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1.
Genes Genomics ; 43(11): 1269-1276, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34449065

RESUMO

BACKGROUND: Botrytis-induced Kinase 1 (BIK1) is a receptor-like cytoplasmic kinase (RLCK) involved in the defense, growth, and development of higher plants. It interacts with various receptor-like kinases (RLKs) such as Brassinosteroid Insensitive 1 (BRI1), Flagellin Sensitive 2 (FLS2), and Perception of the Arabidopsis Danger Signal Peptide 1 (PEPR1), but little is known about signaling downstream of BIK1. OBJECTIVE: In this study, we aimed to identify Arabidopsis thaliana BIK1 (AtBIK1) and Brassica rapa BIK1 (BrBIK1) interacting proteins, which is downstream signaling components in Arabidopsis. In addition, the effect of BIK1 phosphorylation on their interaction were examined. METHODS: For yeast two hybrid (Y2H) screening, a B. rapa cDNA activation domain (AD) library and an A. thaliana cDNA library were used. Reverse reaction (LR) recombinations of appropriate open reading frames (AtBIK1, BrBIK1, AtRGP2, AtPATL2, AtPP7) in either pDONR207 or pDONR/zeo were performed with the split-YFP destination vectors pDEST-GWVYNE and pDEST-GWVYCE to generate N- or C-terminal fusions with the N- and C-terminal yellow fluorescent protein (YFP) moieties, respectively. Recombined vectors were transformed into Agrobacterium strain GV3101. The described GST-AtBIK1, Flag-AtBIK1, and Flag-BrBIK1 constructs were used as templates for site-directed mutagenesis with a QuikChange XL Site-Directed Mutagenesis Kit (Stratagene). RESULTS: In results, A. thaliana BIK1 (AtBIK1) displays strong autophosphorylation kinase activity on tyrosine and threonine residues, whereas B. rapa BIK1 (BrBIK1) does not exhibit autophosphorylation kinase activity in vitro. Herein, we demonstrated that four proteins (RGP2, PATL2, PP7, and SULTR4.1) interact with BrBIK1 but not AtBIK1 in a Y2H system. To confirm interactions between BIK1 and protein candidates in Nicotiana benthamiana, BiFC analysis was performed and it was found that only BrBIK1 bound the three proteins tested. Three phosphosites, T90, T362, and T368, based on amino acid sequence alignment between AtBIK1 and BrBIK1, and performed site-directed mutagenesis (SDM) on AtBIK1 and BrBIK. S90T, P362T, and A369T mutations in BrBIK1 restored autophosphorylation kinase activity on threonine residues comparable to AtBIK1. However, T90A, T362P, and T368A mutations in AtBIK1 did not alter autophosphorylation kinase activity on threonine residues compared with wild-type AtBIK1. BiFC results showed that BIK1 mutations restored kinase activity led to the loss of the binding activity to RGP2, PATL2, or PP7 proteins. CONCLUSION: Phospho-BIK1 might be involved in plant innate immunity, while non-phospho BIK1 may regulate plant growth and development through interactions with RGP2, PATL2, and PP7.

2.
Plants (Basel) ; 10(7)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34371589

RESUMO

Red radish (Raphanus sativus L.) cultivars are a rich source of health-promoting anthocyanins and are considered a potential source of natural colorants used in the cosmetic industry. However, the development of red radish cultivars via conventional breeding is very difficult, given the unusual inheritance of the anthocyanin accumulation trait in radishes. Therefore, molecular markers linked with radish color are needed to facilitate radish breeding. Here, we characterized the RsTT8 gene isolated from four radish genotypes with different skin and flesh colors. Sequence analysis of RsTT8 revealed a large number of polymorphisms, including insertion/deletions (InDels), single nucleotide polymorphisms (SNPs), and simple sequence repeats (SSRs), between the red-fleshed and white-fleshed radish cultivars. To develop molecular markers on the basis of these polymorphisms for discriminating between radish genotypes with different colored flesh tissues, we designed four primer sets specific to the RsTT8 promoter, InDel, SSR, and WD40/acidic domain (WD/AD), and tested these primers on a diverse collection of radish lines. Except for the SSR-specific primer set, all primer sets successfully discriminated between red-fleshed and white-fleshed radish lines. Thus, we developed three molecular markers that can be efficiently used for breeding red-fleshed radish cultivars.

