Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families

Sikorskaitė, Sidona; Rajamäki, Minna-Liisa; Baniulis, Danas; Stanys, V.; Valkonen, Jari P. T.

doi:10.1186/1746-4811-9-31

Cited by 77 publications

(75 citation statements)

References 44 publications

(62 reference statements)

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“…Western blot detection of chloroplast-derived proteins in the nucleus is extremely challenging because it is difficult to obtain nuclear extracts completely free of chloroplast contamination (Sikorskaite et al, 2013). Therefore, to distinguish between these two modes of accumulation, we designed an experimental strategy that uses a nuclear export sequence (NES) from human immunodeficiency virus (HIV) Rev protein fused to NRIP1-Cerulean (Figure 6A).…”

Section: Resultsmentioning

confidence: 99%

Chloroplast Stromules Function during Innate Immunity

et al. 2015

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Summary Inter-organellar communication is vital for successful innate immune responses that confer defense against pathogens. However, little is known about how chloroplasts, which are a major production site of pro-defense molecules, communicate and coordinate with other organelles during defense. Here we show that chloroplasts send out dynamic tubular extensions called stromules during innate immunity or exogenous application of the pro-defense signals, hydrogen peroxide (H2O2) and salicylic acid (SA). Interestingly, numerous stromules surround nuclei during defense response and these connections correlate with an accumulation of chloroplast-localized NRIP1 defense protein and H2O2 in the nucleus. Furthermore, silencing and knockout of CHloroplast Unusual Positioning 1 (CHUP1) that encodes a chloroplast outer envelope protein constitutively induces stromules in the absence of pathogen infection and enhances programmed cell death (PCD). These results support a model in which stromules aid in the amplification and/or transport of pro-defense signals into the nucleus and other subcellular compartments during immunity.

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Section: Resultsmentioning

confidence: 99%

Chloroplast Stromules Function during Innate Immunity

et al. 2015

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“…Chromatin was extracted from Nicotiana benthamiana plants using a method adapted from Huang et al (2009). As this protocol used animal cells, nuclei were first extracted using the method adapted for N. tabacum from Sikorskaite et al (2013), chromatin was then isolated from these nuclei, using the Huang et al (2009) method.…”

Section: Agrobacterium Mediated Transient Assays Protein Extractionmentioning

confidence: 99%

Functional analysis of a Wheat Homeodomain protein, TaR1, reveals that host chromatin remodelling influences the dynamics of the switch to necrotrophic growth in the phytopathogenic fungus Zymoseptoria tritici

et al. 2015

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Summary A distinguishing feature of Septoria leaf blotch disease in wheat is the long symptomless growth of the fungus amongst host cells followed by a rapid transition to necrotrophic growth resulting in disease lesions. Global reprogramming of host transcription marks this switch to necrotrophic growth. However no information exists on the components that bring about host transcriptional reprogramming. Gene‐silencing, confocal‐imaging and protein–protein interaction assays where employed to identify a plant homeodomain (PHD) protein, TaR1 in wheat that plays a critical role during the transition from symptomless to necrotrophic growth of Septoria. TaR1‐silenced wheat show earlier symptom development upon Septoria infection but reduced fungal sporulation indicating that TaR1 is key for prolonging the symptomless phase and facilitating Septoria asexual reproduction. TaR1 is localized to the nucleus and binds to wheat Histone 3. Trimethylation of Histone 3 at lysine 4 (H3K4) and lysine 36 (H3K36) are found on open chromatin with actively transcribed genes, whereas methylation of H3K27 and H3K9 are associated with repressed loci. TaR1 specifically recognizes dimethylated and trimethylated H3K4 peptides suggesting that it regulates transcriptional activation at open chromatin. We conclude that TaR1 is an important component for the pathogen life cycle in wheat that promotes successful colonization by Septoria.

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“…However, continuing technical improvements have largely overcome these difficulties in various explants, including the embryo [Masuda et al, 1991;Yamaguchi et al, 1992;Busk and Pages, 1997], endosperm [Ferreira et al, 2006;Li et al, 2008], seed [Riggs et al, 1989], seed coat [Renouard et al, 2012], leaf [e.g. Cushman, 1995;Zhang et al, 1995;Abdalla et al, 2009Abdalla et al, , 2010Sikorskaite et al, 2013], and root meristem [Silva et al, 2010], and in in vitro cultured cells [Willmitzer and Wagner, 1981]. Currently used extraction protocols directed at nuclear proteins use mechanical homogenization, filtration to remove large detritus, pelleting, suspension in a non-ionic detergent, and finally separation by density gradient centrifugation [for review see Narula et al, 2013].…”

mentioning

confidence: 99%

Proteomic Analysis of Barley Cell Nuclei Purified by Flow Sorting

Petrovská

Jeřábková

Chamrád

et al. 2014

Cytogenet Genome Res

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Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.

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Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families

Cited by 77 publications

References 44 publications

Chloroplast Stromules Function during Innate Immunity

Chloroplast Stromules Function during Innate Immunity

Functional analysis of a Wheat Homeodomain protein, TaR1, reveals that host chromatin remodelling influences the dynamics of the switch to necrotrophic growth in the phytopathogenic fungus Zymoseptoria tritici

Proteomic Analysis of Barley Cell Nuclei Purified by Flow Sorting

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