SCNFTGTTLTTTDPSCF TCDFGTTFSSDPSCPF SCNFGTSTIKTDPSTWL SCNFGGQLTSTDPSCHF DCDFSGTGITSSDPSCSF SCNFDGTITISDPSCHF SCYFKGMITELDPSCQY SCNFNSTFITQTDPSFCY SCTFGGTGMLVTVDPSCHF SCDFGGSLVNTNPSCIY CNFNNNLTSLNPSCKY NCDFNGVLTVQDPSFTF Supp- Figure 5 lignment of the X8 domain amino acid sequences for proteins with structural similarity to PDCB.

pdcb pdcb3 M +ve (kbp) P P3 +ve Col P P P3 -RT gen Col P -RT gen PDCB/3 0.6kbp.5 0.5 Gene specific OBE.6kbp -RT +ve.5 OBE Supp- Figure 6 RN analysis for pdcb and pdcb3 mutant lines.

transcripts from the mutated genes (upper panels). Negative (-RT; reaction lacking RT) and positive (+ve; specific plasmid DN) control were run in parallel.

In this qualitative assay, gels were stained with ethidium bromide. The 0.

6 pdcb pdcb3 M +ve (kbp) P P3 +ve Col P P P3 -RT gen Col P -RT gen PDCB/3 0.6kbp Gene specific OBE.6kbp -RT +ve.5 OBE Supp- Figure 6 RN analysis for pdcb and pdcb3 mutant lines. RNs from three independent plants (P-3) each for pdcb and pdcb3, and RN and genomic DN (gen) from wild type Col-0 (Col) were analysed by RT-PCR using gene specific primers for the accumulation of transcripts from the mutated genes (upper panels). Negative (-RT; reaction lacking RT) and positive (+ve; specific plasmid DN) control were run in parallel. In addition the samples were analysed for the expression of the OBERON gene (Saiga et al., 008) using specific primers (lower panels). In this qualitative assay, gels were stained with ethidium bromide. The 0.6 kbp diagnostic fragment for PDCB or PDCB3 RNs was not detected from any of mutant plants tested (pdcb: three biological and two technical replicates, pdcb3: three biological and one technical replicate). M shows the migration of DN size markers in kbp. Reference: Saiga, S., Furumizu, C., Yokoyama, R., Kurata, T., Sato, S., Kato, T., Tabata, S., Suzuki, M. and Komeda, Y. (008). The rabidopsis OBERON and OBERON genes encode plant homeodomain finger proteins and are required for apical meristem maintenance. Development 35:

7 Mean number of cells Mean number of cells C (50) WT (49) (5) pdcb Plant type (50) WT pdcb pdcb3 Plant type B Mean number of cells D Mean number of cells (48) (5) WT pdcb3 Plant type (34) (34) WT 35Spro:YFP. FL Plant type Supp. Figure 7 GFP trafficking is unchanged in pdcb and -3 knockout lines and in a transgenic line expressing the unrelated GPI-anchor protein FL3. GFP diffusion assessed at 48 h post-bombardment in WT and pdcb lines. B GFP diffusion assessed at 48 h postbombardment in WT and pdcb3 lines. C GFP diffusion assessed at 48 h postbombardment in WT and the pdcb pdcb3 lines. D GFP diffusion assessed in WT and 35Spro:YFP.FL3 lines. Numbers in parentheses indicate the numbers of bombardment sites assayed. Bars = +SE. There was no significant difference in GFP movement in any of the treatments.