3.
Nat Commun ; 12(1): 4194, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234144

RESUMO

Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARK AND YELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Proteínas Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brassinosteroides/metabolismo , Clorofila/biossíntese , Cloroplastos/metabolismo , Estiolamento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/isolamento & purificação , Luz , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/efeitos da radiação , Mutação , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Plântula/crescimento & desenvolvimento , Transdução de Sinais/fisiologia
4.
J Exp Bot ; 72(15): 5494-5507, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34021330

RESUMO

Open Stomata 1 (OST1)/SnRK2.6 is a critical component connecting abscisic acid (ABA) receptor complexes and downstream components, including anion channels and transcription factors. Because OST1 is a serine/threonine kinase, several autophosphorylation sites have been identified, and S175 is known to be critical for its kinase activity. We previously reported that BAK1 interacts with and phosphorylates OST1 to regulate ABA signaling. Here, we mapped additional phosphosites of OST1 generated by autophosphorylation and BAK1-mediated transphosphorylation in Arabidopsis. Many phosphosites serve as both auto- and transphosphorylation sites, especially those clustered in the activation loop region. Phospho-mimetic transgenic plants containing quadruple changes in Y163, S164, S166, and S167 rescued ost1 mutant phenotypes, activating ABA signaling outputs. Moreover, we found that OST1 is an active tyrosine kinase, autophosphorylating the Y182 site. ABA induced tyrosine phosphorylation of Y182 in OST1; this event is catalytically important for OST1 activity in plants. ABA-Insensitive 1 (ABI1) and its homologs ABI2 and HAB1, PP2C serine/threonine phosphatases that are known to dephosphorylate OST1 at S175, function as tyrosine phosphatases acting on the phosphorylated Y182 site. Our results indicate that phosphorylation cycles between OST1 and ABI1, which have dual specificity for tyrosine and serine/threonine, coordinately control ABA signaling in Arabidopsis.


Assuntos
Ácido Abscísico , Proteínas de Arabidopsis , Proteínas Quinases , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases , Serina , Treonina
5.
Front Plant Sci ; 11: 559365, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33193484

RESUMO

The expression of multiple proteins and high-throughput vector assembly system are highly relevant in the field of plant genetic engineering and synthetic biology. Deployment of the self-cleaving 2A peptide that mediates polycistronic gene expression has been an effective strategy for multigene expression, as it minimizes issues in coordinated transgene regulation and trait staking in plants. However, efficient vector assembly systems optimized for 2A peptide-mediated polycistronic expression are currently unavailable. Furthermore, it is unclear whether protein expression levels are influenced by the transgene position in the polycistronic expression cassette. In this article, we present Golden Gate cloning-compatible modular systems allowing rapid and flexible construction of polycistronic expression vectors applicable for plants. The genetic modules comprised 2A peptides (T2A and P2A)-linked tricistron expression cassette and its acceptor backbones, named pGO-DV1 and pGO-DV2. While both acceptor backbones were binary T-DNA vectors, pGO-DV2 was specially designed to function as a DNA replicon enhancing gene expression levels. Using the Golden Gate cloning, a set of six tricistronic vectors was constructed, whereby three transgenes encoding fluorescent proteins (mCherry, eYFP, and eGFP) were combinatorially placed along the expression cassette in each of the binary vectors. Transient expression of the construct in tobacco leaves revealed that the expression levels of three fluorescent proteins were comparable each other regardless of the gene positions in the tricistronic expression cassette. pGO-DV2-based constructs were able to increase protein expression level by up to 71%, as compared to pGO-DV1-based constructs.

