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1.
Sci Rep ; 11(1): 14501, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262119

RESUMO

Cynara cardunculus L. or cardoon is a plant that is used as a source of milk clotting enzymes during traditional cheese manufacturing. This clotting activity is due to aspartic proteases (APs) found in the cardoon flower, named cyprosins and cardosins. APs from cardoon flowers display a great degree of heterogeneity, resulting in variable milk clotting activities and directly influencing the final product. Producing these APs using alternative platforms such as bacteria or yeast has proven challenging, which is hampering their implementation on an industrial scale. We have developed tobacco BY2 cell lines as an alternative plant-based platform for the production of cardosin B. These cultures successfully produced active cardosin B and a purification pipeline was developed to obtain isolated cardosin B. The enzyme displayed proteolytic activity towards milk caseins and milk clotting activity under standard cheese manufacturing conditions. We also identified an unprocessed form of cardosin B and further investigated its activation process. The use of protease-specific inhibitors suggested a possible role for a cysteine protease in cardosin B processing. Mass spectrometry analysis identified three cysteine proteases containing a granulin-domain as candidates for cardosin B processing. These findings suggest an interaction between these two groups of proteases and contribute to an understanding of the mechanisms behind the regulation and processing of plant APs. This work also paves the way for the use of tobacco BY2 cells as an alternative production system for active cardosins and represents an important advancement towards the industrial production of cardoon APs.


Assuntos
Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tabaco/metabolismo , Animais , Ácido Aspártico Endopeptidases/isolamento & purificação , Caseínas/metabolismo , Cisteína Proteases/metabolismo , Concentração de Íons de Hidrogênio , Leite , Células Vegetais , Extratos Vegetais/química , Proteínas de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Tabaco/citologia , Tabaco/genética
2.
Plant Cell Rep ; 40(7): 1247-1267, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34028582

RESUMO

KEY MESSAGE: PSV infection changed the abundance of host plant's transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and cytosol, affecting photosynthesis, translation, transcription, and splicing. Virus infection is a process resulting in numerous molecular, cellular, and physiological changes, a wide range of which can be analyzed due to development of many high-throughput techniques. Plant RNA viruses are known to replicate in the cytoplasm; however, the roles of chloroplasts and other cellular structures in the viral replication cycle and in plant antiviral defense have been recently emphasized. Therefore, the aim of this study was to analyze the small RNAs, transcripts, proteins, and phosphoproteins affected during peanut stunt virus strain P (PSV-P)-Nicotiana benthamiana interactions with or without satellite RNA (satRNA) in the context of their cellular localization or functional connections with particular cellular compartments to elucidate the compartments most affected during pathogenesis at the early stages of infection. Moreover, the processes associated with particular cell compartments were determined. The 'omic' results were subjected to comparative data analyses. Transcriptomic and small RNA (sRNA)-seq data were obtained to provide new insights into PSV-P-satRNA-plant interactions, whereas previously obtained proteomic and phosphoproteomic data were used to broaden the analysis to terms associated with cellular compartments affected by virus infection. Based on the collected results, infection with PSV-P contributed to changes in the abundance of transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and the cytosol, and the most affected processes were photosynthesis, translation, transcription, and mRNA splicing. Furthermore, sRNA-seq and phosphoproteomic analyses indicated that kinase regulation resulted in decreases in phosphorylation levels. The kinases were associated with the membrane, cytoplasm, and nucleus components.


Assuntos
Cucumovirus/patogenicidade , Biologia de Sistemas/métodos , Tabaco/citologia , Tabaco/virologia , Núcleo Celular/genética , Núcleo Celular/virologia , Cloroplastos/genética , Cloroplastos/virologia , Citoesqueleto/genética , Citoesqueleto/virologia , Citosol/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno/fisiologia , MicroRNAs , Nitrogênio/metabolismo , Fosfoproteínas/metabolismo , Células Vegetais/virologia , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas/genética , RNA Satélite , Tabaco/genética
3.
Biosensors (Basel) ; 11(5)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925584

