Search Results (93)

To elucidate the mode of action of Mpp5Ab1 against Colaphellus bowringi Baly larvae, this study aimed to identify midgut proteins interacting with the toxin. A validated bait plasmid, pBT3-SUC-mpp5Ab1, was used to screen a larval midgut cDNA library via the split-ubiquitin yeast two-hybrid system. A total of 33 positive clones representing five distinct proteins were obtained, among which bioinformatic analyses prioritized three candidates: Cb-RP-L23e, Cb-CTSL, and Cb-TsetseEP. Subsequent bimolecular fluorescence complementation (BiFC) assays in Sf9 cells specifically confirmed interactions between Mpp5Ab1 and both Cb-CTSL and Cb-TsetseEP, whereas no fluorescence signal was observed for Cb-RP-L23e. Molecular docking further supported stable interactions between Mpp5Ab1 and the validated candidate proteins through hydrogen bonds, salt bridges, and hydrophobic interactions. These findings suggest that Cb-CTSL and Cb-TsetseEP may function as candidate interacting proteins associated with the activity of Mpp5Ab1 in the larval midgut of C. bowringi. Overall, this study provides new insight into the molecular interactions of Mpp5Ab1 and establishes a foundation for future functional studies on its insecticidal mechanism and receptor validation. Full article

Gibberellins (GAs) are key endogenous hormones regulating chrysanthemum flowering, and Gibberellin INSENSITIVE DWARF1 (GID1) is the core receptor of the gibberellin (GA) signaling pathway. However, the functional mechanism of CmGID1A remains unelucidated. Here, we constructed CmGID1A-RNAi silencing lines and characterized the biological function of CmGID1A by phenotypic identification, protein interaction assays, qRT-PCR and RNA-seq. The results of RT-qPCR showed that CmGID1A responds to short days and gibberellins. Inhibition of the expression of CmGID1A can significantly promote the transition of chrysanthemum from the vegetative growth stage to the reproductive growth stage and accelerate its flowering process. Bimolecular fluorescence complementation (BiFC) and yeast two-hybrid (Y2H) assays confirmed that CmGID1A interacts with the DELLA protein CmRGL1 in a gibberellin-dependent manner. RNA-seq results revealed that silencing of CmGID1A leads to a significant up-regulation of downstream Ethylene Response Factor 6 (ERF6) expression. Collectively, CmGID1A acts as a GA receptor to mediate GA signal transduction via interacting with CmRGL1, and regulates the expression of CmERF6 and other downstream genes, thereby participating in the regulation of floral transition in chrysanthemum. This study clarifies the core role of CmGID1A in the GA signaling pathway and provides novel experimental data for enriching the molecular regulatory mechanism of GA in floral transition in chrysanthemum. Full article

Soybean mosaic virus (SMV) establishes successful infection through the coordinated action of multiple viral proteins, yet how these components are collectively engaged in host processes remains unclear. Using 10 SMV-encoded proteins as baits, we screened a soybean (Glycine max) cDNA library using the yeast two-hybrid (Y2H) method and obtained 200 positive clones corresponding to 147 nonredundant candidate host proteins. Integrative analyses indicated that these interacting proteins were mainly associated with host processes related to photosynthesis and energy metabolism, as well as protein quality control (PQC), and that their promoters were enriched in light- and stress-responsive elements. Photosynthesis-related genes were more strongly perturbed in the susceptible cultivar, whereas PQC-module genes showed overall downregulation in both resistant and susceptible cultivars upon SMV infection. Y2H, Bimolecular Fluorescence Complementation (BiFC), and transient expression assays identified four soybean resistance factors that interact with SMV proteins. These factors delayed systemic spread and continued to restrict viral proliferation after systemic infection was established. Notably, SMV accumulation partially rebounded when viral proteins were co-expressed with GmSTOP1, GmHrBP1, or GmGIP2, while co-expression of GmSiPPase and NIa-Pro further inhibited viral proliferation. In summary, this study maps the host-targeting profile of SMV across multiple viral components and provides clues to the SMV–soybean interaction network and antiviral gene resources in soybean. Full article