6.
Genes Genomics ; 42(8): 957-969, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32648234

RESUMO

BACKGROUND: Brassinosteroids (BRs) are a class of phytohormones with important roles in regulating physiological and developmental processes. Small RNAs, including small interfering RNAs and microRNAs (miRNAs), are non-protein coding RNAs that regulate gene expression at the transcriptional and post-transcriptional levels. However, the roles of small RNAs in BR response have not been studied well. OBJECTIVE: In this study, we aimed to identify BR-responsive small RNA clusters and miRNAs in Arabidopsis. In addition, the effect of BR-responsive small RNAs on their transcripts and target genes were examined. METHODS: Small RNA libraries were constructed from control and epibrassinolide-treated seedlings expressing wild-type BRI1-Flag protein under its native promoter in the bri1-5 mutant. After sequencing the small RNA libraries, differentially expressed small RNA clusters were identified by examining the expression levels of small RNAs in 100-nt bins of the Arabidopsis genome. To identify the BR-responsive miRNAs, the expression levels of all the annotated mature miRNAs, registered in miRBase, were analyzed. Previously published RNA-seq data were utilized to monitor the BR-responsive expression patterns of differentially expressed small RNA clusters and miRNA target genes. RESULTS: In results, 38 BR-responsive small RNA clusters, including 30 down-regulated and eight up-regulated clusters, were identified. These differentially expressed small RNA clusters were from miRNA loci, transposons, protein-coding genes, pseudogenes and others. Of these, a transgene, BRI1, accumulates small RNAs, which are not found in the wild type. Small RNAs in this transgene are up-regulated by BRs while BRI1 mRNA is down-regulated by BRs. By analyzing the expression patterns of mature miRNAs, we have identified BR-repressed miR398a-5p and BR-induced miR156g. Although miR398a-5p is down-regulated by BRs, its predicted targets were not responsive to BRs. However, SPL3, a target of BR-inducible miR156g, is down-regulated by BRs. CONCLUSION: BR-responsive small RNAs and miRNAs identified in this study will provide an insight into the role of small RNAs in BR responses in plants. Especially, we suggest that miR156g/SPL3 module might play a role in BR-mediated growth and development in Arabidopsis.


Assuntos
Arabidopsis/genética , Brassinosteroides/metabolismo , MicroRNAs/genética , RNA Interferente Pequeno/genética , Bases de Dados de Ácidos Nucleicos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , MicroRNAs/isolamento & purificação , Reguladores de Crescimento de Plantas/metabolismo , RNA Interferente Pequeno/isolamento & purificação , Plântula/genética
7.
Int J Mol Sci ; 21(8)2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32326209

RESUMO

Reddish purple Chinese cabbage (RPCC) is a popular variety of Brassica rapa (AA = 20). It is rich in anthocyanins, which have many health benefits. We detected novel anthocyanins including cyanidin 3-(feruloyl) diglucoside-5-(malonoyl) glucoside and pelargonidin 3-(caffeoyl) diglucoside-5-(malonoyl) glucoside in RPCC. Analyses of transcriptome data revealed 32,395 genes including 3345 differentially expressed genes (DEGs) between 3-week-old RPCC and green Chinese cabbage (GCC). The DEGs included 218 transcription factor (TF) genes and some functionally uncharacterized genes. Sixty DEGs identified from the transcriptome data were analyzed in 3-, 6- and 9-week old seedlings by RT-qPCR, and 35 of them had higher transcript levels in RPCC than in GCC. We detected cis-regulatory motifs of MYB, bHLH, WRKY, bZIP and AP2/ERF TFs in anthocyanin biosynthetic gene promoters. A network analysis revealed that MYB75, MYB90, and MYBL2 strongly interact with anthocyanin biosynthetic genes. Our results show that the late biosynthesis genes BrDFR, BrLDOX, BrUF3GT, BrUGT75c1-1, Br5MAT, BrAT-1, BrAT-2, BrTT19-1, and BrTT19-2 and the regulatory MYB genes BrMYB90, BrMYB75, and BrMYBL2-1 are highly expressed in RPCC, indicative of their important roles in anthocyanin biosynthesis, modification, and accumulation. Finally, we propose a model anthocyanin biosynthesis pathway that includes the unique anthocyanin pigments and genes specific to RPCC.


