Two-hybrid Research Articles

Estrogenic analysis of ten Thai medicinal plants used for treating menstrual irregularity by ERα and ERβ yeast two-hybrid systems

IntroductionSeveral traditional Thai medicinal plants have historically been utilised to alleviate menstrual irregularities. While it is hypothesised that phytoestrogens are pivotal in their medicinal efficacy, more empirical evidence is needed to verify this assumption. Furthermore, our understanding of the estrogenic effects mediated by estrogen receptors (ERα and ERβ) and their alterations post-intestinal enzymatic digestion is limited. This study aims to assess the estrogenic activity of 10 medicinal plants before and after in vitro simulated gastrointestinal digestion using yeast two-hybrid (Y2H) systems. MethodsY2H systems were constructed based on the interaction between the ligand-binding domain of estrogen receptors and the receptor interaction domain of coactivators. The medicinal plants were extracted in water at 95 °C for 1 hour or in 70% ethanol for 24 hours (1: 8 w/v). Their estrogenic activities before and after in vitro gastrointestinal digestion were determined using Y2H systems. ResultsThe majority of ethanol extracts exhibited higher estrogenic activity compared to water extracts, with detection notably facilitated by the ERβ-Y2H system. This study represents the first documentation of the estrogenic activities of Dendrolobium lanceolatum and Morinda coreia, both prominently identified by the ERβ-Y2H system. Specifically, the D. lanceolatum extract demonstrated the highest estrogenic activity, showing a significant enhancement following gastrointestinal enzymatic digestion, suggesting the presence of active metabolites. ConclusionsThese findings advance our understanding of the estrogenic properties of medicinal plants for treating menstrual irregularities, clarifying their effects on estrogen receptor subtypes and post-hydrolysis activities. Such insights are vital for determining their potential as alternative medicines.

StSnRK1.1 protein kinase positively regulates tuber dormancy release of potato

SnRK1 is a plant-specific serine/threonine protein kinase, that plays a vital role in maintaining the energy balance, nutrient distribution, growth, and development of organisms for survival. The period of tuber dormancy seriously affects potato production. To elucidate the effect of the StSnRK1.1 gene on dormancy release, the gene was cloned from the potato cultivar 'Desiree', and the overexpression and interfering expression lines were obtained through genetic transformation. StSnRK1.1 promoted early dormancy release and sprouting of potato tubers in the overexpression line, while it delayed tuber sprouting in the StSnRK1.1-interfering expression line. In addition, it was discovered that abscisic acid (ABA) in the StSnRK1.1 overexpression line was significantly reduced compared with the wild type plants, while the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased. Conversely, the opposite result was found in the StSnRK1.1 gene interference expression line, which indicated that the dormancy release was accelerated, the reactive oxygen species metabolism efficiency was high, and the reactive oxygen species could be quickly removed and the accumulation of reactive oxygen species could be avoided during the dormancy release process. The positive regulatory genes StSnRK2.2 and StABI5 of ABA signal transduction were lower than those of wild type in the overexpression line and higher than that of the wild type in the interfering expression line, while the negative regulatory genes StPP2CA1 and StPP2CA3 of ABA signal transduction were higher than those of the wild type in the overexpression line and lower than that of the wild type in the interfering expression line. In summary, StSnRK1.1 affects ABA signaling by mediating the expression of StSnRK2.2, StABI5, StPP2CA1 and StPP2CA3, which provides a theoretical basis for the molecular mechanism of StSnRK1.1 to positively regulate the release of potato tuber dormancy. Through yeast two-hybrid (YH2) screening, two interacting proteins, StCAT3 and StFBA1, were identified. The interaction between StCAT3, StFBA1 and StSnRK1.1 was confirmed through bimolecular fluorescence complementation. These results provide novel information for further study on the molecular mechanism of StSnRK1.1 regulating dormancy and germination of potato tubers.

BCC0 collaborates with IMC32 and IMC43 to form the Toxoplasma gondii essential daughter bud assembly complex.

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 Induce Differential Gene Expression in Human Glioblastoma Cells and Protein-Protein Interactions in a Yeast Two-Hybrid System.

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.

