Results Of Yeast Two-hybrid Assay Research Articles

Genome-Wide Identification, Expression and Interaction Analyses of PP2C Family Genes in Chenopodium quinoa.

Plant protein phosphatase 2Cs (PP2Cs) function as inhibitors in protein kinase cascades involved in various processes and are crucial participants in both plant development and signaling pathways activated by abiotic stress. In this study, a genome-wide study was conducted on the CqPP2C gene family. A total of putative 117 CqPP2C genes were identified. Comprehensive analyses of physicochemical properties, chromosome localization and subcellular localization were conducted. According to phylogenetic analysis, CqPP2Cs were divided into 13 subfamilies. CqPP2Cs in the same subfamily had similar gene structures, and conserved motifs and all the CqPP2C proteins had the type 2C phosphatase domains. The expansion of CqPP2Cs through gene duplication was primarily driven by segmental duplication, and all duplicated CqPP2Cs underwent evolutionary changes guided by purifying selection. The expression of CqPP2Cs in various tissues under different abiotic stresses was analyzed using RNA-seq data. The findings indicated that CqPP2C genes played a role in regulating both the developmental processes and stress responses of quinoa. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis of six CqPP2C genes in subfamily A revealed that they were up-regulated or down-regulated under salt and drought treatments. Furthermore, the results of yeast two-hybrid assays revealed that subfamily A CqPP2Cs interacted not only with subclass III CqSnRK2s but also with subclass II CqSnRK2s. Subfamily A CqPP2Cs could interact with CqSnRK2s in different combinations and intensities in a variety of biological processes and biological threats. Overall, our results will be useful for understanding the functions of CqPP2C in regulating ABA signals and responding to abiotic stress.

Genome-wide identification of apple auxin receptor family genes and functional characterization of MdAFB1

The auxin receptor (TIR1/AFBs) family encodes the F-box protein subunit, which is involved in the formation of the E3 ubiquitin ligase SCFTIR1/AFBs complex, a key component of the auxin signaling pathway. However, there are few studies on the auxin receptor family in apple (Malus × domestica). In this study, eight MdAFBs were identified, and phylogenetic analysis showed that they were classified into four groups and distributed on eight chromosomes. Herein, a comprehensive analysis of the MdAFB gene family was conducted to identify cis-acting elements, gene structures, protein structures, aligned sequences, conserved motifs, conserved amino acids, and the protein–protein interaction network. The results of yeast two-hybrid assays showed that MdAFB1 interacted with three auxin repressor proteins. The results of qRT-PCR showed that MdAFB1 responded to osmotic and salt stress. The overexpression of MdAFB1 increased osmotic and salt resistance in apple calli, and the ectopic expression of MdAFB1 enhanced osmotic and salt tolerance in Arabidopsis. This study provided a basis for the identification of auxin receptor genes in apple and their functions in mediating osmotic and salt stress.

BELL1 interacts with CRABS CLAW and INNER NO OUTER to regulate ovule and seed development in pomegranate.

Pomegranate (Punica granatum) flowers are classified as bisexual flowers and functional male flowers. Functional male flowers have sterile pistils that show abnormal ovule development. In previous studies, we identified INNER NO OUTER (INO), CRABS CLAW (CRC) and BELL1 (BEL1), which were specifically expressed in bisexual and functional male flowers. However, the functions of ovule identity genes and the mechanism underlying ovule sterility in pomegranate remain unknown. Here, we found that the integument primordia formed and then ceased developing in the ovules of functional male flowers with a vertical diameter of 8.1-13.0 mm. Megaspore mother cells were observed in bisexual flowers when the vertical diameters of flowers were 10.1-13.0 mm, but not in functional male flowers. We analyzed the expression patterns of ovule-related genes in pomegranate ovule sterility and found that PgCRC mRNA was highly expressed at a critical stage of ovule development in bisexual flowers. Ectopic expression of PgCRC and PgINO was sufficient to increase seed number in transgenic lines. PgCRC partially complemented the Arabidopsis (Arabidopsis thaliana) crc mutant, and PgINO successfully rescued the seeds set in the Arabidopsis ino mutant. The results of Y2H assays, BiFC assays and genetic data analyses showed that PgCRC and PgINO directly interact with PgBEL1. Our results also showed that PgCRC and PgINO could not interact directly with MADS-box proteins and that PgBEL1 interacted with SEPALLATA (PgSEP) proteins. We report the function of PgCRC and PgINO in ovule and seed development and show that PgCRC and PgINO interact with PgBEL1. Thus, our results provide understanding of the genetic regulatory networks underlying ovule development in pomegranate.