Assuntos
Brassica/genética , Perfilação da Expressão Gênica , Pigmentação/genética , Transcriptoma , Antocianinas/biossíntese , Antocianinas/genética , Brassica/química , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Folhas de Planta/química , Regiões Promotoras Genéticas , Fatores de Transcrição/genética
8.
Int J Mol Sci ; 21(5)2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32143305

RESUMO

Steroid hormones are important signaling molecules in plants and animals. The plant steroid hormone brassinosteroids were first isolated and characterized in the 1970s and have been studied since then for their functions in plant growth. Treatment of plants or plant cells with brassinosteroids revealed they play important roles during diverse developmental processes, including control of cell expansion, cell division, and vascular differentiation. Molecular genetic studies, primarily in Arabidopsis thaliana, but increasingly in many other plants, have identified many genes involved in brassinosteroid biosynthesis and responses. Here we review the roles of brassinosteroids in cell expansion, cell division, and vascular differentiation, comparing the early physiological studies with more recent results of the analysis of mutants in brassinosteroid biosynthesis and signaling genes. A few representative examples of other molecular pathways that share developmental roles with brassinosteroids are described, including pathways that share functional overlap or response components with the brassinosteroid pathway. We conclude by briefly discussing the origin and conservation of brassinosteroid signaling.


Assuntos
Arabidopsis/genética , Botânica/história , Brassinosteroides/metabolismo , Divisão Celular , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/metabolismo , Arabidopsis/citologia , Proteínas de Arabidopsis/metabolismo , Bioensaio , Técnicas de Cultura de Células , Ciclo Celular , Citosol/metabolismo , História do Século XX , História do Século XXI , Ligantes , Biologia Molecular , Mutação , Fenótipo , Fosforilação , Células Vegetais/metabolismo , Desenvolvimento Vegetal , Transdução de Sinais
9.
Genes Genomics ; 42(3): 347-354, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31902106

RESUMO

BACKGROUND: Brassinosteroids (BR) are essential growth hormone in plants. Various components involved in signal transduction pathway have been identified as targets of 14-3-3 phospho-binding proteins. Previously, we showed that 14-3-3 proteins directly interact with the Brassinosteroid Insensitive 1 (BRI1), the BR receptor kinase, and are also subject to phosphorylation in a BR-dependent manner. OBJECTIVE: In this study, we aimed to examine a potential interplay between 14-3-3 proteins and BRI1 in plant growth. METHODS: Morphological phenotypes of a T-DNA insertion mutant line, 14-3-3ψφε, defective in three 14-3-3 isoforms, psi, phi and epsilon, were characterized and compared with bri1-5 and two transgenic lines for BRI1, BRI1-Flag and BRI1-Flag (14-3-3ψφε). We also generated complementation lines carrying each of the three 14-3-3 genes and determined their differences in rosette growth. RESULTS: No significant differences between the wild-type and 14-3-3ψφε seedlings were observed regardless of BR applications. However, BRI1-Flag (14-3-3ψφε) showed a significantly reduced cold tolerance and BR sensitivity in hypocotyl and root development when compared to BRI1-Flag. In addition, narrower leaf shape and smaller rosette size were observed in BRI1-Flag (14-3-3ψφε), while the mutant phenotypes were partially restored in the complementation lines, two of which with 14-3-3φ and 14-3-3ε showed the rosette growth comparable to BRI1-Flag. CONCLUSION: Taken together, our results suggested that 14-3-3 proteins might positively regulate BRI1 activity and showed that 14-3-3 isoforms have different functional impacts in BR signaling.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Folhas de Planta/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Proteínas 14-3-3/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brassinosteroides/biossíntese , Brassinosteroides/farmacologia , Hipocótilo/efeitos dos fármacos , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/efeitos da radiação , Fenótipo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinases/genética , Transdução de Sinais/genética , Triazóis/farmacologia
10.
Genes Genomics ; 41(5): 499-506, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30830683

