RESUMO
The biogenesis and functionality of pattern recognition receptors (PRRs) are critical for robust plant immune responses. Here, we present methods to determine the N-glycosylation state and ligand-induced activity of these receptors for comparative quantitative analysis. These techniques can be used to identify mutants and chemical inhibitors affecting PRR biogenesis and functionality. When combined, these techniques can provide useful insights on biological processes necessary to synthesize a properly membrane-localized and ligand-responsive PRR.
Assuntos
Arabidopsis/metabolismo , Receptores de Reconhecimento de Padrão/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Western Blotting , Glicosilação , Ligantes , Mutação , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
The functions of the plant body rely on interactions among distinct and nonequivalent cell types. The comparison of transcriptomes from different cell types should expose the transcriptional networks that underlie cellular attributes and contributions. Using laser microdissection and microarray profiling, we have produced a cell type transcriptome atlas that includes 40 cell types from rice (Oryza sativa) shoot, root and germinating seed at several developmental stages, providing patterns of cell specificity for individual genes and gene classes. Cell type comparisons uncovered previously unrecognized properties, including cell-specific promoter motifs and coexpressed cognate binding factor candidates, interaction partner candidates and hormone response centers. We inferred developmental regulatory hierarchies of gene expression in specific cell types by comparison of several stages within root, shoot and embryo.
Assuntos
Padronização Corporal/genética , Perfilação da Expressão Gênica , Oryza/citologia , Oryza/genética , Atlas como Assunto , Sequência de Bases , Análise por Conglomerados , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas/fisiologia , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos/genética , Oryza/embriologia , Oryza/fisiologia , Componentes Aéreos da Planta/citologia , Componentes Aéreos da Planta/embriologia , Componentes Aéreos da Planta/genética , Componentes Aéreos da Planta/crescimento & desenvolvimento , Sementes/citologia , Sementes/genéticaRESUMO
VH1/BRL2 is a receptor-like kinase of the BRI1 family with a role in vascular development. In developing Arabidopsis leaves it is expressed first in ground cells and then becomes restricted to provascular and procambial cells as venation forms. We isolated proteins interacting with the activated (phosphorylated) cytoplasmic domain of VH1/BRL2, and found that most belong to three processes: proteasome activity, vesicle traffic and intracellular signal transduction. Two adaptor proteins are included that we named VIT [VH1-interacting tetratricopeptide repeat (TPR)-containing protein] and VIK (VH1-interacting kinase), which are co-expressed in the same cells as VH1/BRL2 at two distinct time points in vein differentiation. Mutation of either adaptor or of VH1 results in vein pattern defects and in alterations in response to auxin and brassinosteroids. We propose that these two adaptors facilitate the diversification and amplification of a ligand signal perceived by VH1/BRL2 in multiple downstream pathways affecting venation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Folhas de Planta/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Mutagênese Insercional , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas Quinases/genética , Transdução de Sinais , Transcrição GênicaRESUMO
Laser capture microdissection (LCM) is a technique by which individual cells can be harvested from tissue sections while they are viewed under the microscope, by tacking selected cells to an adhesive film with a laser beam. Harvested cells can provide DNA, RNA, and protein for the profiling of genomic characteristics, gene expression, and protein spectra from individual cell types. We have optimized LCM for a variety of plant tissues and species, permitting the harvesting of cells from paraffin sections that maintain histological detail. We show that RNA can be extracted from LCM-harvested plant cells in amount and quality that are sufficient for the comparison of RNAs among individual cell types. The linear amplification of LCM-captured RNA should permit the expression profiling of plant cell types.
