Genomic resources have significantly impacted plant biology research in recent years. Cell biology has been further enabled by an ongoing revolution in visualization technologies. Using fluorescent proteins (FPs), we now have unprecedented views of cellular architecture, and we can study real-time dynamics of cell structure, function, and protein localization. To date, these technologies have been most widely used in Arabidopsis (Arabidopsis thaliana); however, the grasses provide a unique opportunity to study the underlying mechanisms and inter-related controls of cell growth, morphogenesis, and physiology in leading crop models.Here, we present a resource that leverages the emerging maize (Zea mays) genome sequence to develop tools to study protein structure and function in a cellular context. Traditionally, such studies relied on fixed tissue or FP fusions driven by constitutive promoters, which can lead to significant artifacts. The maize genome sequence now provides access to regulatory regions that can be used to drive native expression. We have developed streamlined methods to generate maize FP-tagged lines using these regulatory elements, allowing analysis of tissue-specific expression and localized function. Identification of diverse proteins that function in specific subcellular compartments will provide the tools for understanding basic developmental, biochemical, and physiological processes in maize, with direct application potential for crop improvement. METHODOLOGYWe developed a protocol to generate fusion proteins with yellow (YFP), cyan (CFP), or red (RFP) color variants of FPs driven by native regulatory elements, based on our previous work in Arabidopsis (Tian et al., 2004). In brief, the method uses triple template PCR to generate products of the full genomic sequence with the FP insert, which is flanked by linker peptides to minimize folding interference between the FP and tagged protein. The product is cloned using the Gateway system (Invitrogen) into the donor vector, pDONR207. The tagged gene is transferred into binary destination vectors and ultimately transformed into maize. Full details of the protocols are available at http://maize.jcvi.org/cellgenomics/protocol/maizeTT protocolGFP_111405.shtml.Candidate genes were selected for tagging based on several criteria, including, as first priority, the availability of full genomic sequence plus regulatory regions that included 3 kb upstream and 1 kb downstream of the coding region. A size limit of 8 to 9 kb for the full genomic region with the FP insertion is imposed to ensure good cloning efficiency. Given these size constraints, we next prioritized genes that encoded proteins with robust predicted functions. These decisions were based on homology to other well-studied proteins, known localizations to specific compartments, and/or corroborating antibody or expression data. Genes with available mutations were also given high priority so as to provide a means of functional complementation. Our final criterion was to include candidates that wou...
Fluorescent proteins (FP) have significantly impacted the way that we study plants in the past two decades. In the post-genomics era, these FP tools are in higher demand by plant scientists for studying the dynamics of protein localization, function, and interactions, and to translate sequence information to biological knowledge that can benefit humans. Although FP tools have been widely used in the model plant Arabidopsis, few FP resources have been developed for maize, one of the most important food crops worldwide, and an ideal species for genetic and developmental biology research. In an effort to provide the maize and cereals research communities with a comprehensive set of FP resources for different purposes of study, we generated more than 100 stable transformed maize FP marker lines, which mark most compartments in maize cells with different FPs. Additionally, we are generating driver and reporter lines, based on the principle of the pOp-LhG4 transactivation system, allowing specific expression or mis-expression of any gene of interest to precisely study protein functions. These marker lines can be used not only for static protein localization studies, but will be useful for studying protein dynamics and interactions using kinetic microscopy methods, such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET). All of the constructs and maize marker lines are publicly available through our website, http://maize. jcvi.org/cellgenomics/index.php
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