OsWRKY28 positively regulates salinity tolerance by directly activating OsDREB1B expression in rice.

OsWRKY28 confers salinity tolerance by directly binding to OsDREB1B promoter and increasing its transcriptional activity, and negatively regulates abscisic acid mediated seedling establishment in rice. WRKY transcription factors have been reported to play a vital role in plants growth, development, abiotic and biotic stress responses. In this study, we explored the functions of a transcription factor OsWRKY28 in rice. The transcript level of OsWRKY28 was strikingly increased under drought, chilling, salt and abscisic acid treatments. The OsWRKY28 overexpression lines showed enhanced salinity stress tolerance, whereas the oswrky28 mutants displayed salt sensitivity compared to wild-type plants. Under salt stress treatment, the expression levels of OsbZIP05, OsHKT1;1 and OsDREB1B were significantly lower yet the level of OsHKT2;1 was significantly higher in oswrky28 mutants than those in wide type plants. Our data of yeast one-hybrid assay and dual-luciferase assay supported that OsWRKY28 could directly bind to the promoter of OsDREB1B to enhance salinity tolerance in rice. In addition, OsWRKY28 overexpression lines displayed hyposensitivity and the oswrky28 mutants showed hypersensitivity compared to wild-type plants under exogenous abscisic acid treatment. Based on the results of yeast two-hybrid assay and GAL4-dependent chimeric transactivation assay, OsWRKY28 physically interacts with OsMPK11 and its transcriptional activity could be regulated by OsMPK11. Together, OsWRKY28 confers salinity tolerance through directly targeting OsDREB1B promoter and further activating its transcription in rice.

Molecular Characterization of Retinoic Acid Receptor CgRAR in Pacific Oyster (Crassostrea gigas).

Retinoic acid (RA) signaling pathways mediated by RA receptors (RARs) are essential for many physiological processes such as organ development, regeneration, and differentiation in animals. Recent studies reveal that RARs identified in several mollusks, including Pacific oyster Crassostrea gigas, have a different function mechanism compared with that in chordates. In this report, we identified the molecular characteristics of CgRAR to further explore the mechanism of RAR in mollusks. RT-qPCR analysis shows that CgRAR has a higher expression level in the hemocytes and gonads, indicating that CgRAR may play roles in the processes of development and metabolism. The mRNA expression level of both CgRAR and CgRXR was analyzed by RT-qPCR after injection with RA. The elevated expression of CgRAR and CgRXR was detected upon all-trans-RA (ATRA) exposure. Finally, according to the results of Yeast Two-Hybrid assay and co-immunoprecipitation analysis, CgRAR and CgRXR can interact with each other through the C-terminal region. Taken together, our results suggest that CgRAR shows a higher expression level in gonads and hemocytes. ATRA exposure up-regulates the expression of CgRAR and CgRXR. Besides, CgRAR can interact with CgRXR to form a heterodimer complex.

