Figure 1. SEC3a SEC3b mRNAs coding sequences and 3’-UTRs. A) · 8/11/2016 · Supplemental Figure 1. Schematic representation of SEC3a and SEC3b mRNAs coding sequences and 3’-UTRs

Supplemental Figure 1. Schematic representation of SEC3a and SEC3b mRNAs

coding sequences and 3’-UTRs.

(A) Primer names, location and orientation are indicated. The forward primer (2526) was

common for both genes and was used with different combinations of reverse primers (554,

534, 582, 2527). The efficiency of each primer pair was confirmed by control reaction on

genomic DNA (Figure 1F). For determining SEC3b expression we used two reverse

primers with specificity to SEC3b 3`-UTR: 1) 2527 which is located 18 bases after the stop

codon and 2) 582 which is located 191 bases after the stop codon. The same results were

obtained with either primer pair. (B) A Partial sequence alignment of SEC3a and SEC3b

cDNA sequences including the 3` CDS with 3`-UTRs. TAA stop codons are highlighted.

Nucleotide substitutions found in SEC3a and SEC3b are highlighted in grey. Primer names

and position on sequence and orientation are indicated. T-DNA insertion sites in the sec3a-

1 and sec3b-1 alleles as determined by sequencing are marked.

Supplemental Figure 2. Genotyping of sec3a-1 and sec3b-1.

(A) A schematic representation of SEC3a and SEC3b genes exon-intron organization.

Location of gene specific primers used for the genotyping and determining the T-DNA

insert location are specified. Numbers indicate nucleotide position from the initiation ATG

codon. (B) Genotyping of sulfadiazine (Sd) resistant sec3a-1+/- progeny. (C) Genotyping

of sec3b-1-/- homozygote. (D) Genotyping of sec3a-1+/-/LAT52:SEC3a line2 Sd resistant

progeny. Note that plant number 2 is a sec3a-/- homozygote.

Supplemental Figure 3. Phenotype of sec3b-1 homozygote.

(A) Five-week-old sec3b-1-/- and Col-0 plants grown on soil. (B) sec3b-1-/- and Col-0

seedlings grown for 7 DAG on a 0.5X Murashige Skoog medium plate.

Supplemental Figure 4. Pollen nuclei display similar staining in qrt-/-_ and qrt-/- sec3a-

/+ single and double mutants. DAPI-stained mature tetrads from self-crosses of qrt1-/- or

qrt1-/- /sec3a-1+/-.Yellow and white arrowheads indicate vegetative nucleus and sperm cell

nuclei, respectively. Bar = 20 µm.

Supplemental Figure 5. Distribution of FM4-64 in growing non-transgenic Col-0

pollen tubes. (A) Growing FM4-64 stained pollen tube. Imaging began 5 minutes

following application of the dye (0s). The FM4-64 staining is shown as intensity range. (B

and C) Signal intensity plots of plasma membrane FM4-64 fluorescence in arbitrary units

(a.u.). The asterisks in B and C correspond to the asterisks in A 0s and 210s, respectively.

Bar = 5 µm.

Supplemental Figure 6. Not growing Arabidopsis pollen tube labeled with FM4-64.

Selected time-lapse images. Bar = 5 µm

Supplemental Figure 7. Localization of Arabidopsis and tobacco SEC3a variants in

tobacco pollen tubes. (A) Localization of C-terminal YFP-fusions of SEC3a constructs

transiently expressed in tobacco pollen tubes. (B) Localization of N-terminal YFP-fusions

of SEC3a constructs in non-growing tobacco pollen tubes and localization of YFP-only

vector control. (C-E) Localization (C), time snapshots (D) and kymograph (E) of tobacco

YFP-SEC3a in steady growing tobacco pollen tubes. (F-G) Selected time-lapse images of

SEC3a N-terminal domain (LAT52:YFP-Sec3a-N, F) and SEC3a lacking its N-terminal

domain (LAT52:YFP-Sec3a-ΔN, G). (H) Phenotype of overexpressed YFP-SEC3a and

YFP-AtSEC3a-N. Bar = 5 µM.