GGA1 interacts with the endosomal Na+/H+ exchanger NHE6 governing localization to the endosome compartment

Mutations in the endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome, an X-linked neurological disorder. NHE6 functions in regulation of endosome acidification and maturation in neurons. Using yeast two-hybrid screening with the NHE6 carboxyl terminus as bait, we identify Golgi-associated, gamma adaptin ear-containing, ADP-ribosylation factor (ARF) binding protein 1 (GGA1) as an interacting partner for NHE6. We corroborated the NHE6-GGA1 interaction using: coimmunoprecipitation; overexpressed constructs in mammalian cells; and coimmunoprecipitation of endogenously expressed GGA1 and NHE6 from neuroblastoma cells, as well as from the mouse brain. We demonstrate that GGA1 interacts with organellar NHEs (NHE6, NHE7, and NHE9) and that there is significantly less interaction with cell-surface localized NHEs (NHE1 and NHE5). By constructing hybrid NHE1/NHE6 exchangers, we demonstrate the cytoplasmic tail of NHE6 interacts most strongly with GGA1. We demonstrate the colocalization of NHE6 and GGA1 in cultured, primary hippocampal neurons, using super-resolution microscopy. We test the hypothesis that the interaction of NHE6 and GGA1 functions in the localization of NHE6 to the endosome compartment. Using subcellular fractionation experiments, we show that NHE6 is mislocalized in GGA1 KO cells, wherein we find less NHE6 in endosomes, but more NHE6 transport to lysosomes, and more Golgi retention of NHE6, with increased exocytosis to the surface plasma membrane. Consistent with NHE6 mislocalization, and Golgi retention, we find the intraluminal pH in Golgi to be alkalinized in GGA1-null cells. Our study demonstrates a new interaction between NHE6 and GGA1 which functions in the localization of this intracellular NHE to the endosome compartment.

Screening (ant)agonistic activities of xenobiotics on the retinoic acid receptor alpha (RARα) using in vitro and in silico analysis

Retinoic acid receptors (RARs) are known as crucial endocrine receptors that could mediate a broad diversity of biological processes. However, the data on endocrine disrupting effects of emerging chemicals by targeting RAR (ant)agonism are far from sufficient. Herein, we investigated the RARα agonistic or antagonistic activities for 75 emerging chemicals of concern, and explored their interactions with this receptor. A recombinant two-hybrid yeast assay was used to examine the RARα activities of the test chemicals, wherein 7 showed effects of RARα agonism and 54 exerted potentials of RARα antagonism. The representative chemicals with RARα agonistic activities, i.e. 4-hydroxylphenol (4-HP) and bisphenol AF (BPAF), significantly increased the mRNA levels of CRABP2 and CYP26A1, while 4 select chemicals with RARα antagonistic potentials, including bisphenol A (BPA), tetrabromobisphenol A (TBBPA), 4-tert-octylphenol (4-t-OP), and 4-n-nonylphenol (4-n-NP), conversely decreased the transcriptional levels of the test genes. The in silico molecular docking analysis using 3 different approaches further confirmed the substantial binding between the chemicals with RARα activities and this nuclear receptor protein. This work highlights the promising strategy for screening endocrine-disrupting effects of emerging chemicals of concern by targeting RARα (ant)agonism.

Two Calcium Sensor-Activated Kinases Function in Root Hair Growth.

Plant pollen tubes and root hairs typical polarized tip growth. It is well established that calcium ions (Ca2+) play essential roles in maintaining cell polarity and guiding cell growth orientation. Ca2+ signals are encoded by Ca2+ channels and transporters and are decoded by a variety of Ca2+-binding proteins often called Ca2+ sensors, in which calcineurin B-like protein (CBL) proteins function by interacting with and activating a group of kinases, activate CBL-interacting protein kinases (CIPKs). Some CBL-CIPK complexes, such as CBL2/3-CIPK12/19, act as crucial regulators of pollen tube growth. Whether these calcium decoding components regulate the growth of root hairs, another type of plant cell featuring Ca2+-regulated polarized growth, remains unknown. In this study, we identified CIPK13 and CIPK18 as genes specifically expressed in Arabidopsis (Arabidopsis thaliana) root hairs. The cipk13 cipk18 double mutants showed reduced root hair length and lower growth rates. The calcium oscillations at the root hair tip were attenuated in the cipk13 cipk18 mutants as compared to the wild-type plants. Through yeast two-hybrid screens, CBL2 and CBL3 were identified as interacting with CIPK13 and CIPK18. cbl2 cbl3 displayed a shortened root hair phenotype similar to cipk13 cipk18. This genetic analysis, together with biochemical assays showing activation of CIPK13/18 by CBL2/3, supported the conclusion that CBL2/3 and CIPK13/18 may work as Ca2+-decoding modules in controlling root hair growth. Thus, the findings that CIPK12/19 and CIPK13/18 function in pollen tube and root hair growth, respectively, illustrate a molecular mechanism in which the same CBLs recruit distinct CIPKs in regulating polarized tip growth in different types of plant cells.

