Supporting Information
Figure S1. Pollen tubes of N. tabacum (a-h) and N. obtusifolia (i-p) in normal, TT-ablated, TTE
injected and Fraction 2 (F2) injected N. tabacum styles 40 h post pollination. a-d and i-l show
pollen grains (open arrow head a-d and i-l) on the cut style surface and pollen tube growth in the
upper 10% of the style. e-h and m-p show the pollen tube growth front. Black horizontal lines
indicate the pollen tube growth front where the pollen tube length was measured. Line arrows
indicate pollen tubes and open arrows indicate vascular bundles. The TTE was injected at a
protein concentration of 30 µg/µl and F2 was injected at 12 µg/µl (ref. Fig. 4a). Pollen tube
morphology of both N. tabacum and N. obtusifolia appeared similar in all style treatments but the
pollen tube length of N. obtusifolia was significantly affected by style treatments (Fig. 4a).
Figure S2. Interspecific pollen tube growth response of N. obtusifolia to temperature. Average
pollen tube length of N. obtusifolia at temperature incubations from 5-45°C. Nicotiana
obtusifolia mean pollen tube length ± S.E. (5 styles replicated twice in time) in normal (black
bars) and TT-ablated (grey bars) N. tabacum styles. a. Pollinated styles incubated at 5, 15, 25,
30, 35, 40, and 45°C for 40 h. b. Pollinated styles incubated at 35°C for 16 h and 27 h. All
pollinations were performed after stigma removal. Significant differences in mean pollen tube
length between normal and TT-ablated styles at each temperature are indicated by asterisks * p <
0.05, ** p < 0.01. At 35°C N. obtusifolia pollen tubes reached the end of the normal style 40 h
post-pollination (a), but the pollen tube length in the normal style remained shorter than the
length in the TT-ablated style at 16 and 27 h post-pollination (b).
Figure S3. Inhibitory activity of TTE toward N. obtusifolia pollen tube growth at three different
protein concentrations. Mean±S.E. pollen tube length of N. obtusifolia in normal, TT-ablated,
and TT-ablated injected styles 40 h post-pollination. Injections were MCM (control) and TTE.
Trials 1 (a) and 2 (b) represent two different TTE preparations and pollinations were separated in
time. Undiluted protein concentration of the TTE was 41 μg/μl for Trial 1 (a) and 37 μg/μl for
Trial 2 (b). TTE from each trial was diluted 1:1 and 1:2 with MCM followed by injection. Mean
pollen tube length represents the average of five pollinated styles for each treatment, replicated
once in time. Trials were significantly different and could not be pooled. Mean separations are
based on Tukey’s HSD at α=0.05 within each trial. In both trials, the undiluted TTE treatments
had pollen tube lengths that was not significantly different from the normal style, but were
significantly shorter than in the TT-ablated and MCM-injected styles. Both of the 1:2 dilution
treatments, trial 1-TTE (10 μg/μl) and trial 2-TTE (9.3 μg/μl), lost inhibitory activity toward N.
obtusifolia pollen tube growth. The pollen tube lengths were significantly longer in the TTEinjected styles, but not significantly different from the TT-ablated MCM-injected style. The
inhibitory activity of TTE is dependent on total protein concentration.
Table S1. Proteins identified from tandem mass spectrometry analysis of in-gel trypsin digested
samples F2 (high molecular weight; HMW & low molecular weight; LMW) and F3 (high
molecular smear; HMS) (cf. Fig 4b). Extracted, trypsin-digested, peptide solution was loaded on
an Orbitrap Velos (Thermo Scientific, Bremen, Germany) for LC-MS/MS analysis. Resulting
Spectra were analyzed using Protein Pilot software version 4.0 with search parameter for
biological modification, iodoacetamide cysteine alkylation, detected protein threshold [Unused
ProtScore (Conf)] > 0.05 (10%), and a competitor error margin (ProtScore) of 2.00 (AbSciex,
Framingham, MA). Proteins identified in both F2 and F3 are shaded in grey for each sample. For
F2 proteins, HMW indicates protein was identified in F2-HMW and LMW indicates protein was
identified in F2-LMW. When a protein was found in both the F2-HMW and F2-LMW the
identification with the higher score is listed first. Score is a Protein Pilot algorithm score (unused
score) for protein identification confidence. A high score corresponds to high protein
identification confidence. Peptide # is the number of peptides identified (including duplicates)
with 95% confidence that map to the identified protein sequence and can be used as a rough
approximation of abundance of a given protein. %Cov is the percent of the identified protein
sequence covered by peptide spectral data. %Cov(95) is the percent coverage for peptide spectral
data with ≥95% identification score. Accession corresponds to the NCBI protein accession
identification. Five proteins (PELPIII (MG14 and MG15), TTS, Sp41, and cellulase) were
identified in both F2 and F3. PELPIII was identified with high confidence in both F2 and F3, in
agreement with the protein immunoblot. The database used for spectra identification was the
NCBI nr viridiplantae database, released on 12/17/2011 (NCBI, 2012). Identified proteins were
given an unused score threshold of ≥4.0 with at least two peptides with a ≥95% confidence.