Supplemental Figure 8. Phylogenetic analysis of SEC3 N-terminal domain and SEC3

domain. Both trees represent the protein maximum likelihood (ML) phylogeny of

particular genes. Numbers at nodes correspond to he approximate likelihood ratio test with

SH-like (Shimodaira-Hasegawa-like) from ML (top) and posterior probabilities from

Bayesian analysis (bottom). Circles represent support above 90% by both methods,

Missing values indicate support below 50%. Branches were collapsed if inferred topology

was not supported by both methods. Atha – Arabidopsis thaliana, Anig – Aspergillus niger,

Bdis – Brachypodium distachyon, Calb – Candida albicans, Ccin – Coprinopsis cinerea,

Cele – Caenorhabditis elegans, Cneo – Cryptococcus neoformans, Crei – Chlamydomonas

reinhardtii, Dhan – Debaryomyces hansenii, Dmel – Drosophila melanogaster, Dpul –

Daphnia pulex, Drer - Danio rerio, Gzea – Gibberella zeae, Hsap – Homo sapiens, Lbic –

Laccaria bicolor, Mcir – Mucor circinelloides, Ncra – Neurospora crassa, Nsyl –

Nicotiana sylvestris, Ntab – Nicotiana tabacum, Ntom – Nicotiana tomentosiformis, Osat

– Oryza sativa, Ppat – Physcomitrella patens, Ptri – Populus trichocarpa, Pytr -

Pyrenophora tritici, Sbic – Sorghum bicolor, Scer – Saccharomyces cerevisiae, Slyc –

Solanum lycopersicum, Spom – Schizosaccharomyces pombe, Smoe – Selaginella

moellendorffi, Sthe - Sporotrichum thermophile, Stub – Solanum tuberosum, Ylip –

Yarrowia lipolytica, Umay – Ustilago maydis, Vcar - Volvox carteri, Xtro – Xenopus

tropicalis

Supplemental Figure 9. Multiple sequence alignment of SEC3 N-terminal domain for

selected organisms. The alignment was constructed as described in Pleskot et al. (2012).

Atha – Arabidopsis thaliana, Dmel – Drosophila melanogaster, Hsap – Homo sapiens,

Ntab – Nicotiana tabacum, Osat – Oryza sativa, Scer – Saccharomyces cerevisiae, Spom

– Schizosaccharomyces pombe.

Supplemental Movies

Supplemental Movie 1. Localization of GFP-SEC3a in growing Arabidopsis pollen tube.

GFP signal shown as range of intensities. Time: 3.5 min, 60 frames.

Supplemental Movie 2. Localization of GFP-SEC3a in growing Arabidopsis pollen tube.

GFP signal shown as range of intensities. Time: 3.5 min, 60 frames.

Supplemental Movie 3. Localization of GFP-SEC3a in non-growing Arabidopsis pollen

tube. GFP signal shown as range of intensities. Time: 3.1 min, 54 frames.

Supplemental Movie 4. Localization of GFP-SEC3a in multiple tip sec3a-/-; GFPSEC3a

pollen. Note that GFP-SEC3a is only detected at the tip PM of the growing tube and is

absent from the growth arrested tubes. The movie shows the same pollen tube which is

presented in Figure 6I in the main text. Time: 5min, 30 frames

Supplemental Movie 5. Localization of GFP-SEC3a in multiple tip sec3a-/-; GFPSEC3a

pollen. Note that GFP-SEC3a is only detected at the tip PM of the growing tube and is

absent from the growth arrested tubes. The pollen tube in this movie is not shown in the

main text. Time: 6 minutes, 20 frames.

Supplemental Movie 6. Localization of GFP-SEC3a KRKR/A in growing Arabidopsis

pollen tube. GFP signal shown as range of intensities. Time: 5.5 min, 95 frames.

Supplemental Movie 7. Localization of GFP-SEC3a KRKR/A in non-growing (first 5

min) pollen tube that starts to grow (point attention that GFP-SEC3a KRKR/A defines that

position of growth) . GFP signal shown as range of intensities. Time: 10 min, 180 frames.