The AaBBX21-AaHY5 module mediates light-regulated artemisinin biosynthesis in Artemisia annua L.

The sesquiterpene lactone artemisinin is an important anti-malarial component produced by the glandular secretory trichomes of sweet wormwood (Artemisia annua L.). Light was previously shown to promote artemisinin production, but the underlying regulatory mechanism remains elusive. In this study, we demonstrate that ELONGATED HYPOCOTYL 5 (HY5), a central transcription factor in the light signaling pathway, cannot promote artemisinin biosynthesis on its own, as the binding of AaHY5 to the promoters of artemisinin biosynthetic genes failed to activate their transcription. Transcriptome analysis and yeast two-hybrid screening revealed the B-box transcription factor AaBBX21 as a potential interactor with AaHY5. AaBBX21 showed a trichome-specific expression pattern. Additionally, the AaBBX21-AaHY5 complex cooperatively activated transcription from the promoters of the downstream genes AaGSW1, AaMYB108, and AaORA, encoding positive regulators of artemisinin biosynthesis. Moreover, AaHY5 and AaBBX21 physically interacted with the A. annua E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). In the dark, AaCOP1 decreased the accumulation of AaHY5 and AaBBX21 and repressed the activation of genes downstream of the AaHY5-AaBBX21 complex, explaining the enhanced production of artemisinin upon light exposure. Our study provides insights into the central regulatory mechanism by which light governs terpenoid biosynthesis in the plant kingdom.

Identification of Apple Flower Development-Related Gene Families and Analysis of Transcriptional Regulation.

Apple (Malus domestica Borkh.) stands out as a globally significant fruit tree with considerable economic importance. Nonetheless, the orchard production of 'Fuji' apples faces significant challenges, including delayed flowering in young trees and inconsistent annual yields in mature trees, ultimately resulting in suboptimal fruit yield due to insufficient flower bud formation. Flower development represents a pivotal process influencing plant adaptation to environmental conditions and is a crucial determinant of successful plant reproduction. The three gene or transcription factor (TF) families, C2H2, DELLA, and FKF1, have emerged as key regulators in plant flowering regulation; however, understanding their roles during apple flowering remains limited. Consequently, this study identified 24 MdC2H2, 6 MdDELLA, and 6 MdFKF1 genes in the apple genome with high confidence. Through phylogenetic analyses, the genes within each family were categorized into three distinct subgroups, with all facets of protein physicochemical properties and conserved motifs contingent upon subgroup classification. Repetitive events between these three gene families within the apple genome were elucidated via collinearity analysis. qRT-PCR analysis was conducted and revealed significant expression differences among MdC2H2-18, MdDELLA1, and MdFKF1-4 during apple bud development. Furthermore, yeast two-hybrid analysis unveiled an interaction between MdC2H2-18 and MdDELLA1. The genome-wide identification of the C2H2, DELLA, and FKF1 gene families in apples has shed light on the molecular mechanisms underlying apple flower bud development.

HY5 and COP1 function antagonistically in the regulation of nicotine biosynthesis in Nicotiana tabacum

Nicotine constitutes approximately 90% of the total alkaloid content in leaves within the Nicotiana species, rendering it the most prevalent alkaloid. While the majority of genes responsible for nicotine biosynthesis express in root tissue, the influence of light on this process through shoot-to-root mobile ELONGATED HYPOCOTYL 5 (HY5) has been recognized. CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), a key regulator of light-associated responses, known for its role in modulating HY5 accumulation, remains largely unexplored in its relationship to light-dependent nicotine accumulation. Here, we identified NtCOP1, a COP1 homolog in Nicotiana tabacum, and demonstrated its ability to complement the cop1-4 mutant in Arabidopsis thaliana at molecular, morphological, and biochemical levels. Through the development of NtCOP1 overexpression (NtCOP1OX) plants, we observed a significant reduction in nicotine and flavonol content, inversely correlated with the down-regulation of nicotine and phenylpropanoid pathway. Conversely, CRISPR/Cas9-based knockout mutant plants (NtCOP1CR) exhibited an increase in nicotine levels. Further investigations, including yeast-two hybrid assays, grafting experiments, and Western blot analyses, revealed that NtCOP1 modulates nicotine biosynthesis by targeting NtHY5, thereby impeding its transport from shoot-to-root. We conclude that the interplay between HY5 and COP1 functions antagonistically in the light-dependent regulation of nicotine biosynthesis in tobacco.