RESUMO

The plant cell mechanics, including turgor pressure and wall mechanical properties, not only determine the growth of plant cells, but also reflect the functional and structural changes of plant cells under biotic and abiotic stresses. However, there are currently no appropriate techniques allowing to monitor the complex mechanical properties of living plant cells non-invasively and continuously. In this work, quartz crystal microbalance with dissipation (QCM-D) monitoring technique with overtones (3-9) was used for the dynamic monitoring of adhesions of living tobacco BY-2 cells onto positively charged N,N-dimethyl-N-propenyl-2-propen-1-aminiumchloride homopolymer (PDADMAC)/SiO2 QCM crystals under different concentrations of mannitol (CM) and the subsequent effects of osmotic stresses. The cell viscoelastic index (CVIn) (CVIn = ΔD⋅n/ΔF) was used to characterize the viscoelastic properties of BY-2 cells under different osmotic conditions. Our results indicated that lower overtones of QCM could detect both the cell wall and cytoskeleton structures allowing the detection of plasmolysis phenomena; whereas higher overtones could only detect the cell wall's mechanical properties. The QCM results were further discussed with the morphological changes of the BY-2 cells by an optical microscopy. The dynamic changes of cell's generated forces or cellular structures of plant cells caused by external stimuli (or stresses) can be traced by non-destructive and dynamic monitoring of cells' viscoelasticity, which provides a new way for the characterization and study of plant cells. QCM-D could map viscoelastic properties of different cellular structures in living cells and could be used as a new tool to test the mechanical properties of plant cells.


Assuntos
Técnicas de Microbalança de Cristal de Quartzo , Tabaco , Adesão Celular , Microscopia , Osmose/fisiologia , Dióxido de Silício , Tabaco/citologia , Viscosidade
4.
Int J Mol Sci ; 22(8)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924478

RESUMO

The encoding genes of plant intracellular nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain receptors (NLRs) often exist in the form of a gene cluster. Several recent studies demonstrated that the truncated Toll/interleukin-1 receptor-NBS (TIR-NBS) proteins play important roles in immunity. In this study, we identified a large TN gene cluster on Arabidopsis ecotype Col-0 chromosome 1, which included nine TN genes, TN4 to TN12. Interestingly, this cluster also contained two typical TIR-NBS-LRR genes: At1g72840 and At1g72860 (hereinafter referred to as TNL40 and TNL60, respectively), which formed head-to-head genomic arrangement with TN4 to TN12. However, the functions of these TN and TNL genes in this cluster are still unknown. Here, we showed that the TIR domains of both TNL40 and TNL60 associated with TN10 specifically. Furthermore, both TNL40TIR and TNL60TIR induced cell death in Nicotiana tabacum leaves. Subcellular localization showed that TNL40 mainly localized in the cytoplasm, whereas TNL60 and TN10 localized in both the cytoplasm and nucleus. Additionally, the expression of TNL40, TNL60, and TN10 were co-regulated after inoculated with bacterial pathogens. Taken together, our study indicates that the truncated TIR-NBS protein TN10 associates with two clustered TNL immune receptors, and may work together in plant disease resistance.


Assuntos
ADP-Ribosil Ciclase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas NLR/metabolismo , Imunidade Vegetal , Receptores Imunológicos/metabolismo , ADP-Ribosil Ciclase/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Morte Celular , Genes de Plantas , Modelos Biológicos , Família Multigênica , Folhas de Planta/metabolismo , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Pseudomonas syringae/fisiologia , Frações Subcelulares/metabolismo , Tabaco/citologia
5.
Plant Signal Behav ; 16(4): 1873586, 2021 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-33427565

