MADS-Box Protein Complex VvAG2, VvSEP3 and VvAGL11 Regulates the Formation of Ovules in Vitis vinifera L. cv. ‘Xiangfei’

The phenomenon of multi-carpel and multi-ovule exists in the grapevine cultivar ‘Xiangfei’, but the mechanism of ovule formation is seldom reported. In this study, we observed the ovule formation process by using ‘Xiangfei’ grapes. The role of the VvAG2 (VvAGAMOUS) gene in ovule formation was identified, and we explored the relationship between VvAG2, VvSEP3(VvMADS4) and VvAGL11(VvMADS5) proteins. The results showed that the ovule primordium appeared when the inflorescence length of ‘Xiangfei’ grapes were 4–5 cm long; the relative expression levels of VvAG2, VvAGL11 and VvSEP3 genes were higher during ovule formation, and the expression levels of VvAG2 gene was the highest. Transgenic tomato (Solanum lycopersicum) plants expressing VvAG2 produced higher numbers of ovules and carpels than the wild type. Moreover, yeast two-hybrid and yeast three-hybrid experiments demonstrated that VvSEP3 acts as a bridge and interacts with VvAG2 and VvAGL11 proteins, respectively. Meanwhile, a homodimer can be formed between VvSEP3 and VvSEP3, but there was no interaction between VvAG2 and VvAGL11. These findings suggest that the VvAG2 gene is involved in the formation of ovules, and VvAG2/VvSEP3 together with VvAGL11/VvSEP3 can form a tetrameric complex. In summary, our data showed that VvAG2 along with VvSEP3 and VvAGL11 jointly regulate the ovule formation of ‘Xiangfei’ grapes.

Download Full-text

The role of Clt1-regulated-Xylan metabolism in the melanin and toxin formation for the pathogenicity of Curvularia lunata in Maize

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-20-0235-r ◽

2021 ◽

Author(s):

Jinxin Gao ◽

Jie Chen

Keyword(s):

Toxin Production ◽

Bimolecular Fluorescence Complementation ◽

Curvularia Lunata ◽

Yeast Two Hybrid ◽

Acetyl Xylan Esterase ◽

Btb Domain ◽

Intermediate Metabolites ◽

Regulative Mechanism ◽

Two Hybrid

We previously reported that the BTB domain-containing protein Clt1 regulates melanin and toxin synthesis, conidiation, and pathogenicity in Curvularia lunata, but the interacting proteins and regulative mechanism of Clt1 are unclear. In this research, we identified two proteins, which respectively correspond to xylanase (Clxyn24) and acetyl xylan esterase (Claxe43) from C. lunata were regulated by Clt1. Yeast two-hybrid (Y2H), and bimolecular ﬂuorescence complementation assays were conducted to verify the interaction of Clt1 with full-length Clxyn24 and Claxe43. Furthermore, the Y2H assay revealed that Clt1 physically interacted with Clxyn24 and Claxe43 through its BTB domain to degrade xylan which was used as a carbon source for C. lunata growth. The utilization of xylan provides acetyl-CoA for the synthesis of melanin and toxin, as well as energy and other intermediate metabolites for conidiation. Furthermore, transcriptome analysis revealed that PKS18 and its 13 flanking genes are found clustered in a region spanning 57.89 kb on scaffold 9 of the C. lunata CX-3 genome were down-regulated in toxin production deficient mutant T806, and this cluster is possibly responsible for toxin biosynthesis of C. lunata.

Download Full-text

Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome

Molecular and Cellular Biology ◽

10.1128/mcb.23.6.1968-1982.2003 ◽

2003 ◽

Vol 23 (6) ◽

pp. 1968-1982 ◽

Cited By ~ 21

Author(s):

Irfan Saadi ◽

Adisa Kuburas ◽

Jamison J. Engle ◽

Andrew F. Russo

Keyword(s):

Dominant Negative ◽

Hill Coefficient ◽

Homeodomain Protein ◽

Yeast Two Hybrid ◽

Autosomal Dominant Disorder ◽

Rieger Syndrome ◽

A Charge ◽

Two Hybrid

ABSTRACT Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.