The first report of a C-type lectin contains a CLIP domain involved in antibacterial defense in Macrobrachium nipponense

We identified a novel C-type lectin (CTL) from Macrobrachium nipponense, designated as Mn-clip-Lec. It consists of 1315 bp with an open reading frame of 1098 bp, encoding a polypeptide of 365 amino acids. Mn-clip-Lec contains 6 exons and 5 introns. Mn-clip-Lec possessed a CLIP domain at the N-terminal and two carbohydrate recognition domains at the C-terminal. Interaction between Mn-clip-Lec and MnLec was found by Yeast two-hybrid analysis. The expressions of Mn-clip-Lec, MnLec, prophenoloxidase (proPO)-activating system-associated genes (MnPPAF, MnPPAE, and MnPO), and antimicrobial peptides (AMPs) (MnALF and MnCRU) were up-regulated after the challenge with Staphylococcus aureus. RNA interference (RNAi)-mediated suppression of the Mn-clip-Lec and MnLec genes in S. aureus-challenged prawns reduced the transcripts of MnPPAF, MnPPAE, MnPO, MnALF and MnCRU. Knockdown of Mn-clip-Lec and MnLec resulted in decrease in PO activity in M. nipponense infected with S. aureus. The recombinant Mn-clip-Lec (rMn-clip-Lec) protein bound all tested bacteria and agglutinated S. aureus. A sugar-binding assay revealed that rMn-clip-Lec could bind to LPS or PGN. rMn-clip-Lec accelerated the clearance of S. aureus in vivo. Our findings suggest that Mn-clip-Lec and its interacting MnLec play important roles in the induction of the proPO system and AMPs expression in M. nipponense during bacterial infection.

Expression Analysis and Interaction Protein Screening of CoGI, the Key Factor in Photoperiod Regulation of Flowering in Camellia oleifera Abel

Photoperiod is a pivotal regulatory factor in the flowering of Camellia oleifera Abel. (C. oleifera). GIGANTEA (GI) serves as a pivotal regulator, not only orchestrating the intricate circadian rhythm but also governing photoperiod-dependent flowering. In order to explore the function of GI in C. oleifera (CoGI), we obtained a CoGI gene-coding sequence and analyzed a CoGI protein sequence using bioinformatics. Furthermore, we conducted a spatiotemporal expression analysis of CoGI. And a yeast two-hybridization assay was used to screen the interacting proteins of CoGI. Evolutionary analysis revealed high conservation of the CoGI protein, which clustered with the GI protein from Camellia sinensis (CsGI) on a common evolutionary branch. The expression of CoGI was different in each part, and a tissue expression analysis revealed that the relative expression level of the CoGI gene is highest in the leaves of C. oleifera, while it is at its lowest in the seed coats. Transgenic Arabidopsis thaliana (Arabidopsis) overexpressing CoGI exhibited early flowering under long-day conditions. In addition, the yeast two-hybrid library screening revealed interactions between seven C. oleifera proteins and CoGI: CoACR9, CoLAO, CoDExH12-like, CoIT1K-like, CoUPF0481, CoIDM3, and CoAt4g27190-like. The findings demonstrated that CoGI is crucial to C. oleifera’s flowering.

MaHOS15 negatively regulates plant immune defense induced by banana fusarium wilt via salicylic acid pathway