RESUMO

Innate immune signaling of plants is initiated by pattern recognition receptors (PRRs) at the plasma membrane. Upon pathogen attack, PRRs recognize pathogen-associated molecular patterns (PAMPs) via ectodomain and lead to signaling cascade via cytoplasmic kinase domain. PAMP-triggered immunity (PTI) activates basal defense responses sufficient to confer broad-spectrum disease resistance by inhibiting pathogen entry and growth. On the other hand, one of the major virulence factors in plant-pathogenic bacteria is type III secretion system, which can deliver effector proteins into the host cell and modulate host cellular processes. Most type III effectors are implicated in PTI suppression, and PRRs have been identified as targets of multiple type III effectors. Mutants defective in T3SS lack pathogenicity in many bacterial species, revealing that T3SS-mediated PTI suppression is critical for host colonization and subsequent disease development. This review summarizes molecular basis of bacterial pathogen perception by plant PRRs and also interaction between PRRs and type III effectors during early stages of plant-pathogen interaction.


Assuntos
Imunidade Vegetal/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/fisiologia , Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Imunidade Inata/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Transdução de Sinais , Sistemas de Secreção Tipo III/metabolismo , Fatores de Virulência
11.
PeerJ ; 6: e6074, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30581670

RESUMO

Tyrosine phosphorylation has emerged as an important regulator of plasma membrane-localized immune receptors activity. Here, we investigate the role of tyrosine phosphorylation in the regulation of rice XANTHOMONAS RESISTANCE 21 (XA21)-mediated immunity. We demonstrate that the juxtamembrane and kinase domain of Escherichia coli-expressed XA21 (XA21JK) autophosphorylates on tyrosine residues. Directed mutagenesis of four out of the nine tyrosine residues in XA21JK reduced autophosphorylation. These sites include Tyr698 in the juxtamembrane domain, and Tyr786, Tyr907, and Tyr909 in the kinase domain. Rice plants expressing XA21-GFP fusion proteins or proteins with these tyrosine residues individually mutated to phenylalanine (XA21YF-GFP), which prevents phosphorylation at these sites, maintain resistance to Xanthomonas oryzae pv. oryzae. In contrast, plants expressing phosphomimetic XA21 variants with tyrosine mutated to aspartate (XA21YD-GFP) were susceptible. In vitro purified XA21JKY698F, XA21JKY907F, and XA21JKY909F variants are catalytically active, whereas activity was not detected in XA21JKY768F and the four XA21JKYD variants. We previously demonstrated that interaction of XA21 with the co-receptor OsSERK2 is critical for biological function. Four of the XA21JKYF variants maintain interaction with OsSERK2 as well as the XA21 binding (XB) proteins XB3 and XB15 in yeast, suggesting that these four tyrosine residues are not required for their interaction. Taken together, these results suggest that XA21 is capable of tyrosine autophosphorylation, but the identified tyrosine residues are not required for activation of XA21-mediated immunity or interaction with predicted XA21 signaling proteins.

12.
Physiol Plant ; 163(4): 450-458, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29315590

RESUMO

Plants must constantly adjust their growth and defense responses to deal with the wide variety of stresses they encounter in their environment. Among phytohormones, brassinosteroids (BRs) are an important group of plant steroid hormones involved in numerous aspects of the plant lifecycle including growth, development and responses to various stresses including insect attacks. Here, we show that BRs regulate glucosinolate (GS) biosynthesis and function in insect herbivory. Preference tests and larval feeding experiments using the generalist herbivore, diamondback moth (Plutella xylostella), revealed that the larvae prefer to feed on Arabidopsis thaliana brassinosteroid insensitive 1 (bri1-5) plants over wild-type Ws-2 or BRI1-Flag (bri1-5 background) transgenic plants, which results in an increase in larval weight. Analysis of GS contents showed that 3-(methylsulfinyl) propyl GS (C3) levels were higher in bri1-5 than in Ws2 and BRI1-Flag transgenic plants, whereas sinigrin (2-propenylglucosinolate), glucoerucin (4-methylthiobutylglucosinolate) and glucobrassicin (indol-3-ylmethylglucosinolate) levels were lower in this mutant. We investigated the effect of brassinolide (BL) on GS biosynthesis in Arabidopsis and radish (Raphanus sativus L.) by monitoring the expression levels of GS biosynthetic genes, including MAM1, MAM3, BCAT4 and AOP2, which increased in a BL-dependent manner. These results suggest that BRs regulate GS profiles in higher plants, which function in defense responses against insects.