Supplemental Movie 8. Localization of GFP-SEC3a-∆N in growing Arabidopsis pollen


Supplemental Movie 9. Localization of GFP-SEC3a-∆N in growing Arabidopsis pollen


1

Sulfadiazine selection marker Basta selection marker

Reciprocal out-crosses N sensitive +/+

resistant sec3a-1 +/-

sec3a-1-/-

χ2 P N* sensitive resistant LAT52: SEC3A

χ2 P

Pollen source: sec3a-1 +/- Pollen recipient: Col-0

408 100% 0% 408 <0.0001

Pollen source: Col-0 Pollen recipient: sec3a-1 +/-

483 50.5% 49.5% 0.05 >0.8 NS

Expected

50% 50%

Pollen source: sec3a-1 +/-/ LAT52:SEC3a line1+/- Pollen recipient: Col-0

228

72% 28% 2.8 <0.1 64 1.6% 98.4% 0.02 >0.9 NS

Pollen source: sec3a-1 +/-/ LAT52:SEC3a line2+/- Pollen recipient: Col-0

142 81% 19% 13.1 <0.001 27 0% 100% 0 =1 NS

Pollen source: sec3a-1 +/-/ LAT52:GFP-SEC3a line1+/- Pollen recipient: Col-0

198 80% 20% 15.3 <0.001 40 0% 100% 0 =1 NS

Expected #

66.6% 33.3% 0% 100%

Pollen source: Col-0 Pollen recipient: sec3a-1 +/-/ LAT52:SEC3a line1+/-

99 53% 47% 0.36 >0.5 NS n/a

Pollen source: Col-0 Pollen recipient: sec3a-1 +/-/ LAT52:SEC3a line2 +/-

188 52% 48% 0.30 >0.5 NS 71 55% 45% 0.71 >0.4 NS

Pollen source: Col-0 Pollen recipient: sec3a-1 +/-/ LAT52:GFP-SEC3a line1+/-

112 51% 49% 0.04 >0.8 NS 51 61% 39% 2.47 >0.1 NS

Expected

50% 50% 50% 50%

Supplemental Table 1: Reciprocal out crosses of sec3a-1-/+ and first progeny of

LAT52:SEC3a and LAT52:GFP-SEC3a complementation lines to Col-0 background.

Genotyping was performed based on Sd (sec3a-1) allele and Basta (LAT52:SEC3a,

LAT52:GFP-SEC3a) selection markers. The χ2 test was applied for statistical analysis,

P≤0.05 indicate that observed data differs significantly from the expected data.

# - Expected ratio of 2:1 in crosses with pollen sec3a-1 +/-/ LAT52:SEC3a+/- or sec3a-1 +/-/ LAT52:GFP-SEC3a+/- is due to the fact that mutant pollen which does not contain

complementation construct will not germinate. The difference from expected in such crosses

could be explained by variable expression levels of the complementation proteins in

individual pollens. N – number of progeny that was assayed, N* - number of Sd resistant

progeny that was analyzed. NS not significantly differs from expected.

2

Table S2: Segregation of sec3a-1+/- mutant and complementation lines based on Sd

resistance.

* As in case of reciprocal out crosses difference from expected values could be explained by

variable expression levels of the complementation constructs in individual pollens, which

compete form germination with wild type pollen grains. # - Low percent of sensitive plants

was probably due to contamination with wild type seeds.