Fusarium wilt of banana (Musa acuminata) poses a significant threat to the banana industry globally. Salicylic acid (SA) serves as a crucial signaling molecule activating the plant's defense response against pathogen infections while also regulating various aspects of plant growth and development. High Expression of Osmotically Responsive Genes 15 (HOS15) acts as a transcriptional corepressor and an E3 ubiquitin ligase complex substrate receptor, playing an important role in regulating biological stress processes. However, the exact immune mechanism of MaHOS15 in the SA signaling pathway remains unclear. In this study, we utilized RT-qPCR, yeast two-hybridization, subcellular localization and allogeneic expression of Arabidopsis to explore MaHOS15's function against banana fusarium wilt. The expression changes of SA pathway-related factors MaNPR3, MaNPR4, MaNPR11, MaTGA8 and MaHOS15 in Foc TR4-resistant and Foc TR4-susceptible bananas infected with Foc TR4 were compared and analyzed. The results showed that the MaHOS15 expression trend in the two cultivars was opposite, while the expression trend of MaNPR4 and MaNPR11 was contrary to that of MaHOS15. Moreover, heterologous overexpression of MaHOS15 in Arabidopsis reduced the variance in SA-related signaling factors. The expression of MaHOS15 increased after defense activation, inhibiting the expression of SA-related immune factors, the immunoregulation of MaHOS15 and SA exhibited contrasting results. Collectively, these findings suggest that MaHOS15 negatively regulates plant immune defenses against banana Fusarium wilt via the salicylic acid pathway, offering potential applications in improving plant resistance to banana Fusarium wilt.

BcWRKY1 confers Botrytis cinerea susceptibility via inhibiting JA biosynthesis.

WRKYs play important roles in plant stress resistance. However, the role of WRKYs in non-heading Chinese cabbage (Brassica campestris ssp. chinensis) against Botrytis cinerea (B. cinerea) remains poorly understood. Herein, the expression of BcWRKY1 was induced by B. cinerea. Further, the role of BcWRKY1 in B. cinerea infection was identified. Silencing of BcWRKY1 in non-heading Chinese cabbage enhanced plant resistance to B. cinerea. After B. cinerea inoculation, BcWRKY1-silencing plants exhibited lower reactive oxygen species (ROS) content, higher jasmonic acid (JA) content, and the expression level of JA biosynthesis genes, BcOPR3, BcLOX3-1 and BcLOX3-2 were upregulated. Overexpression of BcWRKY1 in Arabidopsis exhibited a complementary phenotype. By directly targeting W-boxes in the promoter of BcLOX3-2, BcWRKY1 inhibited the transcription of this gene. In addition, 13 candidate interacting proteins of BcWRKY1 were identified by yeast two-hybrid (Y2H) screening, and the interaction between BcWRKY1 and BcCaM6 weakened the inhibition of BcLOX3-2. In summary, our findings suggest that BcWRKY1 interacts with BcCaM6 to negatively regulate disease resistance.

Genome-wide identification of Rosa roxburghii CML family genes identifies an RrCML13-RrGGP2 interaction involved in calcium-mediated regulation of ascorbate biosynthesis

Calmodulin-like proteins (CMLs) are an essential family of calcium sensors involved in multiple Ca2+-mediated cellular processes in plants. Rosa roxburghii Tratt, known for the abundance of L-ascorbic acid (AsA) in its fruits, is widely distributed in calcium-rich soil of the karst region in southwestern China. The aim of this study was to identify key CMLs that respond to exogenous Ca2+ levels and regulate AsA biosynthesis in R. roxburghii. A genome-wide scan revealed the presence of 41 RrCML genes with 1–4 EF-hand motif (s) unevenly distributed across the 7 chromosomes of R. roxburghii. qRT-PCR analysis revealed that RrCML13, RrCML10, and RrCML36 responded significantly to exogenous Ca2+ treatment, and RrCML13 was positively correlated with GDP-L-galactose phosphorylase encoding gene (RrGGP2) expression and AsA content in the developing fruit. Overexpression of RrCML13 in fruits and roots significantly promoted the transcription of RrGGP2 and the accumulation of AsA, while virus-induced silencing of RrCML13 reduced the transcription of RrGGP2 and the content of AsA. Furthermore, Moreover, the yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analysis confirmed the interaction between RrCML13 and RrGGP2 proteins, indicating that RrCML13 plays a regulatory role in calcium-mediated AsA biosynthesis. This study enhances our understanding of R. roxburghii CMLs and sheds light on the calcium-mediated regulation of AsA biosynthesis.