Assuntos
Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Glucosinolatos/biossíntese , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosinolatos/genética , Glucosinolatos/metabolismo , Indóis/metabolismo , Mutação , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Raphanus/genética , Raphanus/metabolismo , Transaminases/genética , Transaminases/metabolismo
13.
Molecules ; 23(1)2018 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-29361797

RESUMO

Protein post-translational modification by phosphorylation is essential for the activity and stability of proteins in higher plants and underlies their responses to diverse stimuli. There are more than 300 leucine-rich repeat receptor-like kinases (LRR-RLKs), a major group of receptor-like kinases (RLKs) that plays an important role in growth, development, and biotic stress responses in higher plants. To analyze auto- and transphosphorylation patterns and kinase activities in vitro, 43 full-length complementary DNA (cDNA) sequences were cloned from genes encoding LRR-RLKs. Autophosphorylation activity was found in the cytoplasmic domains (CDs) of 18 LRR-RLKs; 13 of these LRR-RLKs with autophosphorylation activity showed transphosphorylation in Escherichiacoli. BRI1-Associated Receptor Kinase (BAK1), which is critically involved in the brassinosteroid and plant innate immunity signal transduction pathways, showed strong auto- and transphosphorylation with multi-specific kinase activity within 2 h of induction of Brassica oleraceae BAK1-CD (BoBAK1-CD) in E. coli; moreover, the carboxy-terminus of LRR-RLKs regulated phosphorylation and kinase activity in Arabidopsis thaliana and vegetative crops.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Brassica/enzimologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Brassica/genética , Biologia Computacional/métodos , Mutação , Fosforilação , Filogenia , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
14.
Front Plant Sci ; 8: 1273, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28824659

RESUMO

The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1) functions as a co-receptor with several receptor kinases including the brassinosteroid (BR) receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR), respectively, which are involved in immunity. BAK1 is a dual specificity protein kinase that can autophosphorylate on serine, threonine and tyrosine residues. It was previously reported that phosphorylation of Tyr-610 in the carboxy-terminal domain of BAK1 is required for its function in BR signaling and immunity. However, the functional role of Tyr-610 in vivo has recently come under scrutiny. Therefore, we have generated new BAK1 (Y610F) transgenic plants for functional studies. We first produced transgenic Arabidopsis lines expressing BAK1 (Y610F)-Flag in the homozygous bak1-4 bkk1-1 double null background. In a complementary approach, we expressed untagged BAK1 and BAK1 (Y610F) in the bak1-4 null mutant. Neither BAK1 (Y610F) transgenic line had any obvious growth phenotype when compared to wild-type BAK1 expressed in the same background. In addition, the BAK1 (Y610F)-Flag plants responded similarly to plants expressing BAK1-Flag in terms of brassinolide (BL) inhibition of root elongation, and there were only minor changes in gene expression between the two transgenic lines as monitored by microarray analysis and quantitative real-time PCR. In terms of plant immunity, there were no significant differences between plants expressing BAK1 (Y610F)-Flag and BAK1-Flag in the growth of the non-pathogenic hrpA- mutant of Pseudomonas syringae pv. tomato DC3000. Furthermore, untagged BAK1 (Y610F) transgenic plants were as responsive as plants expressing BAK1 (in the bak1-4 background) and wild-type Col-0 plants toward treatment with the EF-Tu- and flagellin-derived peptide epitopes elf18- and flg22, respectively, as measured by reactive oxygen species production, mitogen-activated protein kinase activation, and seedling growth inhibition. These new results do not support any involvement of Tyr-610 phosphorylation in either BR or immune signaling.