RESUMO

Phragmoplasts, which comprise microtubules, actin filaments, and membrane vesicles, are responsible for cell plate formation and expansion during plant cytokinesis. Our previous research using the actin polymerization inhibitor latrunculin B (LatB) to investigate the role of actin filaments suggested the existence of two types of microtubules: 1) initial microtubules sensitive to LatB but unassociated with NACK1 kinesin and 2) later LatB-insensitive, NACK1-associated microtubules. The organization of initial phragmoplast microtubules might have been disrupted by the LatB treatment; this hypothesis remained unverified, however, as the exact timing of cell plate membrane accumulation could not be determined. In the present study, we further investigated the timing of cell plate formation during LatB treatment. We monitored chromosome separation during anaphase as well as accumulation of FM4-64-stained cell plate membranes in dividing transgenic tobacco BY-2 cells expressing RFP-tagged histone H2B. We observed that LatB treatment prolonged the time between the slowdown of daughter chromosome migration and the accumulation of cell plate membranes. This result suggests that disruption of actin filaments resulted in delayed cell plate formation possibly by perturbation of initial phragmoplast microtubules or cell plate assembly.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Segregação de Cromossomos , Citocinese , Cromossomos de Plantas/metabolismo , Fatores de Tempo , Tabaco/citologia
6.
Plant Cell Rep ; 40(1): 127-142, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33068174

RESUMO

KEY MESSAGE: The HbCAld5H1 gene cloned from Hevea brasiliensis regulates the cambial activity, xylem differentiation, syringyl-guaiacyl ratio, secondary wall structure, lignification pattern and xylan distribution in xylem fibres of transgenic tobacco plants. Molecular characterization of lignin biosynthesis gene coniferaldehyde-5-hydroxylase (CAld5H) from Hevea brasiliensis and its functional validation was performed. Both sense and antisense constructs of HbCAld5H1 gene were introduced into tobacco through Agrobacterium-mediated genetic transformation for over expression and down-regulation of this key enzyme to understand its role affecting structural and cell wall chemistry. The anatomical studies of transgenic tobacco plants revealed the increase of cambial activity leading to xylogenesis in sense lines and considerable reduction in antisense lines. The ultra-structural studies showed that the thickness of secondary wall (S2 layer) of fibre had been decreased with non-homogenous lignin distribution in antisense lines, while sense lines showed an increase in S2 layer thickness. Maule color reaction revealed that syringyl lignin distribution in the xylem elements was increased in sense and decreased in antisense lines. The immunoelectron microscopy revealed a reduction in LM 10 and LM 11 labelling in the secondary wall of antisense tobacco lines. Biochemical studies showed a radical increase in syringyl lignin in sense lines without any significant change in total lignin content, while S/G ratio decreased considerably in antisense lines. Our results suggest that CAld5H gene plays an important role in xylogenesis stages such as cambial cell division, secondary wall thickness, xylan and syringyl lignin distribution in tobacco. Therefore, CAld5H gene could be considered as a promising target for lignin modification essential for timber quality improvement in rubber.


Assuntos
Parede Celular/química , Oxigenases de Função Mista/genética , Proteínas de Plantas/genética , Tabaco/genética , Xilema/citologia , Acroleína/análogos & derivados , Acroleína/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Lignina/genética , Lignina/metabolismo , Oxigenases de Função Mista/metabolismo , Fenótipo , Células Vegetais/metabolismo , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Caules de Planta/anatomia & histologia , Caules de Planta/genética , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Tabaco/citologia , Tabaco/metabolismo , Xilanos/genética , Xilanos/metabolismo , Xilema/metabolismo
7.
J Integr Plant Biol ; 63(2): 353-364, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33085164

RESUMO

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV-AC ), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.


Assuntos
Imageamento Tridimensional , Membranas Intracelulares/metabolismo , Doenças das Plantas/virologia , Vírus de Plantas/fisiologia , Vacúolos/metabolismo , Cucumovirus/fisiologia , Cucumovirus/ultraestrutura , Tomografia com Microscopia Eletrônica , Membranas Intracelulares/ultraestrutura , Epiderme Vegetal/citologia , Epiderme Vegetal/ultraestrutura , Epiderme Vegetal/virologia , Vírus de Plantas/ultraestrutura , Frações Subcelulares/metabolismo , Tabaco/citologia , Tombusviridae/fisiologia , Tombusviridae/ultraestrutura , Vacúolos/ultraestrutura , Proteínas Virais/metabolismo , Replicação Viral/fisiologia
8.
Plant J ; 105(3): 600-618, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33119146

