Progress 08/15/02 to 08/14/04
Outputs
This project was to develop a genetic system to investigate the cellular factors that regulate cell-to-cell trafficking of proteins through plasmodesmata (PD). We use trafficking of the Cucumber mosaic virus 3a movement protein (CMV 3a MP) fused to green fluorescent protein (GFP) in Arabidopsis thaliana as a reporter for mutant screening. We originally proposed to use A. thaliana (WS) as our material because we had a transgenic line of this ecotype expressing CMV 3a MP:GFP. Some reviewers suggested that we develop a Columbia line for analyses because it will be easier for mapping and gene cloning in the long run. We took this suggestion and started again by making transgenic Arabidopsis (Col) expressing CMV 3a MP:GFP under the control of the CaMV 35S promoter. We eventually succeeded in getting homozygous lines. Presence of green fluorescent dots in the cell walls of these plants serves as a visual marker for 3a MP:GFP targeting to PD. Homozygous transgenic plants were
subjected to EMS-mutagenesis and fluorescence microscopic screening. The plants showing absence of green fluorescent dots from the cell walls were collected as putatively defective in targeting of 3a MP:GFP to PD. We have obtained four lines of such putative mutants. DNA sequencing, protein expression, immunolabeling and crossing experiments showed that the mutations occur in plant genes and affect CMV 3a MP:GFP targeting to PD. One mutant has altered leaf morphology and segregation analyses so far suggested that the phenotype is associated with the mutation. We have tentatively mapped one of these mutations to chromosome 2. Meanwhile, to corroborate the genetic analyses, we conducted structural studies on PD in Arabidopsis. Published work in tobacco showed that simple PD undergo structural modifications by branching, via mechanisms still poorly understood, during leaf development and that the branching is accompanied with significant changes in protein transport functions. To learn
about the mechanisms of PD branching and its general biological significance, we analyzed structural modifications of PD in relation to protein targeting in leaves of Arabidopsis. Our studies showed that, during branching of simple PD, new branches could be formed via entrapment of endoplasmic reticulum strands when Golgi vesicles fuse to increase local cell wall thickness. These new findings allowed us to develop a model of multi-order branching of PD. We also showed that in transgenic Arabidopsis plants expressing the CMV 3a MP:GFP, the fusion protein was targeted to branched, but not simple, PD. These results suggest that a deliberate system exists in cells to regulate branching of PD to redefine trafficking functions during development. A manuscript reporting these results was recently submitted for publication (Ma et al., 2007).
Impacts
Intercellular communication is critical for cell differentiation, organogenesis, and physiological functions. How plant cells communicate with one another is still poorly understood. Regulated intercellular protein traffic may be a mechanism for such communication. Our pioneering genetic work to identify cellular factors that regulate protein trafficking are expected to shed light on the mechanisms and functions of intercellular communication in plant growth and development as well as host factors for viral movement. In the long term, knowledge of plasmodesmal traffic could aid in genetic engineering of crops with improved or modified carbon allocation patterns, other desirable physiological or developmental traits, resistance to viral infection, and even better utilization of plants as bioreactors to produce and compartmentalize value-added products.
Publications
- Ma, F., Itaya, A., and Ding, B. (2007) Multi-order branching of plasmodesmata and protein targeting during leaf development in Arabidopsis thaliana (Brassicaceae). Am. J. Bot. (In revision)
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Progress 10/01/02 to 09/30/03
Outputs
We have characterized development of plasmodesmal structure in Arabidopsis leaves of successive developmental stages. We have generated new transgenic Arabidopsis lines, in the Col, Ler and Ws backgrounds, that express the cucumber mosaic virus movement protein fused to green fluorescent protein. We have obtained homozygous and single-copy insertion transgenic lines for mutational analysis of the cellular factors that regulate cell-cell protein trafficking. We are also in the process of characterizing the previous mutants we isolated.
Impacts
The genetic analysis of cellular factors involved in intercellular protein trafficking will lead to biochemical and molecular identification of such factors. Once such factors are identified, their functions in controlling trafficking of plant proteins to regulate development and in interacting with viral proteins to facilitate viral spread can be studied.
Publications
- Ding, B., Itaya, A. and Qi, Y. (2003) Symplasmic protein and RNA traffic: regulatory points and regulatory factors. Curr. Opin. Plant Biol. 6:596-602.
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