15.
PLoS One ; 10(11): e0142255, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26588465

RESUMO

Among several receptor-like kinases (RLKs), leucine-rich-repeat receptor-like kinases (LRR-RLKs) are a major group of genes that play crucial roles in growth, development and stress responses in plant systems. Given that they have several functional roles, it is important to investigate their roles in Brassica rapa. In the present study, 303 LRR-RLKs were identified in the genome of B. rapa and comparative phylogenetic analysis of 1213 combined LRR-RLKs of B. rapa, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa helped us to categorize the gene family into 15 subfamilies based on their sequence and structural similarities. The chromosome localizations of 293 genes allowed the prediction of duplicates, and motif conservation and intron/exon patterns showed differences among the B. rapa LRR-RLK (BrLRR-RLK) genes. Additionally, computational function annotation and expression analysis was used to predict their possible functional roles in the plant system. Biochemical results for 11 selected genes showed variations in phosphorylation activity. Interestingly, BrBAK1 showed strong auto-phosphorylation and trans-phosphorylation on its tyrosine and threonine residues compared with AtBAK1 in previous studies. The AtBAK1 receptor kinase is involved in plant growth and development, plant innate immunity, and programmed cell death, and our results suggest that BrBAK1 might also be involved in the same functions. Another interesting result was that BrBAK1, BrBRI1, BrPEPR1 and BrPEPR2 showed activity with both anti-phosphotyrosine and anti-phosphothreonine antibodies, indicating that they might have dual-specificity kinase activity. This study provides comprehensive results for the BrLRR-RLKs, revealing expansion of the gene family through gene duplications, structural similarities and variations among the genes, and potential functional roles according to gene ontology, transcriptome profiling and biochemical analysis.


Assuntos
Proteínas de Arabidopsis/genética , Brassica rapa/genética , Evolução Molecular , Processamento de Proteína Pós-Traducional/genética , Proteínas Serina-Treonina Quinases/genética , Arabidopsis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Família Multigênica , Oryza/genética , Filogenia , Populus/genética
16.
Front Plant Sci ; 6: 562, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26284086

RESUMO

BRI1 becomes highly phosphorylated in vivo upon perception of the ligand, brassinolide, as a result of autophosphorylation and transphosphorylation by its co-receptor kinase, BAK1. Important autophosphorylation sites include those involved in activation of kinase activity and those that are inhibitory, such as Ser-891. The inhibitory sites are autophosphorylated after kinase activation has been achieved and are postulated to contribute to deactivation of the kinase. The function of phosphosites is usually tested by substituting a non-phosphorylatable residue or an acidic residue that can act as a phosphomimetic. What has typically not been examined is substitution of a Thr for a Ser phosphosite (or vice versa) but given that Thr and Ser are not equivalent amino acids this type of substitution may represent a new approach to engineer regulatory phosphorylation. In the present study with BRI1, we substituted Thr at the Ser-891 phosphosite to generate the S891T directed mutant. The recombinant Flag-BRI1 (S891T) cytoplasmic domain protein (the S891T protein) was catalytically active and phosphorylation occurred at the engineered Thr-891 site. However, the S891T recombinant protein autophosphorylated more slowly than the wild-type protein during expression in E. coli. As a result, activation of peptide kinase activity (measured in vitro) was delayed as was transphosphorylation of bacterial proteins in situ. Stable transgenic expression of BRI1 (S891T)-Flag in Arabidopsis bri1-5 plants did not fully rescue the brassinosteroid (BR) phenotype indicating that BR signaling was constrained. Our working model is that restricted signaling in the S891T plants occurs as a result of the reduced rate of activation of the mutant BRI1 kinase by autophosphorylation. These results provide the platform for future studies to critically test this new model in vivo and establish Ser-Thr substitutions at phosphosites as an interesting approach to consider with other protein kinases.

