High-Throughput Fluorescent Tagging of Full-Length Arabidopsis Gene Products in Planta

Guo-wei, Tian; Mohanty, Amitabh; Chary, S. Narasimha; Li, Shijun; Paap, Brigitte; Drakakaki, Georgia; Kopec, Charles D.; Li, Jianxiong; Ehrhardt, David; Jackson, David; Rhee, Seung Y.; Raikhel, Natasha V.; Citovsky, Vitaly

doi:10.1104/pp.104.040139

Cited by 233 publications

(255 citation statements)

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“…The full-length LTPG1 cDNA was used to amplify the LTPG1 cDNA1 and LTPG1 cDNA2 fragments using the LTPG1 cDNA F2/LTPG1 P1 and LTPG1 P2/LTPG1 cDNA R2 primers, respectively. A triple template PCR was then performed to produce an LTPG1 cDNA with the EYFP following the method described by Tian et al (2004). The triple template PCR products were digested with SmaI/BamHI enzymes and ligated into SmaI/BamHI-digested p35S-EYFP-Flag/Strep plasmid.…”

Section: Eyfp Tagging and Subcellular Localization Of Ltpg1mentioning

confidence: 99%

Disruption of Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Gene Altered Cuticular Lipid Composition, Increased Plastoglobules, and Enhanced Susceptibility to Infection by the Fungal Pathogen Alternaria brassicicola

et al. 2009

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All aerial parts of vascular plants are covered with cuticular waxes, which are synthesized by extensive export of intracellular lipids from epidermal cells to the surface. Although it has been suggested that plant lipid transfer proteins (LTPs) are involved in cuticular lipid transport, the in planta evidence is still not clear. In this study, a glycosylphosphatidylinositol-anchored LTP (LTPG1) showing higher expression in epidermal peels of stems than in stems was identified from an Arabidopsis (Arabidopsis thaliana) genome-wide microarray analysis. The expression of LTPG1 was observed in various tissues, including the epidermis, stem cortex, vascular bundles, mesophyll cells, root tips, pollen, and early-developing seeds. LTPG1 was found to be localized in the plasma membrane. Disruption of the LTPG1 gene caused alterations of cuticular lipid composition, but no significant changes on total wax and cutin monomer loads were seen. The largest reduction (10 mass %) in the ltpg1 mutant was observed in the C29 alkane, which is the major component of cuticular waxes in the stems and siliques. The reduced content was overcome by increases of the C29 secondary alcohols and C29 ketone wax loads. The ultrastructure analysis of ltpg1 showed a more diffuse cuticular layer structure, protrusions of the cytoplasm into the vacuole in the epidermis, and an increase of plastoglobules in the stem cortex and leaf mesophyll cells. Furthermore, the ltpg1 mutant was more susceptible to infection by the fungus Alternaria brassicicola than the wild type. Taken together, these results indicated that LTPG1 contributed either directly or indirectly to cuticular lipid accumulation.During growth and development, plants are subjected to various environmental stresses, including drought, cold, exposure to UV light, and pathogen attack. The first barrier between plants and environmental stresses is the cuticle, which is composed of a lipophilic cutin polymer matrix and waxes (Holloway, 1982;Jeffree, 1996;Kunst et al., 2005;Nawrath, 2006). The cuticular waxes, which consist of very long chain fatty acids (VLCFAs; C20 to C34) and their derivatives, are embedded within and encase the cutin polymer matrix, a polyester framework composed of hydroxy fatty acids (C16 and C18) and glycerol monomers (

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Section: Eyfp Tagging and Subcellular Localization Of Ltpg1mentioning

confidence: 99%

Disruption of Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Gene Altered Cuticular Lipid Composition, Increased Plastoglobules, and Enhanced Susceptibility to Infection by the Fungal Pathogen Alternaria brassicicola

et al. 2009

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“…All PCR conditions were as described (Tian et al, 2004). The resulting AtCRT2-YFP gene was verified by DNA sequencing and transferred into pBIN-GW by Gateway recombination as described (Tian et al, 2004). In addition, we transferred the TMV MP-GFP expression cassette from the pCd vector into the Asp-718-XbaI sites of pBIN19 (accession no.…”

Section: Expression Of Fluorescently Tagged Calreticulin and Tmv Mpmentioning

confidence: 99%

“…Our construct included the entire intergenic region (1,235 bp) upstream of the AtCRT2 translation initiation codon and 974 bp downstream of the stop codon. All PCR conditions were as described (Tian et al, 2004). The resulting AtCRT2-YFP gene was verified by DNA sequencing and transferred into pBIN-GW by Gateway recombination as described (Tian et al, 2004).…”

Section: Expression Of Fluorescently Tagged Calreticulin and Tmv Mpmentioning

confidence: 99%

“…For expression in transgenic plants, the full-length AtCRT2 gene (AGI code At1g09210) with its native regulatory elements was internally (30 bp upstream of the stop codon) tagged with the citrine variant of YFP (Griesbeck et al, 2001) using the fluorescent tagging of full-length proteins technique (Tian et al, 2004) with P1, P2, P3, and P4 primers: 5#-GCTCGATCCACCTAGG-CTTTTGGGAGCTCTGTTCTGATCCT-3#, 5#-CACAGCTCCACCTCCACCT-CCAGGCCGGCCGTCTTTCTCAGAGGTTTCCTCGCTAT-3#, 5#-TGCTGGT-GCTGCTGCGGCCGCTGGGGCCGCCACCGCTCATGTATGCTA-3#, and 5#-CGTAGCGAGACCACAGGATACAAGAAGTGGGAGGGAAAATA-3#, respectively. Our construct included the entire intergenic region (1,235 bp) upstream of the AtCRT2 translation initiation codon and 974 bp downstream of the stop codon.…”

Section: Expression Of Fluorescently Tagged Calreticulin and Tmv Mpmentioning

confidence: 99%

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Effects of Calreticulin on Viral Cell-to-Cell Movement

et al. 2005

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Cell-to-cell tobacco mosaic virus movement protein (TMV MP) mediates viral spread between the host cells through plasmodesmata. Although several host factors have been shown to interact with TMV MP, none of them coresides with TMV MP within plasmodesmata. We used affinity purification to isolate a tobacco protein that binds TMV MP and identified it as calreticulin. The interaction between TMV MP and calreticulin was confirmed in vivo and in vitro, and both proteins were shown to share a similar pattern of subcellular localization to plasmodesmata. Elevation of the intracellular levels of calreticulin severely interfered with plasmodesmal targeting of TMV MP, which, instead, was redirected to the microtubular network. Furthermore, in TMV-infected plant tissues overexpressing calreticulin, the inability of TMV MP to reach plasmodesmata substantially impaired cell-to-cell movement of the virus. Collectively, these observations suggest a functional relationship between calreticulin, TMV MP, and viral cell-to-cell movement.Following initial infection (usually by mechanical inoculation), many plant viruses, such as tobacco mosaic virus (TMV), spread from cell to cell through plasmodesmata (for review, see Heinlein, 2002;Waigmann et al., 2004), presumably utilizing the existing cellular pathways of plasmodesmal transport. In the case of TMV, this virus encodes a specific cell-to-cell movement protein (MP), which mediates the transport of the viral genomic RNA through plasmodesmata (Deom et al., 1987). To date, MP has been shown to bind TMV RNA (Citovsky et al., 1990(Citovsky et al., , 1992, associate with the cytoskeleton (Heinlein et al., 1995;McLean et al., 1995;Boyko et al., 2000) and endoplasmic reticulum (ER; Heinlein et al., 1998;Reichel and Beachy, 1999), target to plasmodesmata within plant cell walls (Tomenius et al., 1987;Ding et al., 1992a), increase plasmodesmal permeability (Wolf et al., 1989;Waigmann et al., 1994), and undergo negative regulation by phosphorylation (Citovsky et al., 1993;Waigmann et al., 2000;Trutnyeva et al., 2005). To perform many of these functions, TMV MP most likely cooperates with different cellular factors, some of which, such as cell wall pectin methylesterases (PME; Dorokhov et al., 1999;Chen et al., 2000), microtubules (MTs) and actin filaments (Heinlein et al., 1995;McLean et al., 1995;Boyko et al., 2000), and a RIO protein kinase (Yoshioka et al., 2004), are known, while others remain to be discovered. It would be especially interesting to determine whether cellular proteins exist that not only recognize TMV MP but also coreside with it within plasmodesmata.Using TMV MP as specific ligand in a biochemical purification protocol, we showed that it interacts with plant calreticulin. Calreticulin is a Ca 21 -sequestering protein that is highly conserved between different species, including plants ( Nelson et al., 1997; Borisjuk et al., 1998, and refs. therein;Michalak et al., 1998). Functionally, calreticulin is involved in Ca 21 storage and signaling, chaperone activity, cell ad...

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“…As an analytical tool, confocal microscopy has rapidly evolved from being a challenging technique with limited accessibility to a high-throughput tool providing quantitatively precise localization data for 30% of the proteome in the model plant Arabidopsis (Arabidopsis thaliana; Chalfie et al, 1994;Cutler et al, 2000;Tian et al, 2004;Heazlewood et al, 2007). The capacity to visualize a protein relies on excitation of an autofluorescent protein (AFP), such as the GFP derived from the jellyfish Aqueora victoriae, fused to a plant protein of interest.…”

mentioning

confidence: 99%