RESUMO

Senescence is an integrative final stage of plant development that is governed by internal and external cues. The NAM, ATAF1/2, CUC2 (NAC) transcription factor (TF) family is specific to plants and membrane-tethered NAC TFs (MTTFs) constitute a unique and sophisticated mechanism in stress responses and development. However, the function of MTTFs in oilseed rape (Brassica napus L.) remains unknown. Here, we report that BnaNAC60 is an MTTF associated with the endoplasmic reticulum (ER) membrane. Expression of BnaNAC60 was induced during the progression of leaf senescence. Translocation of BnaNAC60 into nuclei was induced by ER stress and oxidative stress treatments. It binds to the NTLBS motif, rather than the canonical NAC recognition site. Overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, induces significant reactive oxygen species (ROS) accumulation and hypersensitive response-like cell death in both tobacco (Nicotiana benthamiana) and oilseed rape protoplasts. Moreover, ectopic overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, in Arabidopsis also induces precocious leaf senescence. Furthermore, screening and expression profiling identified an array of functional genes that are significantly induced by BnaNAC60 expression. Further it was found that BnaNAC60 can activate the promoter activities of BnaNYC1, BnaRbohD, BnaBFN1, BnaZAT12, and multiple BnaVPEs in a dual-luciferase reporter assay. Electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative PCR assays revealed that BnaNAC60 directly binds to the promoter regions of these downstream target genes. To summarize, our data show that BnaNAC60 is an MTTF that modulates cell death, ROS accumulation, and leaf senescence.


Assuntos
Brassica napus/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Apoptose , Arabidopsis/genética , Arabidopsis/fisiologia , Brassica napus/citologia , Brassica napus/efeitos dos fármacos , Membrana Celular/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Células Vegetais , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Tabaco/citologia , Tabaco/genética
9.
J Integr Plant Biol ; 63(2): 365-377, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32725938

RESUMO

Filamentous fungal pathogens secrete effectors that modulate host immunity and facilitate infection. Fusarium graminearum is an important plant pathogen responsible for various devastating diseases. However, little is known about the function of effector proteins secreted by F. graminearum. Herein, we identified several effector candidates in the F. graminearum secretome. Among them, the secreted ribonuclease Fg12 was highly upregulated during the early stages of F. graminearum infection in soybean; its deletion compromised the virulence of F. graminearum. Transient expression of Fg12 in Nicotiana benthamiana induced cell death in a light-dependent manner. Fg12 possessed ribonuclease (RNase) activity, degrading total RNA. The enzymatic activity of Fg12 was required for its cell death-promoting effects. Importantly, the ability of Fg12 to induce cell death was independent of BAK1/SOBIR1, and treatment of soybean with recombinant Fg12 protein induced resistance to various pathogens, including F. graminearum and Phytophthora sojae. Overall, our results provide evidence that RNase effectors not only contribute to pathogen virulence but also induce plant cell death.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/patogenicidade , Células Vegetais/microbiologia , Ribonucleases/metabolismo , Morte Celular , Resistência à Doença , Fusarium/classificação , Filogenia , Phytophthora/fisiologia , Doenças das Plantas/microbiologia , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Sinais Direcionadores de Proteínas , Proteômica , RNA de Plantas/metabolismo , Soja/microbiologia , Tabaco/citologia , Regulação para Cima , Virulência
10.
Plant Signal Behav ; 16(3): 1861768, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33356829

RESUMO

Autophagy, a bulk degradation system conserved among most eukaryotes, is also involved in responses to viral infection in plant. In our previous study, a new host factor P3IP was identified to interact with RSV (rice stripe virus) p3 and mediate its autophagic degradation to limit the viral infection. Here, we further discovered that P3IP of Nicotiana benthamiana (NbP3IP) participated in regulation of autophagy. Overexpression of NbP3IP induced autophagy and down-regulation of NbP3IP reduced autophagy. Combined the functions of autophagy-mediated plant defense against plant virus and regulation autophagy, we indicate that P3IP participates in the regulation of autophagy.