Download Full-text

LsHSP70 is induced by high temperature to interact with calmodulin, leading to higher bolting resistance in lettuce

Scientific Reports ◽

10.1038/s41598-020-72443-3 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Ran Liu ◽

Zhenqi Su ◽

Huiyan Zhou ◽

Qian Huang ◽

Shuangxi Fan ◽

...

Keyword(s):

High Temperature ◽

High Temperatures ◽

Morphological Changes ◽

High Temperature Stress ◽

Stem Length ◽

Hybrid Technique ◽

Yeast Two Hybrid ◽

Expression Levels ◽

Two Hybrid ◽

Lower Expression

Abstract High temperatures have significant impacts on heat-tolerant bolting in lettuce. In this study, it was found that high temperatures could facilitate the accumulation of GA in lettuce to induce bolting, with higher expression levels of two heat shock protein genes LsHsp70-3701 and LsHsp70-2711. By applying VIGS technology, these two Hsp70 genes were incompletely silenced and plant morphological changes under heat treatment of silenced plants were observed. The results showed that lower expression levels of these two genes could enhance bolting stem length of lettuce under high temperatures, which means these two proteins may play a significant role in heat-induced bolting tolerance. By using the yeast two-hybrid technique, it was found that a calmodulin protein could interact with LsHsp70 proteins in a high-temperature stress cDNA library, which was constructed for lettuce. Also, the Hsp70-calmodulin combination can be obtained at high temperatures. According to these results, it can be speculated that the interaction between Hsp70 and calmodulin could be induced under high temperatures and higher GA contents can be obtained at the same time. This study analyses the regulation of heat tolerance in lettuce and lays a foundation for additional studies of heat resistance in lettuce.

Download Full-text

NOCTURNIN Gene Diurnal Variation in Healthy Volunteers and Expression Levels in Shift Workers

BioMed Research International ◽

10.1155/2019/7582734 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Massimo Bracci ◽

Alfredo Copertaro ◽

Veronica Ciarapica ◽

Mariella Barbaresi ◽

Stefano Esposito ◽

...

Keyword(s):

Diurnal Variation ◽

Healthy Volunteers ◽

Peripheral Blood ◽

Human Peripheral Blood ◽

Peripheral Blood Lymphocytes ◽

Shift Workers ◽

Expression Levels ◽

Blood Lymphocytes ◽

The Relationship

Objective. The NOCTURNIN gene links nutrient absorption and metabolism to the circadian clock. Shift workers are at a heightened risk of overweight and of developing obesity and metabolic syndrome. This study investigates the diurnal variation of NOCTURNIN in healthy volunteers and its expression levels in rotational shift and daytime workers. Methods. NOCTURNIN expression levels were evaluated in peripheral blood lymphocytes from 15 healthy volunteers at 4-hour intervals for 24 h. Metabolic parameters and NOCTURNIN expression were measured in workers engaged in shift and daytime work. Results. In the group of volunteers NOCTURNIN expression showed diurnal variation, with a peak at 8:00 AM. NOCTURNIN expression was higher in shift workers than in daytime workers. Multivariate analysis confirmed the role of shift work as an independent factor affecting NOCTURNIN expression. Notably, its level correlated directly with body mass index and inversely with total energy expenditure. Conclusions. Measuring NOCTURNIN expression levels in human peripheral blood lymphocytes can improve investigations on the relationship between changes in circadian rhythm and metabolic disorders. Shift workers show higher NOCTURNIN levels than daytime workers.

Download Full-text

A Quantitative Tri-fluorescent Yeast Two-hybrid System: From Flow Cytometry to In cellula Affinities

Molecular & Cellular Proteomics ◽

10.1074/mcp.tir119.001692 ◽

2020 ◽

Vol 19 (4) ◽

pp. 701-715 ◽

Cited By ~ 1

Author(s):

David Cluet ◽

Ikram Amri ◽

Blandine Vergier ◽

Jérémie Léault ◽

Astrid Audibert ◽

...