Interaction between wheat translationally controlled tumor protein TCTP and SNF1-related protein kinase SnRK1

Translationally controlled tumor proteins (TCTP) and SNF1- related protein kinase (SnRK1) are conserved and widely present in eukaryotic cells. TCTP regulates cell division, plant growth and development, and mediates plant resistance against pathogen infection. SnRK1 participates in a range of physiological processes including sugar metabolism and resistance to abiotic and biotic stresses. Previous work in our laboratory demonstrated that wheat TCTP can respond to Puccinia triticina infection and induce host defense responses. In order to further investigate the mechanism of TaTCTP in wheat resistance to Puccinia triticina infection, we used TAP (tandem affinity purification) and mass spectrometry to screen the potential interactants of TaTCTP. A SNF1- related protein kinase (SnRK1) was identified as a potential interacting protein of TaTCTP. The results of yeast two-hybrid assay showed that TCTP could interact with SnRK1 in yeast, and the yeast carrying TCTP and SnRK1 could grow on SD/-Leu/-Trp/-His/-Ade (SD/-LWHA) medium. The fluorescence signal of the interaction between TCTP and SnRK1 was found to be distributed in the cytoplasm in the Bi-fluorescense complementation experiment. Co-IP experiments further showed that TCTP and SnRK1 could interact in plant cells. This study lays an important foundation for further studying the mechanism of TaTCTP in the interaction between wheat and Puccinia triticina, and it play a great influence on further improving the molecular mechanism of wheat resistant to Puccinia triticina.

Mapping the Binding Site on Small Ankyrin 1 for Obscurin

Small ankyrin 1 (sAnk1), an integral protein of the sarcoplasmic reticulum encoded by the ANK1 gene, binds with nanomolar affinity to the C terminus of obscurin, a giant protein surrounding the contractile apparatus in striated muscle. We used site-directed mutagenesis to characterize the binding site on sAnk1, specifically addressing the role of two putative amphipathic, positively charged helices. We measured binding qualitatively by blot overlay assays and quantitatively by surface plasmon resonance and showed that both positively charged sequences are required for activity. We showed further that substitution of a lysine or arginine with an alanine or glutamate located at the same position along either of the two putative helices has similar inhibitory or stimulatory effects on binding and that the effects of a particular mutation depended on the position of the mutated amino acid in each helix. We modeled the structure of the binding region of sAnk1 by homology with ankyrin repeats of human Notch1, which have a similar pattern of charged and hydrophobic residues. Our modeling suggested that each of the two positively charged sequences forms pairs of amphipathic, anti-parallel alpha-helices flanked by beta-hairpin-like turns. Most of the residues in homologous positions along each helical unit have similar, though not identical, orientations. CD spectroscopy confirmed the alpha-helical content of sAnk1, approximately 33%, predicted by the model. Thus, structural and mutational studies of the binding region on sAnk1 for obscurin suggest that it consists of two ankyrin repeats with very similar structures.

A chaperone‐like HrpG protein acts as a suppressor of HrpV in regulation of the Pseudomonas syringae pv. syringae type III secretion system

The cloned hrp/hrc cluster of Pseudomonas syringae pv. syringae 61 (Pss61) contains 28 proteins, and many of those are assembled into a type III secretion system (TTSS) that is responsible for eliciting the hypersensitive response (HR) in non-host plants and causing diseases on host plants (Huang et al., 1995). hrpG, the second gene in the hrpC operon, encodes a 15.4 kDa cytoplasmic protein whose predicted structure is similar to SicP (E-value: 0.19), a TTSS chaperone of Salmonella typhimurium. Two non-polar hrpG mutants, Pss61-N826 and Pss61-N674, were produced to investigate the biological function of hrpG gene. Pss61-N826, generated by replacing the coding sequence of hrpG with an nptII gene lacking both the promoter and the terminator, was found to be capable of eliciting the wild-type HR; whereas Pss61-N674 generated by replacement of a terminatorless nptII gene in the hrpG coding sequence showed the delayed HR phenotype. Northern and Western blotting analyses showed that the expression of hrpZ, hrcJ and hrcQb genes residing on two different operons in Pss61-N674 was reduced due to the nptII promoter-driven constitutive expression of hrpV that codes for a negative regulator. Interestingly, a plasmid-borne hrpG can derepress the hrp expression in Pss61-N674 and in Pss61 overexpressing HrpV without decreasing the hrpV transcript. Moreover, results of yeast two-hybrid assay, pull-down assay and far Western analysis show that HrpG and HrpV interact with each other in vivo and in vitro. Additionally, HrpV interacts with a positive regulator HrpS according to analysis of a yeast two-hybrid system. Based on the results presented in this study, we propose that HrpG acts as a suppressor of the negative regulator HrpV mediated via protein-protein interaction, leading to modulation of hrp/hrc expression subsequently freeing HrpS to promote the activation of other downstream hrp/hrc genes.

