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1.
Plant Physiol ; 183(1): 331-344, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32165446

RESUMO

A wide variety of intrinsic and extrinsic cues lead to cell death with unclear mechanisms. The infertility of some death mutants often hurdles the classical suppressor screens for death regulators. We have developed a transient RNA interference (RNAi)-based screen using a virus-induced gene silencing approach to understand diverse cell death pathways in Arabidopsis (Arabidopsis thaliana). One death pathway is due to the depletion of a MAP kinase (MAPK) cascade, consisting of MAPK kinase kinase 1 (MEKK1), MKK1/2, and MPK4, which depends on a nucleotide-binding site Leu-rich repeat (NLR) protein SUMM2. Silencing of MEKK1 by virus-induced gene silencing resembles the mekk1 mutant with autoimmunity and defense activation. The RNAi-based screen toward Arabidopsis T-DNA insertion lines identified SUMM2, MEKK2, and Calmodulin-binding receptor-like cytoplasmic kinase 3 (CRCK3) to be vital regulators of RNAi MEKK1-induced cell death, consistent with the reports of their requirement in the mekk1-mkk1/2-mpk4 death pathway. Similar with MEKK2, overexpression of CRCK3 caused dosage- and SUMM2-dependent cell death, and the transcripts of CRCK3 were up-regulated in mekk1, mkk1/2, and mpk4 MEKK2-induced cell death depends on CRCK3. Interestingly, CRCK3-induced cell death also depends on MEKK2, consistent with the biochemical data that MEKK2 complexes with CRCK3. Furthermore, the kinase activity of CRCK3 is essential, whereas the kinase activity of MEKK2 is dispensable, for triggering cell death. Our studies suggest that MEKK2 and CRCK3 exert concerted functions in the control of NLR SUMM2 activation and MEKK2 may play a structural role, rather than function as a kinase, in regulating CRCK3 protein stability.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 1/metabolismo , MAP Quinase Quinase Quinase 2/genética , MAP Quinase Quinase Quinase 2/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estabilidade Proteica , Interferência de RNA/fisiologia
2.
Methods Mol Biol ; 1287: 235-41, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25740369

RESUMO

Several VIGS protocols have been established for high-throughput functional genomic screens as it bypasses the time-consuming and laborious process of generation of transgenic plants. The silencing efficiency in this approach is largely hindered by a technically demanding step in which the first pair of newly emerged true leaves at the 2-week-old stage are infiltrated with a needleless syringe. To further optimize VIGS efficiency and achieve rapid inoculation for a large-scale functional genomic study, here we describe a protocol of an efficient VIGS assay in Arabidopsis using Agrobacterium-mediated rubbing infection. The Agrobacterium inoculation is performed by simply rubbing the leaves with Filter Agent Celite(®) 545. The highly efficient and uniform silencing effect was indicated by the development of a visibly albino phenotype due to silencing of the Cloroplastos alterados 1 (CLA1) gene in the newly emerged leaves. In addition, the albino phenotype could be observed in stems and flowers, indicating its potential application for gene functional studies in the late vegetative development and flowering stages.


Assuntos
Agrobacterium/virologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Folhas de Planta/fisiologia , Vírus de Plantas/genética , Agrobacterium/genética , Agrobacterium/fisiologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Vetores Genéticos/genética , Fenótipo , Folhas de Planta/genética , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas/genética
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