Table I.

Key points for live-cell imaging in plants

High-throughput protein localization is critical to the success of comprehensive functional genomics projects (Tian et al., 2004; Koroleva et al., 2005; Bhat et al., 2006; Matsuyama et al., 2006; Pepperkok and Ellenberg, 2006).

Current algorithms for predicting protein localization are often incapable of making accurate determinations (Fig. 2B). Thus live-cell imaging of chimeric AFP protein fusions are essential to characterize proteins whose function or localization cannot be predicted using computational methods (Li et al., 2006).

Gateway-cloning technology is currently the most effective means to integrate high-throughput proteomics, localization, and gene-silencing projects since the same set of entry clones could be used in all studies (Brasch et al., 2004; Earley et al., 2006). Many new binary vectors are being constructed using Gateway technology, thereby enhancing exchange of resources between labs (Chung et al., 2005; Earley et al., 2006; Chakrabarty et al., 2007).

Of any comparable system, the pSAT vectors offer the widest choices of (1) AFPs, (2) promoters to drive expression, and (3) flexibility in constructing binary vectors containing more than one expression cassette (Chung et al., 2005; Tzfira et al., 2005).

Due to the inability to efficiently predict the effect of AFPs on the stability of fusion partners, high-throughput protein localization should be performed using both N- and C-terminal fusions (Simpson et al., 2001; Pepperkok and Ellenberg, 2006). Alternatively, cloning of AFPs into internal sites can be performed (Tian et al., 2004; Li et al., 2006).

More than simply markers for protein localization, next generation AFPs (1) provide a greater diversity of colors, (2) can be photoactivated or converted facilitating protein tracking studies, (3) have been optimized for several advanced imaging techniques, and (4) can be used for characterizing protein-protein interactions (Patterson and Lippincott- Schwartz, 2004; Habuchi et al., 2005; Chudakov et al., 2006; Gurskaya et al., 2006).

A. tumefaciens (agroinfiltration) can be used to transiently express proteins in virus-infected plants to provide data similar to that generated with transgenic plants.