Assuntos
Autofagia , Proteínas de Plantas/metabolismo , Tabaco/citologia , Oryza/metabolismo , Plantas Geneticamente Modificadas , Tabaco/genética
11.
Methods Mol Biol ; 2213: 89-98, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33270195

RESUMO

Ca2+-based second messenger signaling is used by many signal perception mechanisms to modulate specific cellular responses. The well-characterized phytohormone auxin elicits a very rapid Ca2+ signal, but the molecular players involved in auxin-induced Ca2+ signaling are still largely unknown. The complicated and often redundant nature of the plant Ca2+ signaling machinery makes the use of mutants and transgenic lines a painstaking process, which makes a pharmacological approach an attractive alternative to study these processes. Here, we describe the development and utilization of a screening assay that can be used to probe a compound library for inhibitors of auxin-induced Ca2+ entry in plant cell suspensions.


Assuntos
Sinalização do Cálcio , Testes Genéticos/métodos , Ácidos Indolacéticos/metabolismo , Cálcio/metabolismo , Linhagem Celular , Reprodutibilidade dos Testes , Tabaco/citologia , Transformação Genética
12.
BMC Plant Biol ; 20(1): 555, 2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33302867

RESUMO

BACKGROUND: The degradation of intracellular proteins plays an essential role in plant responses to stressful environments. ClpS1 and E3 ubiquitin ligase function as adaptors for selecting target substrates in caseinolytic peptidase (Clp) proteases pathways and the 26S proteasome system, respectively. Currently, the role of E3 ubiquitin ligase in the plant immune response to pathogens is well defined. However, the role of ClpS1 in the plant immune response to pathogens remains unknown. RESULTS: Here, wheat (Triticum aestivum) ClpS1 (TaClpS1) was studied and resulted to encode 161 amino acids, containing a conserved ClpS domain and a chloroplast transit peptide (1-32 aa). TaClpS1 was found to be specifically localized in the chloroplast when expressed transiently in wheat protoplasts. The transcript level of TaClpS1 in wheat was significantly induced during infection by Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaClpS1 via virus-induced gene silencing (VIGS) resulted in an increase in wheat resistance against Pst, accompanied by an increase in the hypersensitive response (HR), accumulation of reactive oxygen species (ROS) and expression of TaPR1 and TaPR2, and a reduction in the number of haustoria, length of infection hypha and infection area of Pst. Furthermore, heterologous expression of TaClpS1 in Nicotiana benthamiana enhanced the infection by Phytophthora parasitica. CONCLUSIONS: These results suggest that TaClpS1 negatively regulates the resistance of wheat to Pst.


Assuntos
Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Proteínas de Plantas/genética , Triticum/genética , Sequência de Aminoácidos , Cloroplastos/genética , Cloroplastos/metabolismo , Interações Hospedeiro-Patógeno , Filogenia , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/classificação , Protoplastos/citologia , Protoplastos/metabolismo , Puccinia/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Plântula/genética , Plântula/metabolismo , Plântula/microbiologia , Homologia de Sequência de Aminoácidos , Tabaco/citologia , Tabaco/metabolismo , Triticum/metabolismo , Triticum/microbiologia
13.
Acta Biochim Pol ; 67(4): 449-452, 2020 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-33245659

RESUMO

Callus from Nicotiana tabacum is used as a model in plant developmental research. We tested several phytohormone (Indoleacetic acid - IAA; 2,4-Dichlorophenoxyacetic acid - 2,4-D; kinetin - KIN; 6-Benzylaminopurine - BAP) combinations to compare different approaches to callus induction directly from the seeds of Nicotiana tabacum. Callus formation was observed up to 4 weeks after sowing and the most effective were 0.5 mg/L of 2,4-D with 0.25 mg/L of BAP and 2 mg/L 2,4-D with 1 mg/L of BAP. The calli were green, photosynthetically active and after 6 weeks of growth, no stress symptoms (estimated on the basis of fluorescence of chlorophyll a in photosystem II) were noticed.


