Myosin XI Is Required for Actin-Associated Movement of Plastid Stromules  Natesan Senthil Kumar A. , Sullivan James A. , Gray John C.   Molecular Plant  Volume 2, Issue 6, Pages 1262-1272 (November 2009) DOI: 10.1093/mp/ssp078 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Effect of Cytoskeleton Inhibitors on Stromules. (A) Effect of the myosin ATPase inhibitor 2,3-butanedione 2-monoxime (BDM) on stromules. Lower epidermal peels of tobacco leaves expressing TPT–YFP or GFP–mTn were incubated with 1 mM BDM and extended-focus images were produced at 0 and 120 min. (B) Effect of actin-microfilament inhibitors, latrunculin B (LatB) and cytochalasin D (CytD), on stromules. Lower epidermal peels of tobacco leaves expressing TPT–YFP or GFP–mTn were incubated with 100 μM cytochalasin D or 10 μM latrunculin and extended-focus images were produced at 0 and 60 min. (C) Effect of microtubule inhibitors, amiprophosmethyl (APM) and oryzalin, on stromules. Lower epidermal peels of tobacco leaves expressing TPT–YFP or GFP–MBD were incubated with 26 μM APM or 36 μM oryzalin and extended-focus images were produced at 0 and 45 min or 0 and 75 min, respectively. Images represent dual channel (chlorophyll, red; YFP, green) for leaves expressing TPT–YFP whereas the images for leaves transiently expressing GFP–mTn or GFP–MBD are represented by a single channel (GFP, green). Molecular Plant 2009 2, 1262-1272DOI: (10.1093/mp/ssp078) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Structure and Phylogenetic Relationships of Myosin XI from N. benthamiana. (A) Diagrammatic representation of the structural features of myosin XI-2. The protein consists of 1512 amino acid residues and the following domains were identified using Pfam, SMART, and coiled-coil predictions: an SH-3-like domain (residues 10–51), the motor domain (residues 56–734), six IQ calmodulin binding motifs (residues 735–876), two coiled-coil domains (residues 878–958 and 973–1049), and the dilute domain (residues 1156–1458). (B) Phylogenetic relationships of tobacco and Arabidopsis proteins of the myosin XI-G subclass (Avisar et al., 2008). C-terminal regions homologous to amino acid residues 1109–1512 of myosin XI-2 from N. benthamiana were aligned in Clustal W 2.0 (Larkin et al., 2007) and analyzed in Phylo_win, using 1000 bootstrap trials (Galtier et al., 1996), with AtXI-I as the out-group. The alignment is shown in Supplemental Figure 1. The sequences were N. tabacum Ntmy175 (Nt175, AB082121), N. benthamiana myosin XI-2 (NbXI-2, DQ875135), Arabidopsis XI-A (AtXI-A, AT1G04600.1), Arabidopsis XI-B (AtXI-B, AT1G04160.1), Arabidopsis XI-D (AtXI-D, AT2G33240.1), Arabidopsis XI-G (AtXI-G, AT2G20290.1), Arabidopsis XI-H (AtXI-H, AT4G28710.1), Arabidopsis XI-I (AtXI-I, AT4G33200.1), and Arabidopsis MYA2 (AtMya2, AT5G43900.1). Molecular Plant 2009 2, 1262-1272DOI: (10.1093/mp/ssp078) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 RNA Interference for Myosin XI in N. benthamiana Plants Expressing Plastid Envelope-Targeted YFP. (A) Confocal images of chloroplasts in non-infiltrated, pROK2 (empty vector)-infiltrated and pMyoRNAi-infiltrated N. benthamiana leaves at 5, 6, 7, and 8 DAI (days after infiltration). Images are overlays of YFP (green) and chlorophyll (red) fluorescence. Images are maximum projections of several confocal images taken along the z-axis. Scale bar 10 μm. (B) Effect of myosin XI RNAi on the percentage of chloroplasts with stromules in leaf epidermal cells. The number of chloroplasts with and without stromules was counted in at least 10 cells from each infiltrated region of a total of 15 leaves; chloroplasts with stromules were expressed as a percentage. All the values are mean ± SEM of 15 independent observations. ▪, control leaves; ▴, pROK2 (empty vector)-infiltrated leaves; O, myosin XI RNAi vector-infiltrated leaves. (C) Down-regulation of myosin XI transcripts in N. benthamiana leaves by RNAi. RT–PCR was performed with 24 cycles for actin and 27 cycles for myosin XI in a duplex RT–PCR reaction and 30 cycles for myosin VIII in a separate reaction, with equal amounts of cDNA from control and RNAi leaves. The products from the duplex reaction for actin and myosin XI were resolved on 1% agarose gel, whereas the products from the reaction for myosin VIII was resolved on 1.5% agarose gel. L, hyper ladder IV; B, blank; 1, pMyoRNAi-infiltrated leaves at 5 DAI; 2, non-infiltrated leaves at 6 DAI; 3, pROK2-infiltrated leaves at 6 DAI; 4, pChlSynRNAi-infiltrated leaves at 6 DAI; 5, pMyoRNAi-infiltrated leaves at 6 DAI; 6, pMyoRNAi-infiltrated leaves at 8 DAI. DAI, days after infiltration. Molecular Plant 2009 2, 1262-1272DOI: (10.1093/mp/ssp078) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Transient Expression of GFP–Myosin XI Cargo Domain and FDH–DsRed Fusion Proteins in Tobacco Leaf Epidermal Cells. (A) Transient expression of GFP–myosin XI cargo domain fusion-protein following microprojectile bombardment with tungsten particles coated with pGFPmXIcd. A small GFP-labeled non-chlorophyll-containing body is indicated by an arrow. The chlorophyll channel also shows out-of-focus chlorophyll fluorescence from underlying mesophyll cells. Scale bar 5 μm. (B) Fluorescence from a single epidermal cell showing GFP localized around the chloroplasts, in smaller bodies and at the cell periphery. Arrow shows a single mitochondrion labeled by DsRed and GFP. Chlorophyll fluorescence is false-colored blue. Scale bar 10 μm. (C) Fluorescence around a single chloroplast within the epidermal cell shown in (B). GFP is green, DsRed is red, and chlorophyll fluorescence is false-colored blue. Images are maximum projections of several confocal images taken along the z-axis. Scale bar 2 μm. Molecular Plant 2009 2, 1262-1272DOI: (10.1093/mp/ssp078) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions