Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Hellens, Roger P.; Allan, Andrew C.; Friel, Ellen N.; Bolitho, Karen M.; Grafton, Karryn; Templeton, Matthew D.; Karunairetnam, Sakuntala; Gleave, Andrew P.; Laing, William A.

doi:10.1186/1746-4811-1-13

Cited by 1,434 publications

(716 citation statements)

References 29 publications

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“…The parent plant transformation vector was pGreenII, providing hygromycin resistance in plants and kanamycin resistance in bacteria [16] (see Supplemental Figure 2 for plasmid sequence)…”

Section: Methodsmentioning

confidence: 99%

Proteomic profiling of tandem affinity purified 14‐3‐3 protein complexes inArabidopsis thaliana

et al. 2009

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In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, a 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a “tandem affinity purification” (TAP) tag and expressed in transgenic plants. Purified complexes were analyzed by tandem mass spectrometry. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (1) Ion transport, such as a K+ channel (GORK), a Cl− channel (CLCg), Ca2+ channels belonging to the glutamate receptor family (GLRs 1.2, 2.1, 2.9, 3.4, 3.7); (2) hormone signaling, such as ACC synthase (isoforms ACS-6, 7 and 8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (3) transcription, such as 7 WRKY family transcription factors; (4) metabolism, such as phosphoenol pyruvate (PEP) carboxylase; and (5) lipid signaling, such as phospholipase D (β, and γ). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.

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“…The parent plant transformation vector was pGreenII, providing hygromycin resistance in plants and kanamycin resistance in bacteria [16] (see Supplemental Figure 2 for plasmid sequence)…”

Section: Methodsmentioning

confidence: 99%

Proteomic profiling of tandem affinity purified 14‐3‐3 protein complexes inArabidopsis thaliana

et al. 2009

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“…We attributed it to the efficiency of our vector design. A spliceable intron was included as a spacer in the RNAi vector to increase the frequency of silencing (44,45). Moreover, we used a long gene fragment, which can facilitate efficient gene silencing by minimizing the variability in efficiency attributed to different sites of genes and the length of the short interfering RNA (siRNA) (46).…”

Section: Discussionmentioning

confidence: 99%

Evidence for a Regulatory Role of Diatom Silicon Transporters in Cellular Silicon Responses

Shrestha

Hildebrand

2015

Eukaryot Cell

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The utilization of silicon by diatoms has both global and small-scale implications, from oceanic primary productivity to nanotechnological applications of their silica cell walls. The sensing and transport of silicic acid are key aspects of understanding diatom silicon utilization. At low silicic acid concentrations (<30 M), transport mainly occurs through silicic acid transport proteins (SITs), and at higher concentrations it occurs through diffusion. Previous analyses of the SITs were done either in heterologous systems or without a distinction between individual SITs. In the present study, we examined individual SITs in Thalassiosira pseudonana in terms of transcript and protein abundance in response to different silicic acid regimes and examined knockdown lines to evaluate the role of the SITs in transport, silica incorporation, and lipid accumulation resulting from silicon starvation. SIT1 and SIT2 were localized in the plasma membrane, and protein levels were generally inversely correlated with cellular silicon needs, with a distinct response being found when the two SITs were compared. We developed highly effective approaches for RNA interference and antisense knockdowns, the first such approaches developed for a centric diatom. SIT knockdown differentially affected the uptake of silicon and the incorporation of silicic acid and resulted in the induction of lipid accumulation under silicon starvation conditions far earlier than in the wild-type cells, suggesting that the cells were artificially sensing silicon limitation. The data suggest that the transport role of the SITs is relatively minor under conditions with sufficient silicic acid. Their primary role is to sense silicic acid levels to evaluate whether the cell can proceed with its cell wall formation and division processes. Silicon, a member of group 14 of the periodic table containing carbon, germanium, and lead, is one of the most abundant elements (27.7%) of the Earth's crust second only to oxygen (46.6%) (1). Dissolved silica in the form of orthosilicic acid, Si(OH) 4 , derived from the weathering of the Earth's crust (2), is an essential nutrient for a few groups of organisms of marine and freshwater environments, such as diatoms, radiolaria, silicoflagellates, and sponges (3). Diatoms require a large quantity of silicon, which is biomineralized to form their unique siliceous cell wall (frustule) in various shapes and morphologies with highly intricate and ornate architectures. Diatoms contribute about 40% of the total ocean organic carbon production, and they dominate ocean Si production (4, 5). Though silicon does not appear to be directly involved in cellular metabolic processes, diatoms require silicon for progression of cell cycle events, such as cell division and DNA replication (5-8). Silicon requirements are tightly coupled to cell cycle progression, a unique feature of diatoms (7). Deprivation of Si arrests the cell cycle at G 1 , G 2 , or M phase, depending upon the diatom species. In Thalassiosira pseudonana, upon Si depletion the ...

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“…Transient dual-luciferase assays in N. benthamiana were performed as described previously (62,69). LUC and REN were assayed by dual-luciferase assay (Promega) as previously described (70,71).…”

Section: Methodsmentioning

confidence: 99%

Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant

Zhong

Wang

Xiao

et al. 2017

J Virol

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Phosphorylation of the ␤C1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-␤C1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana. However, how phosphorylation of TYLCCNB-␤C1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-␤C1 that are phosphorylated by SnRK1. The effects of TYLCCNB-␤C1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-␤C1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-␤C1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-␤C1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-␤C1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor. IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, ␤C1 (TYLCCNB-␤C1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-␤C1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N. benthamiana ASYMMETRIC LEAVES 1. To our knowledge, this is the first report establishing an inhibitory effect of phosphorylation of TYLCCNB-␤C1 on its pathogenic functions as both a VSR and a symptom determinant and to provide a mechanistic explanation of how SNF1-related protein kinase 1 acts as a host defense factor. These findings expand the scope of phosphorylation-mediated defense mechanisms and contribute to further understanding of plant defense mechanisms against geminiviruses.KEYWORDS TYLCCNB-␤C1, Nicotiana benthamiana, SnRK1, geminivirus, host defense factor, posttranscriptional gene silencing, protein phosphorylation, transcriptional gene silencing

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Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Cited by 1,434 publications

References 29 publications

Proteomic profiling of tandem affinity purified 14‐3‐3 protein complexes inArabidopsis thaliana

Proteomic profiling of tandem affinity purified 14‐3‐3 protein complexes inArabidopsis thaliana

Evidence for a Regulatory Role of Diatom Silicon Transporters in Cellular Silicon Responses

Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant

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