Maize (Zea mays L.), a typical thermophilic crop originating from tropical regions, exhibits an inherent sensitivity to low-temperature stress. Cold stress severely restricts maize seed germination, seedling growth, the physiological metabolism, and the final grain yield, which greatly limits its geographical cultivation range and sustainable industrial development. Elucidating the molecular regulatory mechanisms underlying maize cold tolerance and excavating cold-resistant functional genes are essential for the molecular breeding of cold-tolerant maize varieties and expanding maize planting areas in high-latitude and low-temperature-prone regions. In this study, using the strongly cold-tolerant maize inbred line B144 as the experimental material, we cloned the ZmMAPKKKA gene (NCBI accession: LOC103651289) and systematically screened and verified its cold-stress-specific interacting proteins via multiple molecular biological assays. The full-length coding sequence (CDS) of ZmMAPKKKA is 1134 bp, encoding a 377-amino-acid protein with a predicted molecular weight of 40.37 kDa. The quantitative real-time PCR (qRT-PCR) results demonstrated that the ZmMAPKKKA expression was significantly upregulated by 16.56-fold in maize roots after 12 h of low-temperature treatment, indicating a tissue-specific and robust cold response in root tissues. A total of 25 interacting proteins were identified through yeast two-hybrid screening, among which three stress-responsive proteins, including a protein kinase (LOC100286253), a protein phosphatase 2C (PP2C) (LOC542176), and a NAC transcription factor (LOC118474710), were selected for subsequent verification. The Pull-Down, Co-immunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC) assays consistently confirmed that ZmMAPKKKA specifically interacts with these three proteins both in vitro and in vivo under cold stress conditions. This study is the first to construct a ZmMAPKKKA-centered protein interaction module in the maize mitogen-activated protein kinase (MAPK) cascade under cold stress, establishing a novel kinase–phosphatase–transcription factor regulatory cascade that improves the current understanding of cold signal transduction mechanisms in maize. Homologous genes of ZmMAPKKKA in gramineous crops including rice (Oryza sativa) and sorghum (Sorghum bicolor) have been proven to participate in diverse abiotic stress responses, suggesting the conserved functional roles of MAPKKK family genes across gramineous species. Collectively, our findings provide comprehensive insights into the molecular mechanism of the maize MAPK signaling pathway mediating cold stress adaptation and supply valuable functional gene resources for cold-tolerant maize germplasm innovation and molecular breeding. Full article

Maize (Zea mays L.) is a vital global crop whose productivity is severely threatened by abiotic stresses. Epicuticular waxes provide a hydrophobic barrier that protects land plants from environmental stresses. However, the role of key wax biosynthetic enzymes, such as aldehyde decarbonylase CER1, in maize stress adaptation remains unclear. In this study, we performed a functional characterization of ZmCER1 in maize. Our results show that the overexpression of ZmCER1 in both Arabidopsis and maize substantially improved tolerance to these abiotic stresses. Under stress conditions, the transgenic plants displayed better growth performance, elevated activities of antioxidant enzymes, and reduced levels of oxidative damage markers. Additionally, the alkane content—especially that of C29 and C31—was significantly increased in the ZmCER1OE lines. Through a yeast two-hybrid screening (Y2H screening), we identified the peroxisomal membrane protein ZmPEX14 as an interacting partner of ZmCER1, and the interaction was further confirmed by luciferase complementation (LUC) and bimolecular fluorescence complementation (BiFC) assays. We propose a model wherein ZmCER1 enhances stress tolerance not only by reinforcing the cuticular wax barrier but also by potentially regulating reactive oxygen species (ROS) detoxification via association with ZmPEX14. Collectively, our findings establish ZmCER1 as a key regulator of abiotic stress tolerance in maize and a promising candidate for the molecular breeding of stress-resilient crops. Full article

Chlorophylls are the major light-harvesting pigments in photosynthetic organisms. Their biosynthesis requires the coordinated supply of metabolic intermediates from two independent upstream branches: the methylerythritol 4-phosphate (MEP)-derived terpenoid pathway, which supplies the phytyl side chain via geranylgeranyl diphosphate (GGPP), and the tetrapyrrole biosynthesis pathway (TBP), which provides the porphyrin ring. How flux through these two branches is coordinated remains poorly understood. In this study, we report the identification of a direct protein–protein interaction between δ-aminolevulinic acid dehydratase (ALAD), the second enzyme of the TBP, positioned immediately upstream of the first metabolic branch point, and the Type II small subunit of GGPP synthase (SSUII), a key regulator of terpenoid flux toward chlorophyll biosynthesis. ALAD was identified as a candidate SSUII-interacting protein by co-immunoprecipitation coupled with LC-MS analysis of rice leaf tissue, with a sequence coverage of 57.04%. The interactions between OsALAD1 and OsSSUII in rice, and between AtALAD1 and AtSSUII in Arabidopsis thaliana, were validated by yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) in Arabidopsis protoplasts. BiFC imaging demonstrated that the interaction is localized to the chloroplast. Sequence analysis revealed that plant ALAD proteins are highly conserved, with 92% similarity between OsALAD1 and AtALAD1, and 76.9% similarity between OsALAD1 and the green alga Chlamydomonas reinhardtii CrALAD1, indicating cross-species conservation of the ALAD–SSUII interaction. In vitro enzyme activity assays showed that AtSSUII does not directly alter AtALAD1 catalytic activity, suggesting the interaction operates through post-translational rather than direct catalytic mechanisms. Overexpression of AtALAD1 caused severe chlorosis and seedling lethality, while AtSSUII overexpression produced no distinct phenotype; neither transgene altered the transcript level of the other. Together, our results reveal a conserved cross-pathway protein–protein interaction linking the terpenoid regulatory machinery to the early TBP, suggesting a molecular possibility for the coordinated regulation of chlorophyll biosynthesis. Full article