GmHXK2 promotes the salt tolerance of soybean seedlings by mediating AsA synthesis, and auxin synthesis and distribution

BackgroundSalt is an important factor that affects crop productivity. Plant hexokinases (HXKs) are key enzymes in the glycolytic pathway and sugar signaling transduction pathways of plants. In previous studies, we identified and confirmed the roles of GmHXK2 in salt tolerance.ResultsIn this study, we analyzed the tissue-specific expression of GmHXK2 at different growth stages throughout the plant’s life cycle. The results showed that GmHXK2 was expressed significantly in all tissues at vegetative stages, including germination and seedling. However, no expression was detected in the pods, and there was little expression in flowers during the later mature period. Arabidopsis plants overexpressing the GmHXK2 (OE) had more lateral roots. The OE seedlings also produced higher levels of auxin and ascorbic acid (AsA). Additionally, the expression levels of genes PMM, YUC4/YUC6/YUC8, and PIN/LAX1,LAX3, which are involved respectively in the synthesis of AsA and auxin, as well as polar auxin transport, were upregulated in OE plants. This upregulation occurred specifically under exogenous glucose treatment. AtHKT1, AtSOS1, and AtNHX1 were up-regulated in OE plants under salt stress, suggesting that GmHXK2 may modulate salt tolerance by maintaining ion balance within the cells and alleviating damage caused by salt stress. Additionally, we further confirmed the interaction between GmHXK2 and the protein GmPMM through yeast two-hybridization and bimolecular fluorescence complementation assays, respectively.ConclusionThe expression of GmHXK2 gene in plants is organ-specific and developmental stage specific. GmHXK2 not only regulates the synthesis of AsA and the synthesis and distribution of auxin, but also promotes root elongation and induces lateral root formation, potentially enhancing soil water absorption. This study reveals the crosstalk between sugar signaling and hormone signaling in plants, where GmHXK2 acts as a glucose sensor through its interaction with GmPMM, and sheds light on the molecular mechanism by which GmHXK2 gene is involved in salt tolerance in plants.

Decapod iridescent virus 1 (DIV1) 168L can target cuticle protein 8 from Litopenaeus vannamei

Decapod iridescent virus 1 (DIV1) stands as a significant pathogen affecting crustaceans, posing a grave threat to the shrimp industries in aquaculture dependent nations. Within the Iridoviridae family, the conserved envelope protein DIV1-168L plays a pivotal role in virion entry. Nonetheless, the host factors that interact with 168L remain unidentified. To address this gap, we established a cDNA library derived from Litopenaeus vannamei gill tissue and conducted yeast two-hybrid screening to identify host factors that interact with 168L. Additionally, we performed co-immunoprecipitation assays to verify the interaction between cuticle protein 8 (CP8) and 168L. Expression pattern analysis revealed the presence of CP8 transcripts in the gill and epidermis. Furthermore, immunohistochemistry results demonstrated the expression of CP8 in gill cells and its localization in the gill filament epithelium. Fluorescence analysis indicated that full-length CP8 colocalized with 168L in the cytoplasm of Sf9 cells. Removal of the signal peptide from the N-terminal of CP8 eliminated its concentration in the cytoplasm. Additionally, CP8 expression was significantly inhibited during DIV1 infection. Therefore, our research contributes to a better understanding of the entry mechanism of iridovirids.The GenBank accession number for the DIV1 sequence is MF197913.1.

Lectin Receptor-Like Protein Kinase OsNRFG6 is Required for Embryo Sac Development and Fertilization in Neo-Tetraploid Rice

Great yield-enhancing prospects of autotetraploid rice was restricted by various polyploidy-induced reproductive dysfunction. To surmount these challenges, our group has generated a series of valuable fertile tetraploid lines (denoted as neo-tetraploid rice) through 20-year efforts. With this context, a G-type lectin receptor-like kinase, OsNRFG6, was identified as a pivotal factor associated with reproductive regulation in neo-tetraploid rice. Nevertheless, it is still elusive about a comprehensive understanding of its precise functional roles and underlying molecular mechanisms during reproduction of neo-tetraploid rice. Here, we demonstrated that OsNRFG6 executed a constitutive expression pattern and encoded proteins localizing in perinucleus and endoplasmic reticulum. Subsequently, four independent mutant lines of OsNRFG6 within neo-tetraploid rice background were further identified, all displaying low seed-setting rate due to abortive embryo sacs and defective double fertilization. RNA-seq and RT-qPCR revealed a significant down-regulation of OsNRFG6 and female reproductive genes such as OsMEL1 and LOG in ovaries prior to and post-fertilization, attributing this effect to OsNRFG6 mutation. Furthermore, through yeast-two hybrids, bimolecular fluorescence complementation assays, and luciferase complementation imaging assays, it was determined that OsNRFG6 could interact with itself and two female reproductive proteins (LOG and OsDES1) to form protein complexes. These results elucidate the reproductive functions and molecular pathway governed by OsNRFG6 in regulating fertility of neo-tetraploid rice, offering insights into molecular understanding of fertility improvement in polyploid rice.

