PDF file: Annual Report 2002/2003 - Scottish Crop Research Institute

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Mechanisms & Processes The role of Potato mop-top virus proteins in intracellular movement L.Torrance, G.H. Cowan, S. Haupt and A. Ziegler To establish a successful systemic infection, plant viruses must move cell-to-cell from the site of infection to the vascular system where they can be translocated throughout the plant. Intracellular movement involves traversing the infected source cell to reach the plasmodesmata (PD), which are small partially occluded pores in the cell walls. PD are the cells’ gatekeepers and regulate the passage of macromolecules, facilitating intercellular communication. Plant viruses have evolved several different strategies to locate and pass through PD, which involve one or more virus-encoded movement proteins (MP). If viruses could not move out of the initial infected cell then the plant would not succumb to infection so a study of movement processes may lead to novel strategies for disease control. The aim of this work is to understand how the movement proteins of Potato mop-top virus (PMTV) operate and what host factors are involved in the movement process. This research project is done in collaboration with colleagues in cell-cell communication programme. The PMTV genome encodes a conserved module of three partially overlapping reading frames called the triple gene block (TGB). We study the expressed proteins using several different experimental systems including biochemical characterisation, transient expression in living cells to examine subcellular localisation, and interactions with host proteins using the yeast two-hybrid system. We have shown previously that the first protein of the movement module (TGBp1) binds RNA and the other two proteins (TGBp2 and TGBp3) are integral membrane proteins that interact with themselves and with each other when expressed in the yeast system. The current model of intracellular movement is that TGBp1 forms a complex with viral RNA (vRNP), which is transported to the periphery of the cell and localised to the PD by interactions with membrane bound TGBp2 and TGBp3. However, the precise details of this process are unknown. The gene encoding TGBp3 was cloned as a fusion to either green (GFP) or red (RFP) fluorescent protein and transiently expressed in plant cells. A fluorescent network was produced on expression of GFP- or RFP- TGBp3 similar in appearance to the cortical endoplasmic reticulum (ER)(see Figure panel a), fluorescence was also observed associated with the perinuclear envelope and in small punctate spots at the cell periphery suggestive of PD labelling. In addition, TGBp3 formed small spots that could be seen moving along the ER (see Figure panel b), this movement was inhibited by the compound latrunculin A, which is known to depolymerise actin. To try to identify the structures more accurately, RFP-TGBp3 was transiently expressed on transgenic plants where the ER or PD were labelled with GFP. In these experiments, red fluorescence was seen colocalised to the green ER network (Figure panel c). Also, the peripheral punctate red spots produced by RFP-TGBp3 co-localised to green PD (Figure panel a) b) c) d) Figure 1 Confocal microscope images of fluorescent TGBp3 fusion proteins expressed in living cells a) Network of fluorescence and small motile spots obtained on expression of GFP-TGBp3 in Nicotiana benthamiana cells b) Small red fluorescent spots seen moving along ER network in cells of transgenic N. benthamiana (where ER labelled with GFP) c) Red fluorescent network co-localises to green ER network when RFP-TGBp3 is expressed on transgenic N. benthamiana (where ER labelled with GFP) d) Punctate red fluorescent spots co-localise to green plasmodesmata in transgenic N. tabacum (transformed with GFP fused to the 30K MP of Tobacco mosaic virus). 104
Mechanisms & Processes d). This data suggests that TGBp3 is involved in transporting the vRNP from the sites of virus replication associated with perinuclear and ER membranes utilizing the cortical ER/actin network. Also, that TGBp3 contains a signal to enable PD targeting, either directly or indirectly via other host factors. Yeast two-hybrid experiments where TGBp3 was used as bait to search for putative interacting plant proteins have revealed an interaction between TGBp3 and a nuclear envelope protein and further work is in progress to confirm this interaction and understand the role it plays in the movement process. Future work will also examine the role of TGBp2, its coordinate mode of action with TGBp3 and identify and characterise key host proteins and receptors that interact with them. 105
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Scottish Crop Research Institute ®
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The Scottish Crop Research Institut
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Contents Biomathematics and Statist
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Introduction upon the Commissioner
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Introduction ® Scottish Crop Resea
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Report of the Director John R. Hill
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Director’s Report tion from dismi
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Director’s Report paper A revised
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Director’s Report and the relatio
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Director’s Report Member States A
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Director’s Report US subsidies fr
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Director’s Report regions of the
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Director’s Report of silver and m
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Director’s Report tions. Obscenel
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Director’s Report participation.
