Functional analysis of genes controlling protein trafficking through plasmodesmata using Arabidopsis thaliana T-DNA insertion mutants

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Plasmodesmata are cytoplasmic connections between plants cells which are essential for intercellular communication and exchange of molecules and signals. They are highly organised in their structure and function but molecular mechanisms regulating their dynamic changes and transport activities are largely unknown. We have generated a T-DNA insertion bank using transgenic Arabidopsis thaliana plants expressing GFP from a phloem specific promoter. In a screen for reduced trafficking of GFP by symplastic post-phloem transport in the root tissue eight mutants have been isolated. A first PCR-based analysis of the T-DNA insertions has revealed several candidate genes for each mutant. We will identify the genes responsible for the phenotypes and confirm these results by a second mutant allele for each gene and complementation tests. The genes will be characterised by generating promoterreporter lines to analyse the tissue specific expression patterns and by cloning the cDNAs to generate constructs for protein localisation studies and gain-of-function analyses. The loss-of-function and gain-of-function mutants will be analysed for anatomical phenotypes and for GFP movement phenotypes in various tissues and at all developmental stages. Further functional tests will depend on the identified genes and their predicted structure and function.