Yeast two-hybrid (YTH) technology is a powerful tool for studying protein interactions and has been widely used in various fields of molecular biology, including the study of antiviral innate immunity. This chapter presents detailed information and experimental procedures for identifying virus-host protein interactions involved in immune regulation using yeast two-hybrid technology.

CONCLUSIONS: Excess of KRP4 in the developing kernels in rice causes poor filling of the grains possibly through inhibition of CDKA;2 and CDKB;1 activity mediated by its interaction with CDKF;3. The potential yield of the rice varieties producing compact and heavy panicles bearing numerous spikelets is compromised because a high percentage of spikelets remain poorly filled, reportedly because of a high expression of KRPs that causes suppression of endosperm cell proliferation. To test the stated negative relationship between KRP expression and grain filling, Orysa;KRP4 was overexpressed under the control of seed-specific glutelin promoter in IR-64 rice variety that shows good grain filling. The transgenic lines showed more than 15-fold increase in expression of KRP4 in the spikelets concomitant with nearly 50% reduction in grain filling compared with the wild type without producing any significant changes on the other yield-related parameters like panicle length and the spikelets numbers that were respectively 30.23 ± 0.89 cm and 229.25 ± 33.72 per panicle in the wild type, suggesting a highly organ-targeted effect of the genetic transformation. Yeast two-hybrid test revealed CDKF;3 as the interacting partner of KRP4, and CDKF;3 was found to interact with CDKA;2, CDKB;1 and CDKD;1. Significant decrease in grain filling in the transgenic lines compared with the wild type due to overexpression of KRP4 could be because of suppression of the activity of CDKB;1 and CDKA;2 by inhibition of their phosphorylation directly by CDKF;3, or mediated through inhibition of phosphorylation of CDKD;1 by CDKF;3. The study thus indicated that suppression of expression of KRP(s) by genetic manipulation of their promoters could be an important way of improving the yield of the rice varieties bearing compact and heavy panicles.

VQ1 and VQ10 are largely unstructured homologous proteins with a significant potential for protein-protein interactions. Yeast two-hybrid (Y2H) analysis confirmed that both proteins interact not only with themselves and each other but also with other VQ and WRKY proteins. Screening an Arabidopsis Y2H library with VQ1 as bait identified 287 interacting proteins. Validation of the screening confirmed that interactions with VQ1 also occurred with VQ10, supporting their functional homology. Although VQ1 or VQ10 proteins do not localize in plastids, 47 VQ1-targets were found to be plastidial proteins. In planta interaction with the isoprenoid biosynthetic enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) was confirmed by co-immunoprecipitation. DXS oligomerizes through redox-regulated intermolecular disulfide bond formation, and the interaction with VQ1 or VQ10 do not involve their unique C residues. The VQ-DXS protein interaction did not alter plastid DXS localization or its oligomerization state. Although plants with enhanced or reduced VQ1 and VQ10 expression did not exhibit significantly altered levels of isoprenoids compared to wild-type plants, they did display significantly improved or diminished photosynthesis efficiency, respectively.

The localization of the meiotic specific regulatory molecule Moa1 to the centromere is regulated by the kinetochore protein CENP-C, and participates in the cohesion of sister chromatids in the centromere region mediated by the cohesin Rec8. To examine the interaction of these proteins, we analyzed the interactions between Moa1 and Rec8, CENP-C by yeast two-hybrid assays and identified several amino acid residues in Moa1 required for the interaction with CENP-C and Rec8. The results revealed that the interaction between Moa1 and CENP-C is crucial for the Moa1 to participate in the regulation of monopolar attachment of sister kinetochores. However, mutation at S143 and T150 of Moa1, which are required for interaction with Rec8 in the two-hybrid assay, did not show significant defects. Mutations in amino acid residues may not be sufficient to interfere with the interaction between Moa1 and Rec8 in vivo. Further research is needed to determine the interaction domain between Moa1 and Rec8. This study revealed specific amino acid sites at which Moa1 affects the meiotic homologous chromosome segregation, providing a deeper understanding of the mechanism of meiotic chromosome segregation.
减数分裂特异性调控分子Moa1定位到着丝粒受到动粒蛋白CENP-C的调控，同时Moa1参与黏连蛋白Rec8介导的着丝粒区域姐妹染色单体的黏连。为了研究这些蛋白质之间的相互作用，本研究利用酵母双杂交实验(yeast two-hybrid assay)测定分析了Moa1和CENP-C、Rec8之间的相互作用，并通过在Moa1中定点突变鉴定了与CENP-C和Rec8相互作用所需的一些氨基酸残基。实验结果表明，Moa1和CENP-C的相互作用对于Moa1参与调节姐妹动粒的单极附着很重要。然而，双杂交实验中与Rec8相互作用所需的Moa1的S143和T150突变没有显示出Moa1或Rec8功能的显著缺陷。这表明氨基酸残基的突变可能不足以干扰体内Moa1和Rec8之间的相互作用，需要进一步的研究来确定Moa1和Rec8的相互作用域。本研究揭示了影响减数分裂同源染色体分离的Moa1氨基酸位点，为减数分裂的染色体分离机制提供更深入的理解。.