Keyword(s):

Flow Cytometry ◽

Hybrid System ◽

Protein Interactions ◽

Dissociation Constants ◽

Automated Analysis ◽

Technological Advancement ◽

Yeast Two Hybrid ◽

Expression Levels ◽

Yeast Two Hybrid System ◽

Two Hybrid

We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pm to 17 μm. After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.

Download Full-text

Analysis of the Yarrowia lipolytica proteome reveals subtle variations in expression levels between lipogenic and non-lipogenic conditions

FEMS Yeast Research ◽

10.1093/femsyr/foab007 ◽

2021 ◽

Vol 21 (2) ◽

Author(s):

Ryan Sestric ◽

Vic Spicer ◽

Oleg V Krokhin ◽

Richard Sparling ◽

David B Levin

Keyword(s):

Yarrowia Lipolytica ◽

Carbon Flux ◽

Neutral Lipids ◽

Pyruvate Decarboxylase ◽

Atp Citrate Lyase ◽

Expression Levels ◽

Rich Media ◽

Citrate Lyase ◽

The Relationship

ABSTRACT Oleaginous yeasts have the ability to store greater than 20% of their mass as neutral lipids, in the form of triacylglycerides. The ATP citrate lyase is thought to play a key role in triacylglyceride synthesis, but the relationship between expression levels of this and other related enzymes is not well understood in the role of total lipid accumulation conferring the oleaginous phenotype. We conducted comparative proteomic analyses with the oleaginous yeast, Yarrowia lipolytica, grown in either nitrogen-sufficient rich media or nitrogen-limited minimal media. Total proteins extracted from cells collected during logarithmic and late stationary growth phases were analyzed by 1D liquid chromatography, followed by mass spectroscopy. The ATP citrate lyase enzyme was expressed at similar concentrations in both conditions, in both logarithmic and stationary phase, but many upstream and downstream enzymes showed drastically different expression levels. In non-lipogenic conditions, several pyruvate enzymes were expressed at higher concentration. These enzymes, especially the pyruvate decarboxylase and pyruvate dehydrogenase, may be regulating carbon flux away from central metabolism and reducing the amount of citrate being produced in the mitochondria. While crucial for the oleaginous phenotype, the constitutively expressed ATP citrate lyase appears to cleave citrate in response to carbon flux upstream from other enzymes creating the oleaginous phenotype.

Download Full-text

The Rice Tungro Bacilliform Virus Gene II Product Interacts with the Coat Protein Domain of the Viral Gene III Polyprotein

Journal of Virology ◽

10.1128/jvi.74.5.2073-2083.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2073-2083 ◽

Cited By ~ 18

Author(s):

Etienne Herzog ◽

Orlene Guerra-Peraza ◽

Thomas Hohn

Keyword(s):

Coat Protein ◽

Protein Domain ◽

Rice Tungro Bacilliform Virus ◽

Viral Gene ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Genes Encoding ◽

Two Hybrid

ABSTRACT Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly.

Download Full-text

MCAF Mediates MBD1-Dependent Transcriptional Repression

Molecular and Cellular Biology ◽

10.1128/mcb.23.8.2834-2843.2003 ◽

2003 ◽

Vol 23 (8) ◽

pp. 2834-2843 ◽

Cited By ~ 53

Author(s):

Naoyuki Fujita ◽

Sugiko Watanabe ◽

Takaya Ichimura ◽

Yoshiaki Ohkuma ◽

Tsutomu Chiba ◽

...

Keyword(s):

Transcriptional Repression ◽

Histone Deacetylation ◽

Yeast Two Hybrid ◽

Direct Inhibition ◽

Promoter Regions ◽

Chromatin Dynamics ◽

Mechanistic Basis ◽

Two Hybrid ◽

Repression Domain

ABSTRACT DNA methylation is involved in a variety of genome functions, including gene control and chromatin dynamics. MBD1 is a transcriptional regulator through the cooperation of a methyl-CpG binding domain, cysteine-rich CXXC domains, and a transcriptional repression domain. A yeast two-hybrid screen was performed to investigate the role of MBD1 in methylation-based transcriptional repression. We report a mediator, MBD1-containing chromatin-associated factor (MCAF), that interacts with the transcriptional repression domain of MBD1. MCAF harbors two conserved domains that allow it to interact with MBD1 and enhancer-like transactivator Sp1. MCAF possesses a coactivator-like activity, and it seems to facilitate Sp1-mediated transcription. In contrast, the MBD1-MCAF complex blocks transcription through affecting Sp1 on methylated promoter regions. These data provide a mechanistic basis for direct inhibition of gene expression via methylation-dependent and histone deacetylation-resistant processes.