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Director’s Report heads of state
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Director’s Report Philippines (2.
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Director’s Report Food Aid Famine
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Director’s Report adoption of unh
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Director’s Report economy propose
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Director’s Report (herbicides, in
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Director’s Report by a combinatio
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Director’s Report mended a study
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Director’s Report notes, bulletin
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Director’s Report research instit
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Director’s Report Ireland. Also i
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Page 55 and 56: Director’s Report shares of total
Page 57 and 58: Director’s Report tonnes in 1999.
Page 59 and 60: Director’s Report Relative Import
Page 61 and 62: Director’s Report cheap food-stuf
Page 63 and 64: Director’s Report culturally-rela
Page 65 and 66: Director’s Report on large parts
Page 67 and 68: Director’s Report minimum input s
Page 69 and 70: Director’s Report organic farming
Page 71 and 72: Viruses - evolving organisms ? Viru
Page 73 and 74: Viruses - evolving organisms ? CLCu
Page 75 and 76: Viruses - evolving organisms ? a) b
Page 77 and 78: Barley Crop Development formation o
Page 79 and 80: Barley Crop Development Year Total
Page 81 and 82: Barley Crop Development on a year t
Page 83 and 84: GM herbicide-tolerant crops Some pe
Page 85 and 86: GM herbicide-tolerant crops and som
Page 87 and 88: GM herbicide-tolerant crops mitted
Page 89 and 90: GM herbicide-tolerant crops 12 Squi
Page 91 and 92: Mechanisms & Processes Peronospora
Page 93 and 94: Mechanisms & Processes Proteomic an
Page 95 and 96: Mechanisms & Processes High-through
Page 97 and 98: Mechanisms & Processes Studying pla
Page 99 and 100: Mechanisms & Processes Phytoene pea
Page 101 and 102: Mechanisms & Processes New discover
Page 103: Mechanisms & Processes in these cel
Page 107 and 108: Genes to Products ic enzymes (use o
Page 109 and 110: Genes to Products Carotenogenesis i
Page 111 and 112: Genes to Products accumulation in s
Page 113 and 114: Genes to Products Retrotransposons
Page 115 and 116: SH43 SL03 SH44 SH12 B20 SL22 B47 B0
Page 117 and 118: Management of genes and organisms i
Page 119 and 120: Environment Functional diversity in
Page 121 and 122: Environment Plant root biomechanics
Page 123 and 124: Environment Microbial and microfaun
Page 125 and 126: Environment The role and importance
Page 127 and 128: Environment duced from barley mixtu
Page 129 and 130: Environment IOBC GMO Guidelines Pro
Page 131 and 132: Environment Mathematical modelling
Page 133 and 134: Biomathematics and Statistics Scotl
Page 135 and 136: Research services Analytical facili
Page 137 and 138: Research services Division of Finan
Page 139 and 140: Research services Information Techn
Page 141 and 142: Research services Estate, Glasshous
Page 143 and 144: Research services Engineering and M
Page 145 and 146: Research services MRS will: strengt
Page 147 and 148: Research services Health and Safety
Page 149 and 150: Publications Blok, V.C. 2002. Progr
Page 151 and 152: Publications Phenotype/genotype ass
Page 153 and 154: Publications for virus-transmitting
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Publications Muckenschnabel, I., Go
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Publications rich grassland. Procee
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Publications Woodford, J.A.T., Fent
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Statement of health & safety policy
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The Governing Body The Governing Bo
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The Governing Body development and
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Staff Theme 2 - Genes to Products T
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Staff Division of Finance and Admin
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Staff Resignations Name Unit Band M
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Staff Editorial Duties Name Positio
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Staff Name Position Committee or Or
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SCRI Research Programme 2002-2003 S
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Research Projects SCR/587/02 SCR/58
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Meteorological Records D. Petrie &
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List of Abbreviations AAB ACRE ADAS
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