Assuntos
Ácido 2,4-Diclorofenoxiacético/farmacologia , Compostos de Benzil/farmacologia , Ácidos Indolacéticos/farmacologia , Cinetina/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Purinas/farmacologia , Tabaco/efeitos dos fármacos , Clorofila A/biossíntese , Germinação/efeitos dos fármacos , Germinação/fisiologia , Células do Mesofilo/citologia , Células do Mesofilo/efeitos dos fármacos , Células do Mesofilo/metabolismo , Fotossíntese/efeitos dos fármacos , Fotossíntese/fisiologia , Plântula/citologia , Plântula/efeitos dos fármacos , Plântula/metabolismo , Sementes/citologia , Sementes/efeitos dos fármacos , Sementes/metabolismo , Tabaco/citologia , Tabaco/metabolismo
14.
PLoS One ; 15(10): e0241613, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33125444

RESUMO

Pine wilt disease (PWD) is an infectious disease of pines that typically kills affected trees. The causal pathogen of PWD is the pine wood nematode (PWN), Bursaphelenchus xylophilus. Understanding of the disease has advanced in recent years through the use of a highly sensitive proteomics procedure and whole genome sequence analysis; in combination, these approaches have enabled identification of proteins secreted by PWNs. However, the roles of these proteins during the onset of parasitism have not yet been elucidated. In this study, we used a leaf-disk assay based on transient overexpression in Nicotiana benthamiana to allow functional screening of 10 candidate pathogenic proteins secreted by PWNs. These proteins were selected based on previous secretome and RNA-seq analyses. We found that five molecules induced significant cell death in tobacco plants relative to a GFP-only control. Three of these proteins (Bx-TH1, Bx-TH2, and Bx-CPI) may have a role in molecular mimicry and likely make important contributions to inducing hypersensitive responses in host plants.


Assuntos
Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita , Doenças das Plantas/parasitologia , Tabaco/parasitologia , Tylenchida/fisiologia , Animais , Morte Celular , Inibidores de Cisteína Proteinase/metabolismo , Tabaco/citologia , Tabaco/fisiologia
15.
Curr Biol ; 30(24): 4999-5006.e3, 2020 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-33035485

RESUMO

Several plant proteins are preferentially localized to one end of a cell, allowing a polarity to be assigned to the cell. These cell polarity proteins often exhibit coordinated patterns between neighboring cells, termed tissue cell polarity. Tissue cell polarity is widespread in plants and can influence how cells grow, divide, and differentiate [1-5]. However, it is unclear whether cell polarity is established through cell-intrinsic or -extrinsic mechanisms and how polarity is coupled to growth. To address these issues, we analyzed the behavior of a tissue cell polarity protein BASL (BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE) in the simplifying context of cultured cell filaments and in protoplasts before and during regeneration. We show that BASL is polarly localized when ectopically expressed in tobacco BY-2 cell cultures. Ectopic BASL is found preferentially at the developing tips of cell filaments, likely marking a polarized molecular address. Polarity can shift during the cell cycle and is resistant to treatment with microtubule, actin or auxin transport inhibitors. BASL also exhibits polar localization in spherical protoplasts, in contrast to other polarity proteins so far tested. BASL polarity within protoplasts is dynamic and resistant to auxin transport inhibitors. As protoplasts regenerate, polarity remains dynamic in isotropically growing cells but becomes fixed in anisotropic cells and aligns with the axis of cell growth. Our findings suggest that plant cells have an intrinsic ability to polarize and that environmental or developmental cues may act by biasing the direction of this polarity and thus the orientation of anisotropic growth.