Download Full-text

Role of the Carboxy Terminus of Escherichia coli FtsA in Self-Interaction and Cell Division

Journal of Bacteriology ◽

10.1128/jb.182.22.6366-6373.2000 ◽

2000 ◽

Vol 182 (22) ◽

pp. 6366-6373 ◽

Cited By ~ 51

Author(s):

Lucía Yim ◽

Guy Vandenbussche ◽

Jesús Mingorance ◽

Sonsoles Rueda ◽

Mercedes Casanova ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Biological Function ◽

Wild Type ◽

Carboxy Terminus ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Carboxy Terminal ◽

Two Hybrid

ABSTRACT The role of the carboxy terminus of the Escherichia coli cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of theftsA alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362–5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsAΔ1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsAΔ27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsAΔ27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.

Download Full-text

SLFL Genes Participate in the Ubiquitination and Degradation ofS-RNase in Self-Compatible Chinese Peach

10.1101/160267 ◽

2017 ◽

Author(s):

Qiuju Chen ◽

Dong Meng ◽

Wei Li ◽

Zhaoyu Gu ◽

Hui Yuan ◽

...

Keyword(s):

Prunus Persica ◽

Hypervariable Region ◽

Self Incompatibility ◽

S Locus ◽

Yeast Two Hybrid ◽

Peach Genome ◽

General Inhibitor ◽

Two Hybrid

AbstractThe gametophytic self-incompatibility (SI) mediated by S-RNase of Rosaceae, Solanaceae and Plantaginaceae, is controlled by two tightly linked genes located at highly polymorphic S-locus: the S-RNase for pistil specificity and the F-box gene (SFB/SLF) for pollen specificity, respectively. The F-box gene of peach (Prunus persica) isShaplotype-specific F-box (SFB). In this study, we selected 37 representative varieties according to the evolution route of peach and identified their S genotypes. We cloned pollen determinant genes mutantPperSFB1m, PperSFB2m, PperSFB4mand normalPperSFB2, and style determinant genesS1-RNase, S2-RNase, S2m-RNaseandS4-RNase.MutantPperSFBswere translated terminated prematurely because of fragment insertion. Yeast two-hybrid showed that mutant PperSFBs and normal PperSFB2 interacted with all S-RNases. NormalPperSFB2was divided into four parts: box, box-V1, V1-V2 and HVa-HVb. Protein interaction analyses showed that the box portion did not interact with S-RNases, both of the box-V1 and V1-V2 had interactions with S-RNases, while the hypervariable region ofPperSFB2HVa-HVb only interacted with S2-RNase. Bioinformatics analysis of peach genome revealed that there were other F-box genes located at S-locus, and of which three F-box genes were specifically expressed in pollen, namelyPperSLFL1, PperSLFL2andPperSLFL3, respectively. Phylogenetic analysis showed that PperSFBs and PperSLFLs were classified into two different clades. Yeast two-hybrid analysis revealed that as with PperSFBs, the three F-box proteins interacted with PperSSK1. Yeast two-hybrid and BiFC showed that PperSLFLs interacted with S-RNases with no allelic specificity. In vitro ubiquitination assay showed that PperSLFLs could tag ubiquitin molecules to PperS-RNases. In all, the above results suggest that threePperSLFLsare the appropriate candidates for the ‘general inhibitor’, which would inactivate the S-RNases in pollen tubes, and the role of three PperSLFL proteins is redundant, as S-RNase repressors involved in the self-incompatibility of peach.

Download Full-text