Volume 8 issue 1 January 2007
Targeting of TMV movement protein to plasmodesmata requires the actin/ER network; evidence from FRAP
Kathryn M. Wright, Nicola T. Wood, Alison G Roberts, Sean Chapman, Petra Boevink, Katrin M. MacKenzie, and Karl J. Oparka

Video 1: Time series of ER movement. Series of 30 images, taken at 1s 164ms intervals, of ER-GFP epidermal cells showing normal ER movement in tissue pre-treated for 2h with water (control), 500µM colchicine or
20µg ml^-1 oryzalin, and the disrupted structure and lack of movement in tissue treated with 0.02% sodium azide, 25µM latrunculin or 0.1mg ml^-1 cytochalasin B.

Video 1 (.mov)

Video 2: Time series of Golgi apparatus movement. Series of 30 images, taken at 1s 164ms intervals, of STtmd-GFP epidermal cells showing the normal movement of Golgi stacks in tissue pre-treated for 2h with water (control), 500µM colchicine or 20µg ml^-1 oryzalin, and the lack of movement in tissue treated with 0.02% sodium azide, 25µM latrunculin or 0.1mg ml^-1 cytochalasin B.

Video 2 (.mov)

Figure 1: Appearance of cell organelles in Nicotiana epidermal cells. In all images GFP-associated fluorescence is coloured green and chlorophyll-associated autofluorescence is coloured blue.

(A) Appearance of a sodium azide-treated TMV.MP-EGFP.CP lesion on Nicotiana benthamiana away from the leading edge showing that azide does not alter the accumulation of MP on MTs. Bar = 25µm.

(B) TUA-GFP plants showing MTs and apparent accumulation of GFP into aggregates after treatment with
100µg ml^-1 BFA. Bar = 25µm.

(C) Maximum projection of a stack of confocal images taken at the leading edge of a TMV.MP-EGFP.CP lesion on Nicotiana benthamiana (left) showing the targeting of MP to PD, formation of clusters and accumulation on MT after treatment with 100µg ml^-1 BFA. Bar = 25µm.

(D) Alexa 488 phalloidin staining of actin, showing an intact actin network after treatment with 100µg ml^-1 BFA. Bar = 25µm.

(E) TUA-GFP transgenic plants, 4 days after Agrobacterium inoculation with a strain carrying a binary for YFP-HDEL expression. When treated with cytochalasin B, GFP fluorescence (green) in a non-infected cell (left) forms a similar pattern to ER-associated fluorescence (magenta) in the neighbouring cell (upper right) indicating possible accumulation of GFP-tubulin at the ER vertices. Bar = 25µm.

(F) Maximum projection of a stack of confocal images taken at the leading edge of a TMV.MP-EGFP.CP lesion on Nicotiana benthamiana (lower left) showing the targeting of MP to PD and the formation of clusters after treatment with latrunculin. Bar = 25µm.

(G) Anti-ß-tubulin Cy3 conjugate labeling of Nicotiana benthamiana leaf epidermis under control conditions showing an intact MT network. Bar = 10µm.

(H) and (I) TUA-GFP transgenic plants infected with TMV.MP-mRFP.ΔCP. At the edge of the infection the presence of MP clusters (magenta) (darts) does not prevent the complete disruption of the MTs (green) following treatment with 500µM colchicine (H). Under control conditions (I) MP clusters (magenta) (darts) at the edge of the infection can be seen, as well as MTs (green) (arrows). Bar = 25µm.

(J) Maximum projection of a stack of confocal images of a TMV.MP-EGFP.CP lesion on Nicotiana benthamiana showing that the association of MP with MT prevents the disruption of MT when treated with 500µM colchicine.
Bar = 25µm.

(K) and (L). Alexa 488 phalloidin staining of actin, showing no significant change after treatment with colchicine (K) or oryzalin (L). Bar = 25µm.

(M) and (N) ER-GFP transgenic plants showing no obvious alteration to the ER network in the presence of colchicine (M) or oryzalin (N). Bar = 25µm.

(O) and (P) STtmd-GFP transgenic plants treated with colchicine (O) or oryzalin (P) showing no significant effect on Golgi stacks. Bar = 25µm.

Figure 1(.jpg)