8 rabidopsis thaliana (treatment) Stress: heat_ Nutrient: nitrate(45mm)_sucrose(90mm) PCD: senescence Chemical: cycloheximide (+) Nutrient: Nitrate_low Biotic: P. infestans (+) Stress: osmotic_green_late Nutrient: lown_glucose Chemical: syringolin (+) Light intensity: high light 3 Stress: oxidative_roots_early Biotic: P. syringae_3 (+) Biotic: Bemisia tabaci type B (+) Chemical: ozone_ Biotic: B. cinerea (+) Light intensity: high light Nutrient: mannitol_-4-6h Chemical: low CO (+) Nutrient: nitrate(45mm)_sucrose(90mm) Nutrient: P-_long-term Hormone: B_ (+) Nutrient: nitrate(5mm)_sucrose(0) Nutrient: P-_medium-term Chemical: furyl acrylate ester (+) Light quality: lowr-fr Biotic: P. syringae_ (+) Stress: drought_roots_late Chemical: EF-Tu (elf8) Stress: hypoxia (+) Stress: cold_green_early Nutrient: K (-) Hormone: I_ (+) Biotic: P. syringae (avrrpm) (+) Stress: osmotic_green_early Hormone: PC (+) Light intensity: light + low CO Chemical: PCIB (+) Stress: oxidative_roots_late Hormone: MJ_ (+) Hormone: CC_ (+) Chemical: lincomycin_ (+) Nutrient: Cs (+) Chemical: EF-Tu (elf6) Stress: genotoxic_green_late Stress: heat_green Stress: oxidative_green_early Stress: drought_green_late Hormone: B_6 (+) Chemical: chitin (+) Stress: wounding_roots_late Hormone: salicylic acid_ Chemical: gno3 (+) Light intensity: high light Chemical: prohexadione (+) Hormone: ethylene (+) Stress: anoxia (+) Nutrient: nitrate(5mm)_sucrose(0) Biotic: E. orontii (+) Light quality: blue Chemical: zearalenone (+) Chemical: benzyladenine (+) Chemical: 4-thiazolidinone/acetic acid (+) Chemical: NP (+) Stress: genotoxic_roots_late Stress: cold_roots_early Hormone: G3_ (+) Hormone: BL_ (+) Stress: wounding_roots_early Light quality: white Chemical:,4,6 T (+) Stress: drought_green_early Light quality: far red Chemical: norflurazon (+) Biotic: E. cichoracearum (+) Chemical: ibuprofen (+) Hormone: G3_3 (+) Hormone: B_7 (+) Stress: salt_roots_early Light quality: UV- Chemical: hydrogen peroxide (+) Light intensity: light_ Stress: salt_green_early t5g08000 t5g630 tg Light intensity: light_ # rrays(exp/ctrl) Stress: salt_green_early Stress: wounding_green_late Stress: salt_green_late Chemical: brassinazole (brz9) Biotic: P. syringae (hrp) (+) Nutrient: Caesium-37 (+) Nutrient: P-_short-term Biotic: M. persicae (+) Hormone: G3_ (+) Hormone: I_ Chemical: MG3 (+) Light quality: red Stress: genotoxic_green_early Chemical: TIB (+) Hormone: zeatin (+) Chemical: PNO8 (+) Hormone: B_ (+) 7/3 Light quality: UV-B 4/ Nutrient: sucrose (+) /4 Chemical: brassinazole (brz0) Hormone: I_3 Biotic: P. syringae_4 (avrrps4) /4 Stress: heat_roots Stress: genotoxic_roots_early Light quality: lowr-fr_early Chemical: 6-benzyl adenine (+) 9/9 Chemical: daminozide (+) Chemical: paclobutrazole (+) 4/ Stress: salt_roots_late Hormone: OPD_timecourse triploidy Chemical: propiconazole (+) Stress: oxidative_green_late Hormone: B_4 (+) Biotic: nematode_late (+) Hormone: B_5 (+) Biotic: nematode_early (+) Light quality: blue Chemical: VG (+) Chemical: lincomycin_ (+) Stress: wounding_green_early Light intensity: photoperiod 8h Hormone: I_4 (+) 4/ Hormone: J_timecourse Chemical: uniconazole (+) Nutrient: glucose_-4-6h Stress: osmotic_roots_early Light quality: lowr-fr_late Hormone: B_3 (+) 6/3 Biotic: P. syringae_4 (avrrpm) tetraploidy Chemical:,4-dichlorophenoxyacetic acid (+) Light quality: UV_filtered_WG95 Stress: drought_roots_early Stress: osmotic_roots_late Stress: cold_green_late Nutrient: S deprivation /4 Biotic: nematode (+) Nutrient: K deprivation late Stress: cold_4 Stress: cold_3 8/8 Stress: cold_ 3/4 Nutrient: nitrate(0)_sucrose(30mm) Hormone: zeatin (+) Hormone: BL / H3BO3 (+) Hormone: zeatin 3 (+) Hormone: BL_ (+) Nutrient: K deprivation early Chemical: dexamethasone_ Light quality: UV_filtered_WG37 Light quality: UV_filtered_WG305 Biotic: mycorrhiza (+) Nutrient: nitrate(0)_sucrose(90mm) Stress: cold_roots_late Chemical: N (+) Chemical: CO high Chemical: isoxaben (+) 3/ 4/3 Supp- Figure 8 Stress-induced changes in the expression of PDCB genes (arbitrary units). Public dataset ( for the expression for t5g630 (PDCB), t5g08000 (PDCB) and tg8650 (PDCB3), displayed as a heat map. Treatments are ranked with respect to the magnitude of the expression changes from down-regulation (green) to up-

9 Supp- Figure 9 qrt-pcr data for PDCB expression following stress treatments Box plot of qrt-pcr data for heat treated seedlings. B Numerical data as presented in. =treatment; Ref=Reference C Box plot of qrt-pcr data for wounded seedlings. D Numerical data as presented in C. Data from three biological and three technical replicates were analysed using REST PDCB PDCB PDCB3 HSP70 B Gene Type Expression Std. Error 95% C.I. P value Result PDCB PDCB DOWN PDCB eif REF.000 HSP UP C PDCB PDCB PDCB3 LOX D Gene Type Expression Std. Error 95% C.I. P value Result PDCB eif REF.000 PDCB PDCB LOX UP

Supplemental Data. Perrella et al. (2013). Plant Cell /tpc

Supplemental Data. Perrella et al. (2013). Plant Cell /tpc Intensity Intensity Intensity Intensity Intensity Intensity 150 50 150 0 10 20 50 C 150 0 10 20 50 D 0 10 20 Distance (μm) 50 20 40 E 50 F 0 10 20 50 0 15 30 Distance (μm) Supplemental Figure 1: Co-localization