17.
Science ; 343(6178): 1509-12, 2014 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-24625928

RESUMO

Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Pseudomonas syringae/patogenicidade , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas de Arabidopsis/agonistas , Peptídeos/metabolismo , Peptídeos/farmacologia , Fosforilação , Pseudomonas syringae/enzimologia , Receptores de Reconhecimento de Padrão/agonistas , Tirosina/metabolismo
18.
Front Plant Sci ; 5: 16, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24550926

RESUMO

Binding of brassinolide to the brassinosteroid-insenstive 1(BRI1) receptor kinase promotes interaction with its co-receptor, BRI1-associated receptor kinase 1 (BAK1). Juxtaposition of the kinase domains that occurs then allows reciprocal transphosphorylation and activation of both kinases, but details of that process are not entirely clear. In the present study we show that the carboxy (C)-terminal polypeptide of BAK1 may play a role. First, we demonstrate that the C-terminal domain is a strong inhibitor of the transphosphorylation activity of the recombinant BAK1 cytoplasmic domain protein. However, recombinant BAK1 lacking the C-terminal domain is unable to transactivate the peptide kinase activity of BRI1 in vitro. Thus, the C-terminal domain may play both a positive and negative role. Interestingly, a synthetic peptide corresponding to the full C-terminal domain (residues 576-615 of BAK1) interacted with recombinant BRI1 in vitro, and that interaction was enhanced by phosphorylation at the Tyr-610 site. Expression of a BAK1 C-terminal domain truncation (designated BAK1-ΔCT-Flag) in transgenic Arabidopsis plants lacking endogenous bak1 and its functional paralog, bkk1, produced plants that were wild type in appearance but much smaller than plants expressing full-length BAK1-Flag. The reduction in growth may be attributed to a partial inhibition of BR signaling in vivo as reflected in root growth assays but other factors are likely involved as well. Our working model is that in vivo, the inhibitory action of the C-terminal domain of BAK1 is relieved by binding to BRI1. However, that interaction is not essential for BR signaling, but other aspects of cellular signaling are impacted when the C-terminal domain is truncated and result in inhibition of growth. These results increase the molecular understanding of the C-terminal domain of BAK1 as a regulator of kinase activity that may serve as a model for other receptor kinases.

19.
Plant Signal Behav ; 8(12): e27671, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24394563

RESUMO

Several plant CDPKs were recently shown to be dual specificity kinases rather than Ser/Thr kinases as traditionally classified by sequence analysis. In the present study we confirm the autophosphorylation of recombinant soybean His 6-GmCDPKß at the Tyr-24 site using sequence- and modification- specific antibodies. Homology modeling of soybean CDPKß based on recent structures determined for several apicomplexan CDPKs suggested that phosphotyrosine-24 may be inaccessible to phosphatases. However, we report that dephosphorylation of CDPKß by the protein tyrosine phosphatase 1B, PTP1B, was not restricted in the presence of calcium. Thus, despite conformational changes likely associated with calcium binding to the CDPKs, phosphotyrosine sites remain fully accessible to dephosphorylation suggesting the possibility of conformational breathing and flexing.


Assuntos
Cálcio/farmacologia , Fosfotirosina/metabolismo , Proteínas Quinases/metabolismo , Soja/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Proteínas Quinases/química , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Homologia Estrutural de Proteína , Toxoplasma/enzimologia
20.
Front Plant Sci ; 3: 262, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23226150

RESUMO

Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E. coli. We apply this to the study of the cytoplasmic domain of the plant receptor kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which functions in brassinosteroid (BR) signaling. Recombinant BRI1 is catalytically active and both autophosphorylates and transphosphorylates E. coli proteins in situ. Using enrichment approaches followed by LC-MS/MS, phosphosites were identified allowing motifs associated with auto- and transphosphorylation to be characterized. Four lines of evidence suggest that transphosphorylation of E. coli proteins by BRI1 is specific and therefore provides meaningful results: (1) phosphorylation is not correlated with bacterial protein abundance; (2) phosphosite stoichiometry, estimated by spectral counting, is also not related to protein abundance; (3) a transphosphorylation motif emerged with strong preference for basic residues both N- and C-terminal to the phosphosites; and (4) other protein kinases (BAK1, PEPR1, FLS2, and CDPKß) phosphorylated a distinct set of E. coli proteins and phosphosites. The E. coli transphosphorylation assay can be applied broadly to protein kinases and provides a convenient and powerful system to elucidate kinase specificity.

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