The regulation of fruit development is a complex process and a core issue in the fruit tree industry. To investigate the role of PbGIF1 in pear fruit development, we identified a transcription factor PbbHLH137 that regulates pear (Pyrus bretschneideri) fruit development by screening a yeast library constructed from fruit cDNA. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and split luciferase complementation (split-LUC) assays were performed to confirm the PbbHLH137-PbGIF1 interaction. By tracing the complete fruit development process, we found that PbbHLH137 expression was closely related to fruit size and highly involved at the late pear fruit development stage. Transgenic experiments showed that heterologous expression of PbbHLH137 or PbGIF1 promoted fruit enlargement. PbbHLH137 promoted mainly the expansion of fruit cell volume, whereas PbGIF1 mainly increased the number of cells. Further LUC experiments demonstrated that PbGIF1 promoted the transcriptional activation ability of PbbHLH137. Our work identified PbbHLH137 as a transcription factor that regulates fruit development, and showed that PbGIF1 played an ongoing role during fruit development, making it a candidate gene for genetic improvement of pear fruit development.

Anthocyanins are major flavonoid compounds with established health benefits. Although the molecular mechanisms of MYB transcription factors (TFs) within the MYB-basic helix-loop-helix (bHLH)-WD-repeat protein (MBW) complex in anthocyanin biosynthesis have been revealed, the functions of other MYB TFs that are unable to form the MBW complex in this process remain unclear. In this study, we uncovered and extensively characterized an R2R3-MYB TF in onion (Allium cepa L.), named AcMYB96, which was identified as a potential anthocyanin activator. AcMYB96 was classified into subgroup 1 of the R2R3-MYB TF family and lacked the conserved sequences required for interactions with bHLH IIIf TFs. Consistently, yeast two-hybrid assays showed that AcMYB96 did not interact with any bHLH IIIf TFs examined, including AcB2 and AtTT8. The transcription pattern of AcMYB96 correlated with the level of anthocyanin accumulation, and its role in activating anthocyanin biosynthesis was confirmed through overexpression in the epithelial cells of onion bulbs and Arabidopsis. Yeast one-hybrid, electrophoretic mobility shift, and promoter transactivation assays further demonstrated that AcMYB96 promoted anthocyanin biosynthesis by binding to the promoters of the chalcone synthase (AcCHS1), anthocyanidin synthase (AcANS), and UDP-glucose-flavonoid 3-O-glucosyltransferase (AcUFGT) genes, thereby activating their expression independent of bHLH IIIf TFs. These results demonstrate that AcMYB96 activates anthocyanin biosynthesis without forming the MBW complex, providing a theoretical foundation to further enrich the gene resources for promoting anthocyanin accumulation and breeding red onions with high anthocyanin content.