CsERECTA alternative splicing regulates the flowering numbers depending on temperature in Crocus sativus L.

Saffron, utilized for its medicinal properties in its pistils, can significantly boost yield by increasing the flowering numbers. Several strategies have been implemented to enhance saffron yield, but they may not guarantee the consistent inheritance of desirable traits. However, identifying and manipulating the genes responsible for flower production through molecular breeding offers considerable potential for innovating saffron germplasm resources. Here, a comparative transcriptomic analysis of single-flowered and multi-flowered apical buds was performed to mine the key genes. CsERECTA gene (Crocus sativus L. ERECTA, CsER), which has two spliceosomes in saffron: CsER long transcript (CsERlt) and CsER short transcript (CsERst), was identified as a putative regulator of flower production. To elucidate the effect of CsER alternative splicing on the flowering numbers, a series of molecular biology and genetics experiments were conducted. We found the expression of CsERlt decreased by 1.5-fold in multi-flowered apical buds compared to single-flowered apical buds at 22 ℃, while CsERst increased by 6.47-fold in the non-flowering apical buds at 9 ℃. Overexpression of CsERlt and CsERst in Arabidopsis thaliana revealed that CsERlt suppresses flowering numbers and the application of small-molecule inhibitors targeting CsERlt in flowering-competent corms led to a notable enhancement in the flowering numbers, increasing by up to 139.25 %. qRT-PCR and immunohistochemistry assays revealed a widespread expression pattern of CsERlt in various plant structures. The subcellular localization demonstrated that CsERlt predominantly localized at the cell membrane, while CsERst exhibited both at the membrane and cytoplasm. The transcriptomic data and yeast two-hybrid library screening indicated CsER alternative splicing may regulate flowering numbers through the mitogen-activated protein kinase (MAPK) signaling pathway. Taken together, our findings identify a CsER gene associated with flowering numbers in saffron, and its function in modulating flowering numbers is temperature-dependent, and the MAPK pathway may be involved in the regulation of flowering numbers in saffron.

Identification of nuclear membrane SUN proteins and components associated with wheat fungal stress responses

In eukaryotes, the nuclear membrane that encapsulates genomic DNA is composed of an inner nuclear membrane (INM), an outer nuclear membrane (ONM), and a perinuclear space. SUN proteins located in the INM and KASH proteins in the ONM form the SUN-KASH NM-bridge, which functions as the junction of the nucleocytoplasmic complex junction. Proteins containing the SUN domain showed the highest correlation with differentially accumulated proteins (DAPs) in the wheat response to fungal stress. To understand the characteristics of SUN and its associated proteins in wheat responding to pathogen stress, here we investigated and comprehensive analyzed SUN- and KASH-related proteins among the DAPs under fungi infection based on their conserved motifs. In total, four SUN proteins, one WPP domain-interacting protein (WIP), four WPP domain-interacting tail-anchored proteins (WIT), two WPP proteins and one Ran GTPase activating protein (RanGAP) were identified. Following transient expression of Nicotiana benthamiana, TaSUN2, TaRanGAP2, TaWIT1 and TaWIP1 were identified as nuclear membrane proteins, while TaWPP1 and TaWPP2 were expressed in both the nucleus and cell membrane. RT-qPCR analysis demonstrated that the transcription of TaSUN2, TaRanGAP2 and TaWPP1 were strongly upregulated in response to fungal infection. Furthermore, using the bimolecular fluorescence complementation, the luciferase complementation and a nuclear and split-ubiquitin-based membrane yeast two-hybrid systems, we substantiated the interaction between TaSUN2 and TaWIP1, as well as TaWIP1/WIT1 and TaWPP1/WPP2. Silencing of TaSUN2, TaRanGAP2 and TaWPP1 in wheat leaves promoted powdery mildew infection and hyphal growth, and reduced the expression of TaBRI1, TaBAK1 and Ta14-3–3, indicating that these NM proteins play a positive role in resistance to fungal stress. Our study reveals the characteristics of NM proteins and propose the preliminary construction of SUN-WIP-WPP-RanGAP complex in wheat, which represents a foundation for detail elucidating their functions in wheat in future.