Assuntos
Polaridade Celular/fisiologia , Células Vegetais/fisiologia , Protoplastos/fisiologia , Tabaco/crescimento & desenvolvimento , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tabaco/citologia
16.
Int J Mol Sci ; 21(17)2020 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-32842656

RESUMO

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, seriously affects watermelon and other cucurbit crops, resulting in significant economic losses. However, the pathogenicity mechanism of A. citrulli is not well understood. Plant pathogenic bacteria often suppress the plant immune response by secreting effector proteins. Thus, identifying A. citrulli effector proteins and determining their functions may improve our understanding of the underlying pathogenetic mechanisms. In this study, a novel effector, AopN, which is localized on the cell membrane of Nicotiana benthamiana, was identified. The functional analysis revealed that AopN significantly inhibited the flg22-induced reactive oxygen species burst. AopN induced a programmed cell death (PCD) response. Unlike its homologous protein, the ability of AopN to induce PCD was dependent on two motifs of unknown functions (including DUP4129 and Cpta_toxin), but was not dependent on LXXLL domain. More importantly, the virulence of the aopN mutant of A. citrulli in N. benthamiana significantly decreased, indicating that it was a core effector. Further analysis revealed that AopN interacted with watermelon ClHIPP and ClLTP, which responds to A. citrulli strain Aac5 infection at the transcription level. Collectively, these findings indicate that AopN suppresses plant immunity and activates the effector-triggered immunity pathway.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Comamonadaceae/patogenicidade , Doenças das Plantas/microbiologia , Motivos de Aminoácidos , Apoptose , Membrana Celular/metabolismo , Citrullus/microbiologia , Comamonadaceae/genética , Comamonadaceae/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Patógeno , Células Vegetais/microbiologia , Doenças das Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tabaco/citologia , Tabaco/metabolismo , Tabaco/microbiologia , Técnicas do Sistema de Duplo-Híbrido , Virulência
17.
Methods Mol Biol ; 2166: 343-356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32710419

RESUMO

Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms are regulated, it is required to establish imaging methods to visualize the chromatin dynamic in living cells. Here, we provide a protocol for a live plant cell imaging technique based on application of two orthologs of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Streptococcus pyogenes and Staphylococcus aureus. This technique uses the inactive variants of Cas9 combined with different fluorescent proteins (GFP and mRuby) and telomere-specific guide RNA to target telomeric repeats in Nicotiana benthamiana. Our immuno-FISH data revealed that signals arising from the CRISPR/dCas9 method are specifically belonging to telomeric regions.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células Vegetais/metabolismo , Folhas de Planta/citologia , RNA Guia/genética , Telômero/genética , Tabaco/citologia , Proteína 9 Associada à CRISPR/genética , Cromatina/genética , Cromatina/metabolismo , Loci Gênicos , Proteínas de Fluorescência Verde/genética , Microscopia Confocal/métodos , Staphylococcus aureus/genética , Streptococcus pyogenes/genética , Telômero/metabolismo
18.
J Plant Physiol ; 251: 153223, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32645555

RESUMO

The production of volatile organic compounds (VOCs) during programmed cell death (PCD) is still insufficiently studied and their implication in the process is not well understood. The present study demonstrates that the release of VOSCs with presumed antioxidant capacity (methanethiol, dimethylsulfide and dimethyldisulfide) accompanies the cell death in chemical-stressed tobacco BY-2 suspension cultured cells. The cells were exposed to cell death inducers of biotic nature mastoparan (MP, wasp venom) and camptothecin (CPT, alkaloid), and to the abiotic stress agent CdSO4. The VOCs emission was monitored by proton-transfer reaction mass spectrometry (PTR-MS). The three chemicals induced PCD expressing apoptotic-like phenotype. The identified VOSCs were emitted in response to MP and CPT but not in presence of Cd. The VOSCs production occurred within few hours after the administration of the elicitors, peaked up when 20-50 % of the cells were dead and further levelled off with cell death advancement. This suggests that VOSCs with antioxidant activity may contribute to alleviation of cell death-associated oxidative stress at medium severity of cell death in response to the stress factors of biotic origin. The findings provide novel information about cell death defence mechanisms in chemical-challenged BY-2 cells and show that PCD related VOSCs synthesis depends on the type of inducer.