Toxoplasma gondii divides by endodyogeny, in which two daughter buds are formed within the cytoplasm of the maternal cell using the inner membrane complex (IMC) as a scaffold. During endodyogeny, components of the IMC are synthesized and added sequentially to the nascent daughter buds in a tightly regulated manner. We previously showed that the early recruiting proteins IMC32 and IMC43 form an essential daughter bud assembly complex which lays the foundation of the daughter cell scaffold in T. gondii. In this study, we identify the essential, early recruiting IMC protein BCC0 as a third member of this complex by using IMC32 as bait in both proximity labeling and yeast two-hybrid screens. We demonstrate that BCC0\'s localization to daughter buds depends on the presence of both IMC32 and IMC43. Deletion analyses and functional complementation studies reveal that residues 701-877 of BCC0 are essential for both its localization and function and that residues 1-899 are sufficient for function despite minor mislocalization. Pairwise yeast two-hybrid assays additionally demonstrate that BCC0\'s essential domain binds to the coiled-coil region of IMC32 and that BCC0 and IMC43 do not directly interact. This data supports a model for complex assembly in which an IMC32-BCC0 subcomplex initially recruits to nascent buds via palmitoylation of IMC32 and is locked into the scaffold once bud elongation begins by IMC32 binding to IMC43. Together, this study dissects the organization and function of a complex of three early recruiting daughter proteins which are essential for the proper assembly of the IMC during endodyogeny.

Rexinoids are compounds that bind to the rexinoid X receptor (RXR) to modulate gene expression and have been proposed as a new class of therapeutics to treat Alzheimer\'s disease. Different rexinoids will initiate downstream effects that can be quite marked even though such compounds can be structurally similar and have comparable RXR binding affinities. RXR can both homo- and heterodimerize, and these protein-protein interactions and subsequent transactivating potential lead to differential gene expression, depending on the RXR dimeric partner, additional cofactors recruited, and downstream transcription factors that are up- or downregulated. Expression analysis was performed in the U87 human glioblastoma cell line treated with a panel of rexinoids, and our analysis demonstrated that rexinoids with similar RXR EC50 values can have pronounced differences in differential gene expression. Rexinoid binding likely leads to distinctive RXR conformations that cause major downstream gene expression alterations via modulation of RXR interacting proteins. Yeast two-hybrid analysis of RXR bait with two RXR interacting partners demonstrates that rexinoids drive differential binding of RXR to distinctive protein partners. Physiochemical analysis of the rexinoids reveals that the molecules cluster similarly to their gene expression patterns. Thus, rexinoids with similar RXR binding affinities drive differential gene expression by stimulating additional binding patterns in RXR and its homo- and heteropartners, driven by the physicochemical characteristics of these molecules.

bZIP transcription factors can function as homodimers or heterodimers through interactions with their disordered coiled-coil domain. Such dimer assemblies are known to influence DNA-binding specificity and/or the recruitment of binding partners, which can cause a functional switch of a transcription factor from being an activator to a repressor. We recently identified the genomic targets of a bZIP transcription factor called CREB3L1 in rat hypothalamic supraoptic nucleus by ChIP-seq. The objective of this study was to investigate the CREB3L1 protein-to-protein interactome of which little is known. For this approach, we created and screened a rat supraoptic nucleus yeast two-hybrid prey library with the bZIP region of rat CREB3L1 as the bait. Our yeast two-hybrid approach captured five putative CREB3L1 interacting prey proteins in the supraoptic nucleus. One interactor was selected by bioinformatic analyses for more detailed investigation by co-immunoprecipitation, immunofluorescent cellular localisation, and reporter assays in vitro. Here we identify dimerisation hub protein Dynein Light Chain LC8-Type 1 as a CREB3L1 interacting protein that in vitro enhances CREB3L1 activation of target genes.

Alzheimer\'s disease is currently not curable. Almost all attempts to identify disease-modifying drugs failed and the causes of disease etiology are not well understood. Neurofibrillary tangles composed of pathological tau protein belong to the main hallmarks of this disease. Identification of novel physiological and pathological tau interacting proteins may lead to a better understanding of Alzheimer\'s disease pathology and tau physiology and therefore we performed a screening of the brain library by a yeast two-hybrid system intending to identify new tau interaction partners. We identified CHORDC1 (cysteine and histidine-rich domain-containing protein 1) as a novel tau interaction partner by this approach. The CHORDC1-tau interaction was validated by co-immunoprecipitation from rat brain tissues and by in vitro co-localization in the cellular model expressing full-length human tau protein. We believe that our results can be useful for researchers studying tau protein in health and disease.