Self-crosses No

of progeny

sensitive

+/+

resistant

sec3a-1 +/- sec3a-1-/-

χ2 P

sec3a-1+/- 1140 52% 48% 323 <0.0001*

sec3a-1 +/-/ LAT52:SEC3a line1+/+ 507 34% 66% 22.5 <0.0001*

sec3a-1 +/-/ LAT52:SEC3a line2+/+ 651 37.8% 62.2% 56.7 <0.0001*

sec3a-1 +/-/ LAT52:GFP-SEC3a line1+/+ 525 42% 58% 81.8 <0.0001*

Expected 25% 75%

sec3a-1 -/-/ LAT52:SEC3a line1+/+ 265 0.4% # 99.6% 0.003 >0.8 NS

sec3a-1 -/-/ LAT52:SEC3a line2+/+ 442 1.3% # 98.6% 0.081 >0.4NS

sec3a-1 -/-/ LAT52:GFP-SEC3a line1+/+ 343 0% 100% 0.000 1 NS

Expected 0% 100%

3

Percent of aborted seeds

No. of aborted seeds

Total No. of seeds counted

No. of silliques analyzed

Genotype

~1.0 % 12 1219 23 +/+

~1.1 % 10 899 17 sec3a-1+/-

Supplemental Table 3: Abortion of seed development in sec3a-1 heterozygote. Counts were carried out on sec3a-1 +/- and segregating plants without T-DNA insert, as was revealed by PCR genotyping.

4

Supplemental Table 4: Out crosses of sec3a-1+/-/LAT52:SEC3a KRKR/A, sec3a-1+/-

/LAT52:GFP-SEC3a KRKR/A, sec3a-1+/-/LAT52:SEC3a ΔN +/- and sec3a-1+/-/LAT52:GFP-

SEC3a ΔN +/- complementation lines to Col-0. Expected ration of 2:1 in crosses is due to the

fact that mutant pollen which does not contain complementation construct will not germinate.

The difference from expected in such crosses could be explained by variable expression levels

of the complementation constructs in individual pollens. N – number of progeny that was

assayed. NS - not significantly differs from expected.

Sulfadiazine selection marker

Reciprocal out-crosses N % sensitive

+/+

% resistant sec3a-1

+/-sec3a-1-/-

χ2 P

Pollen source: sec3a-1+/-/LAT52:SEC3a KRKR/A line 1+/- Pollen recipient: Col-0

79 62.0 38.0 0.6 >0.5 NS


117 80.3 19.7 6.3 <0.01*


145 73.8 26.2 2.0 >0.1 NS


99 68.7 31.3 0.1 <0.8 NS

Pollen source: sec3a-1+/-/LAT52:GFP-SEC3a KRKR/A line 3+/- Pollen recipient: Col-0

61 95.1 4.9 22.2 <0.001*


93 67.7 32.3 0.05 <0.5 NS


80 83.7 16.3 10.5 >0.001*


56 69.6 30.4 0.2 <0.7 NS

Pollen source: sec3a-1+/-/LAT52:SEC3a ΔN line 2+/- Pollen recipient: Col-0

182 78.0 22.0 10.6 <0.001*


194 78.4 21.6 11.9 <0.001*


190 92.1 7.9 55.4 <0.001*

Pollen source: sec3a-1+/-/LAT52:GFP-SEC3a ΔN line 1+/- Pollen recipient: Col-0

160 96.2 3.8 63.1 <0.001*


166 84.9 15.1 25.0 <0.001*


127 86.6 13.4 22.8 <0.001*


113 100.0 0.0 56.6 <0.001*

Expected # 66.6% 33.3%

Supplemental Table 5. List of primers used in this study.

Primer name Description Sequence (5`-3`)

