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Figure  S1. Localization of Os Tudor-SN and cytoskeleton in developing rice endosperm by indirect immunofluorescence analysis. Os Tudor-SN exists as two types; one tightly associated with microtubules (a–d) and a second form as particles dispersed among microtubules and actin filaments (e, f). (a) Staining of microtubule bundles in two endosperm cells with the cell on the left sectioned through the cortical region and the cell on the right sectioned towards the middle. (b) Same section as in (a) but stained for Os Tudor-SN. (c) Merged images of (a) and (b). (d) Merged image of the distribution of microtubules disrupted by nocodazole (green channel) and Os Tudor-SN (red channel). (e) Merged image depicting the distribution of Os Tudor-SN particles (red channel) in a region enriched for actin filaments (green channel). (f) Merged image depicting the distribution of Os Tudor-SN particles (red channel) and actin filaments (green channel) disrupted by latrunculin B.

Figure  S2. Evidence for microfilament-dependent GFP-Os  Tudor-SN particle movement. Developing endosperm sections were obtained by free-hand and bathed in N6 media supplemented with amino acids and containing DMSO and various cytoskeletal inhibitors. For each treatment, six endosperm sections were analyzed. In most instances, GFP-Os  Tudor-SN particle movement was detected up to 30 min in the control speciments (N6 media and DMSO) and in the presence of the microtubule inhibitors oryzalin and nocodazole. The microfilament inhibitor, latrunculin B, effectively inhibited movement before or at 15 min of incubation except for one instance where movement was detected up to 25 min. Cytochalasin D also suppressed particle movement although it was not as efficient as latrunculin B.

Figure  S3. Distribution of prolamine PBs in wildtype (a) and Gt1-7 RNAi transgenic (b) lines. The bottom panel depicts size distribution of PBs in wildtype (blue) and red (Gt1-7).

Figure  S4. The expression of OsTudor-SN during seed development in the RNAi plant lines Gt1-7 and Gt1-15.  Protein extracts were prepared from ~60 mg of seeds at different stages of development and 20 μg of protein of each sample analyzed for the levels of Os Tudor-SN and PDI by immunoblot analysis. Note the absence or near absence of OsTudor-SN and decrease amounts of prolamine and glutelin in the two RNAi plant lines, Gt1-15 and Gt1-7, in 10 day old developing seeds, whereas 12 day old wildtype seeds contain ample quantities. By contrast, PDI protein levels were relatively unaltered. The wildtype sample was prepared from mature seeds.

Video Clip  S1. A series of images depicting the close spatial relationship between of OsTudor-SN particles and prolamine protein bodies (PBs) in developing endosperm cells.

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