Effects of chlorine on the decrease of estrogenic chemicals

The effects of chlorination on the elimination of three estrogenic chemicals such as 17 β-estradiol, nonylphenol and bis-phenol A were investigated using yeast two-hybrid assay (YTA), estrogen receptor (ER) competition assay (ER-CA), and high-performance liquid chromatography/mass spectrometry (LC/MS). The results of YTA, ER-CA and the analysis of LC/MS indicated that the estrogenic activity of the above-mentioned three endocrine disruptors were significantly reduced as a result of chlorination. The decrease in estrogenic activity paralleled a decrease in estrogenic chemicals under the influence of free chlorine. One common characteristic of estrogenic chemicals is the presence of a phenolic ring. Considering that a phenolic ring is likely to undergo some sort of transformation in an aqueous chlorination solution, the above-mentioned results may be applied to the rest of the estrogenic chemicals in natural waters.

Arabidopsis GARP transcriptional activators interact with the Pro-rich activation domain shared by G-box-binding bZIP factors.

The Pro-rich regions, found in a subset of plant bZIP transcription factors, including G-box-binding factors (GBFs) of Arabidopsis thaliana, are thought to be deeply involved in transcriptional regulation. However, the molecular mechanisms of the Pro-rich region-mediated transcriptional regulation are still largely unknown. Here we report evidence showing that two closely related Arabidopsis proteins, designated GPRI1 and GPRI2, containing a GARP DNA-binding domain, are likely partners of one or more GBFs. The results of yeast two-hybrid assays and in vitro binding assays indicated that GPRI1 can interact with the Pro-rich regions of GBF1 and GBF3. GPRI2 interacted with the Pro-rich region of GBF1. GPRI1 and GPRI2 transactivated transcription in yeast. In GPRI1 the region responsible for this activation was mapped in the N-terminal third of the protein. Transient assays showed that in Arabidopsis cells not only the N-terminal but also the C-terminal regions of GPRI1 can function as a separable activation domain. GPRI1 and GPRI2 may function in some promoters in concert with a GBF through interaction with its Pro-rich region to enhance the transcriptional level of the corresponding genes.

Molecular cloning and characterization of RAD51-like genes from Arabidopsis thaliana.

Homologous recombination is an essential process for the maintenance and variability of the genome. In eukaryotes, the Rad52 epistasis group proteins serve the main role for meiotic recombination and/or homologous recombinational repair. Rad51-like proteins, such as Rad55 and Rad57 in yeast, play a role in assembly or stabilization of multimeric Rad51 that promotes homologous pairing and strand exchange reactions. We cloned two RAD51-like genes named AtXRCC3 and AtRAD51C from Arabidopsis thaliana. Both AtXRCC3 and AtRAD51C expressed two alternatively spliced transcripts, and AtRAD51C produced two different sizes of isoforms, a long (AtRAD51Calpha) and a short one (AtRAD51Cbeta). The predicted protein sequences of these genes showed characteristic features of the RecA/Rad51 family; especially the amino acids around the ATP-binding motifs were well conserved. The transcripts of AtXRCC3 and AtRAD51C were detected in various tissues, with the highest level of expression in flower buds. Expression of both genes was induced by gamma-ray irradiation. The results of yeast two-hybrid assays suggested that Arabidopsis Rad51 family proteins form a complex, which could participate in meiotic recombination and/or homologous recombinational repair.