Myosin XI-K plays an important role in cell expansion and polarized growth, acting as a motor protein that drives organelle trafficking and cytoplasmic streaming. To elucidate the molecular mechanisms of myosin XI-K’s role in the polarized growth of cotton fiber, we investigated the interactions between GhXI-K and Rab GTPases in cotton (Gossypium hirsutum). Protein docking analyses based on AlphaFold3 predicted that GhXI-K interacted with eight Rab GTPases. A total of 37 interaction residues were identified in GhXI-K, of which 5 crucial contact residues were located in the globular tail domain (GTD) and 2 were located in the motor domain. Key interaction residues in the Rab GTPases were also found to be located in conserved regions: switch-I and switch-II. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays confirmed the predictions and showed that these interactions occur primarily in the GTD and the motor domain. Our findings reveal that GhXI-K interacts with Rab GTPases through both the motor and tail domains, suggesting a synergistic mechanism that facilitates polarized vesicle trafficking in cotton fiber cells. Full article

Wheat is one of the most important crops contributing to global food and nutritional security. However, the gradual increase in soil salt content significantly impairs wheat growth and development, ultimately resulting in reduced yields. Therefore, enhancing the salt tolerance of wheat is of significant importance. Salt stress commonly induces oxidative stress in plants, and nucleoredoxin (NRX) has been shown to effectively maintain redox homeostasis under stress conditions. However, the functional role and molecular mechanism of the NRX gene in regulating salt tolerance in wheat remain to be elucidated. The results of this study demonstrated that TaNRX1-2D homologous overexpression (OE) lines exhibited significantly enhanced tolerance to salt stress. The survival rate and antioxidant enzyme activities (including superoxide dismutase and catalase) in the OE lines were higher than those in the wild type (WT). In contrast, the levels of superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and malondialdehyde (MDA) in the OE lines were markedly lower than those in the WT. Conversely, the RNA interference (RNAi) lines displayed opposing trends. The results of yeast one-hybrid (Y1H) and dual luciferase assays (D-LUC) demonstrated that the TaERD15L-3B transcription factor positively regulated the expression of the TaNRX1-2D gene by binding to the ABRERATCAL cis-acting element in the TaNRX1-2D promoter. Through luciferase complementation assay (LCA), bimolecular fluorescence complementation (BiFC) assay, and a “mutation capture strategy”, it was found that TaNRX1-2D (C54, 327S) interacted with TaCAT2-B, indicating that TaCAT2-B was the target protein of TaNRX1-2D. The results of data-independent acquisition (DIA) proteomics analysis indicated that TaNRX1-2D may mediate salt tolerance in wheat through the positive regulation of nsLTP protein abundance and the negative regulation of hexokinase protein abundance. In general, the TaERD15L-3B/TaNRX1-2D regulatory module played a crucial role in conferring salt tolerance in wheat. This study provided an important theoretical basis and identified a potential gene target for developing salt-tolerant wheat varieties through molecular breeding approaches. Full article

Longan (Dimocarpus longan Lour.) is a commercially valuable tropical fruit crop that contains two antagonistic FLOWERING LOCUS T (FT) homologs involved in regulating flowering time. However, how these FT genes interact with flowering regulators FLOWERING LOCUS D (FD) and APETALA1 (AP1) remains unknown. Four flowering-related genes in longan, DlFT1, DlFT2, DlAP1 and DlFD, were successfully isolated. Expression profiling revealed that all four genes were expressed in leaves and buds across different stages of natural and KClO₃-induced floral bud differentiation. Functional characterization through heterologous overexpression in Arabidopsis thaliana showed that DlAP1 significantly promotes early flowering under long-day conditions and induced morphological changes in floral organs and leaves. In contrast, DlFD overexpression had no effect on flowering time. Subcellular localization assays revealed that DlFT1 and DlFT2 localized to both the nucleus and the plasma membrane, while DlAP1 and DlFD localized exclusively to the nucleus. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses revealed a novel regulatory node: DlFT1 directly interacts with DlAP1, a finding that expands the classical FT-FD-AP1 flowering model. Additionally, DlFD interacts more strongly with DlFT1 than with DlFT2, whereas DlFT1 only interacts with DlAP1, but not DlFT2. These results demonstrate that DlFT1 promotes flowering not only via the conserved FD-dependent pathway but also through direct association with AP1. These findings advance our understanding of the regulatory mechanisms of flowering in longan and provide valuable insights into flowering pathways of perennial woody species. Full article

