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...

RAB guanosine triphosphatases (GTPases) are key regulators of vesicle trafficking and are essential to the growth and development of all eukaryotic cells. During evolution, the RAB family has expanded in different patterns to facilitate distinct cellular, developmental and physiological adaptations. Yeast has only 11 family members, whereas mammalian RABs have expanded to 18 RAB subfamilies. Plant RABs have diversified primarily by duplicating members within a single subfamily. Plant RABs are divided into eight subfamilies, corresponding to mammalian RAB1, RAB2, RAB5, RAB6, RAB7, RAB8, RAB11 and RAB18. Functional diversification of these is exemplified by the RAB11s, orthologs of which are partitioned into unique cell compartments in plants where they function to transport vesicles during localized tip growth. Similarly, the RAB2 family in grasses is likely involved in vesicle secretion associated with wall expansion, as determined by analysis of over-expression mutants. We propose that dicots and monocots have also diverged in their RAB profiles to accommodate unique cellular functions between the two groups. Here we present a bioinformatics analysis comparing the RAB sub-families of rice, maize and Arabidopsis. These results will guide future functional studies to test for the role of diversification of subfamilies unique to monocots compared to dicots. Key words: dicot; GTP binding protein; monocot; phylogenetic analysis; RAB guanosine triphosphatase.Zhang J, Hill DR, Sylvester AW (2007). Diversification of the RAB guanosine triphosphatase family in dicots and monocots.Coordination of vesicle trafficking is essential to the growth and development of all eukaryotic cells. Members of the RAB family of guanosine triphosphatase (GTP) binding proteins are central to this process because they shuttle vesicles within specific cellular compartments and enable exocytosis, endocytosis, membrane cycling and other trafficking events (for review see

The inability to sialylate recombinant glycoproteins is a critical limitation of the baculovirusinsect cell expression system. This limitation is due, at least in part, to the absence of detectable sialyltransferase activities and CMP-sialic acids in the insect cell lines routinely used as hosts in this system. SfSWT-1 is a transgenic insect cell line encoding five mammalian glycosyltransferases, including sialyltransferases, which can contribute to sialylation of recombinant glycoproteins expressed by baculovirus vectors. However, sialylation of recombinant glycoproteins requires culturing SfSWT-1 cells in the presence of fetal bovine serum or another exogenous source of sialic acid. To eliminate this requirement and extend the utility of SfSWT-1 cells, we have isolated a new baculovirus vector, AcSWT-7B, designed to express two mammalian enzymes that can convert N-acetylmannosamine to CMP-sialic acid during the early phase of infection. AcSWT-7B was also designed to express a model recombinant glycoprotein during the very late phase of infection. Characterization of this new baculovirus vector showed that it induced high levels of intracellular CMP-sialic acid and sialylation of the recombinant Nglycoprotein upon infection of SfSWT-1 cells cultured in serum-free medium supplemented with N-acetylmannosamine. In addition, co-infection of SfSWT-1 cells with AcSWT-7B plus a conventional baculovirus vector encoding human tissue plasminogen activator resulted in sialylation of this recombinant N-glycoprotein under the same culture conditions. These results demonstrate that AcSWT-7B can be used in two different ways to support recombinant Nglycoprotein sialylation by SfSWT-1 cells in serum-free medium. Thus, AcSWT-7B can be used to extend the utility of this previously described transgenic insect cell line for recombinant sialoglycoprotein production.