Assuntos
Antioxidantes/metabolismo , Morte Celular/fisiologia , Compostos de Enxofre/metabolismo , Tabaco/fisiologia , Compostos Orgânicos Voláteis/metabolismo , Compostos de Cádmio/efeitos adversos , Camptotecina/efeitos adversos , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intercelular/efeitos adversos , Sulfatos/efeitos adversos , Tabaco/citologia , Venenos de Vespas/efeitos adversos
19.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641477

RESUMO

Positive-strand RNA [(+)RNA] viruses are important pathogens of humans, animals, and plants and replicate inside host cells by coopting numerous host factors and subcellular membranes. To gain insights into the assembly of viral replicase complexes (VRCs) and dissect the roles of various lipids and coopted host factors, we have reconstituted Tomato bushy stunt virus (TBSV) replicase using artificial giant unilamellar vesicles (GUVs). We demonstrate that reconstitution of VRCs on GUVs with endoplasmic reticulum (ER)-like phospholipid composition results in a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)RNA viruses. TBSV VRCs assembled on GUVs provide significant protection of the double-stranded RNA (dsRNA) replication intermediate against the dsRNA-specific RNase III. The lipid compositions of GUVs have pronounced effects on in vitro TBSV replication, including (-) and (+)RNA synthesis. The GUV-based assay has led to the discovery of the critical role of phosphatidylserine in TBSV replication and a novel role for phosphatidylethanolamine in asymmetrical (+)RNA synthesis. The GUV-based assay also showed stimulatory effects by phosphatidylinositol-3-phosphate [PI(3)P] and ergosterol on TBSV replication. We demonstrate that eEF1A and Hsp70 coopted replicase assembly factors, Vps34 phosphatidylinositol 3-kinase (PI3K) and the membrane-bending ESCRT factors, are required for reconstitution of the active TBSV VRCs in GUVs, further supporting that the novel GUV-based in vitro approach recapitulates critical steps and involves essential coopted cellular factors of the TBSV replication process. Taken together, this novel GUV assay will be highly suitable to dissect the functions of viral and cellular factors in TBSV replication.IMPORTANCE Understanding the mechanism of replication of positive-strand RNA viruses, which are major pathogens of plants, animals, and humans, can lead to new targets for antiviral interventions. These viruses subvert intracellular membranes for virus replication and coopt numerous host proteins, whose functions during virus replication are not yet completely defined. To dissect the roles of various host factors in Tomato bushy stunt virus (TBSV) replication, we have developed an artificial giant unilamellar vesicle (GUV)-based replication assay. The GUV-based in vitro approach recapitulates critical steps of the TBSV replication process. GUV-based reconstitution of the TBSV replicase revealed the need for a complex mixture of phospholipids, especially phosphatidylserine and phosphatidylethanolamine, in TBSV replication. The GUV-based approach will be useful to dissect the functions of essential coopted cellular factors.


Assuntos
RNA de Cadeia Dupla/genética , RNA Polimerase Dependente de RNA/genética , Tombusvirus/genética , Lipossomas Unilamelares/metabolismo , Proteínas Virais/genética , Bioensaio , Linhagem Celular , Retículo Endoplasmático/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ergosterol/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Células Vegetais/metabolismo , Células Vegetais/virologia , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/metabolismo , Lipossomas Unilamelares/química , Proteínas Virais/metabolismo , Replicação Viral
20.
Methods Mol Biol ; 2149: 89-109, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32617931

RESUMO

Plant tissue cultures are an efficient system to study cell wall biosynthesis in living cells in vivo. Tissue cultures also provide cells and culture medium from which enzymes and cell wall polymers can easily be separated for further studies. Tissue cultures with tracheary element differentiation or extracellular lignin formation have provided useful information related to several aspects of xylem and lignin formation. In this chapter, methods for nutrient medium preparation and callus culture initiation and its maintenance as well as those for protoplast isolation and viability observation are described. As a case study, we describe the establishment of a xylogenic culture of Zinnia elegans mesophyll cells.


Assuntos
Plantas/metabolismo , Técnicas de Cultura de Tecidos/métodos , Asteraceae/citologia , Diferenciação Celular , Divisão Celular , Parede Celular/metabolismo , Células Cultivadas , Germinação , Células do Mesofilo/citologia , Células do Mesofilo/metabolismo , Folhas de Planta/citologia , Protoplastos/metabolismo , Esterilização , Tabaco/citologia
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