SYP534 CDS SEC3a/SEC3b – R TTACATGGAAGCCAGAAGTCCTCTC

SYP554 SEC3a 3ÙTR specific/genotyping sec3a-1 - R CTGCCTTCAGTTATGGTTCTATAC

SYP575 sec3b-1 genotyping - F TCAACCGAATGGTTTTCTGTTTG

SYP576 sec3b-1 genotyping - R AACAGGGTGGACGGAAGTACTAACT

SYP582 SEC3b 3ÙTR specific - R CTACCATGGTGAAGGAGCAGTC

SYP594 SEC3a promoter - F AAAGTCGACGGCCAACTTCTTTGTCTCTGTC

SYP595 SEC3a promoter - R AAAGGTACCTTGTTGTTGTTGCGGATCCAGCA

SYP1417 Genotyping sec3a-1 - F GTTTGTGTTTGCTTCATGTGTTCT

SYP1427 SEC3a cloning to pSY597 - F AAAAAGCAGGCTTGATGGCGAAATCAAGCGCC

SYP1428 SEC3a cloning to pSY597/pSY2538- R GAAAGCTGGGTTTACATGGAAGCCAGAAGTCCTCTC

SYP1429 SEC3a cloning to pSY597 - F GGGGACAAGTTTGTACAAAAAAGCAGGCTTGATGGCG

SYP1430 SEC3a cloning to pSY597/pSY2538 - R GGGGACCACTTTGTACAAGAAAGCTGGGTTTACATGGAAGC

SYP2526 SEC3a/SEC3b mRNA - F GATGGTATTCAGGAGGATTTCTATGC

SYP2527 SEC3b 3ÙTR specific - R AACAACTGCCTTCAGTTTTGGTTCA

SYP2546 SEC3-ΔN cloning to pSY2538 -F AAAAAGCAGGCTTGATGGGGGAGCCTGTTGCTGAATC

SYP2547 SEC3-ΔN cloning to pSY2538 -F GGGGACAAGTTTGTACAAAAAAGCAGGCTTGATGGGG

SYP2552 SEC3a KRKR/A cloning to pSY2525- R GAGTAGTGACTACTGGAGTGTTATCCTTTG

SYP2553 SEC3a KRKR/A cloning to pSY2525- F GATATTGTCGAGATGGCCCTTTGG

LBa1 T-DNA left border (sec3b-1) - R TGGTTCACGTAGTGGGCCATCG

LBb1 T-DNA left border (sec3b-1) - sequencing GCGTGGACCGCTTGCTGCAACT

GABI-PCR T-DNA left border (sec3a-1) - R CCCATTTGGACGTGAATGTAGACAC

GABI-seq T-DNA left border (sec3a-1) - sequencing ATATTGACCATCATACTCATTGC

PPP62 SEC3a-N-YFP cloning - R TATGGGCCCCGCATCTTCTGTACTTCTTTGAGTAGTGACTACTGG

PPP63 YFP-SEC3a-N cloning - R TATGGGCCCTCAATCTTCTGTACTTCTTTGAGTAGTGACTACTGG

PPP64 SEC3a-YFP cloning - R TATGGGCCCCGCCATGGAAGCCAGAAGTCCTCT

PPP65 YFP-SEC3a cloning - R TATGGGCCCTTACATGGAAGCCAGAAGTCCTC

PPP66 SEC3a cloning - F ATAGCCGGCATGGCGAAATCAAGCGC

PPP90 SEC3a-ΔN cloning - F ATAGCCGGCATGGGGGAGCCTGTTGCTGA

PPP126 SEC3a-N K51A R53A cloning GACGGAAAGAGCGAGAACTGCGGGTGCAGCCATTTGTCGACCC

PPP128 SEC3a-N KRKR/A cloning GACTTGCCCCAAACACCTGCACTTGCGGCGACACGTATCGACA

PPP129 GST-SEC3a-N cloning - F ATAGGATCCATGGCGAAATCAAGCG

PPP130 GST-SEC3a-N cloning - R GCTCTCGAGTCAATCTTCTGTACTTCTTTGAGTAGTG

Supplemental Figure 1. Schematic representation of SEC3a and SEC3b mRNAs coding sequences and 3’-UTRs. (A) Primer names, location and orientation are indicated. The forward primer (2526) was common for both genes and was used with different combinations of reverse primers (554, 534, 582, 2527). The efficiency of each primer pair was confirmed by control reaction on genomic DNA (Figure 1F). For determining SEC3b expression we used two reverse primers with specificity to SEC3b 3`-UTR: 1) 2527 which is located 18 bases after the stop codon and 2) 582 which is located 191 bases after the stop codon. The same results were obtained with either primer pair. (B) A Partial sequence alignment of SEC3a and SEC3b cDNA sequences including the 3` CDS with 3`-UTRs. TAA stop codons are highlighted. Nucleotide substitutions found in SEC3a and SEC3b are highlighted in grey. Primer names and position on sequence and orientation are indicated. T-DNA insertion sites in the sec3a-1 and sec3b-1 alleles as determined by sequencing are marked.

Documents

Figure 1. SEC3a SEC3b mRNAs coding sequences and 3’-UTRs. A) · 8/11/2016 · Supplemental Figure 1. Schematic representation of SEC3a and SEC3b mRNAs coding sequences and 3’-UTRs