Most land plants engage in a mutualistic interaction with arbuscular mycorrhizal fungi (AMF), for which Rhizophagus irregularis is a model species. Like plant pathogenic fungi, AMF genomes encode hundreds of putative effector proteins. However, for only a few, the molecular mechanisms by which they alter the host’s physiology are known. Here, we combined several reverse genetic approaches to unravel the role of the RIRG190 effector protein in arbuscular mycorrhiza (AM) symbiosis. Using multiple heterologous tools, evidence is provided that the RIRG190 effector is secreted and localizes to the plant nucleus. Moreover, by means of yeast two-hybrid (Y2H) and ratiometric bimolecular fluorescence complementation (rBIFC) assays, the data demonstrate that RIRG190 interacts with the protein Something About Silencing (SAS10), known to be involved in rRNA biogenesis in the nucleolus of cortical cells. Our findings suggest that rRNA biogenesis is a key process modulated by AMF, potentially to enhance plant metabolic activity, facilitating cell cycle progression, and to support the establishment of the symbiosis. Full article

Actinidia valvata, a promising rootstock for kiwifruit cultivation, demonstrates superior waterlogging tolerance compared with commercial cultivars. Lateral organ boundaries domain (LBD) transcription factors (TFs) are known to be pivotal in plant responses to abiotic stress. Nevertheless, the characterization of the LBD family under waterlogging stress in A. valvata remains limited. In this study, 26 AvLBD genes were identified from a transcriptome dataset, with the majority classified into phylogenetic Class II. Under waterlogging stress, transcript accumulation of most AvLBD41 members, particularly AvLBD41_11 and AvLBD41_7, was markedly increased in roots. Bimolecular fluorescence complementation (BiFC) assays indicated that AvLBD41_7 heterodimerizes with both the AP2/ERF activator AvERF75 and the trihelix repressor AvHRA1, whereas AvLBD41_11 only interacts with AvERF75. Neither AvLBD41 isoform interacts with AvERF73, thereby defining distinct components of a waterlogging-responsive module. Yeast-based assays revealed an absence of transactivation activity for AvLBD41_7, and transient expression analyses confirmed its exclusive nuclear localization. The promoters of both AvLBD41_11 and AvLBD41_7 harbor numerous cis-elements responsive to hormones and abiotic stresses. An AvLBD41_7-derived PCR marker could be used to distinguish A. valvata from A. deliciosa accessions. Collectively, these findings provide a comprehensive functional annotation of the LBD gene family in A. valvata and establish AvLBD41_7 as a potential molecular target for future kiwifruit breeding programs aimed at waterlogging resilience. Full article

The tomato zonate spot virus (TZSV) poses a significant threat to agriculture. Therefore, the elucidation of the functional roles and interactions of its encoded proteins is crucial for the development of effective control strategies. The aim of this study was to investigate the interaction network between the TZSV nucleocapsid (N), the non-structural M-segment (NSm) and the non-structural S-segment (NSs) proteins, with a focus on the functional characterization of the NSm protein. Yeast two-hybrid (Y2H) analysis indicated that both the N protein (N-N) and the NSm protein (NSm-NSm) exhibit self-interaction in vitro, with successful expression of all fusion proteins confirmed by Western blotting. Subsequently, we used bimolecular fluorescence complementation (BiFC) and luciferase complementation imaging (LCI) assays in epidermal cells of Nicotiana benthamiana to confirm that N and NSm proteins self-interact. In addition, heterologous interactions between NSs-N, N-NSm and NSs-NSm were also detected. BiFC and co-localization experiments with fusion proteins elucidated the interaction place of the cell: N-N and NSm-N interactions occurred in both the cytoplasm and nucleus, with NSm-NSm interaction occurring in the nucleus, whereas NSs-N and NSs-NSm interactions only occurred in the cytoplasm. Subcellular localization studies showed that the N protein is distributed in both the cytoplasm and the nucleus, whereas the NSm and NSs proteins are predominantly localized in the cytoplasm. In particular, NSm was found to specifically target plasmodesmata (PD) and co-localize with the known PD marker protein PDLP8. Interestingly, TZSV NSm was demonstrated to mediate the cell-to-cell movement of a cucumber mosaic virus mutant (ΔCMV-GFP) lacking its native movement protein (3a). This was evidenced by the spread of approximately 50 fluorescent foci to neighboring cells observed at 6 dpi. This study comprehensively describes the intricate interaction network between the N, NSm and NSs proteins of TZSV and clarifies their subcellular localizations within plant cells. Crucially, we provide conclusive evidence that the NSm protein of TZSV is a functional movement protein essential for facilitating viral intercellular transport which promotes viral spread within the host during systemic infection. These findings offer important insights into the infection mechanism of TZSV and provide potential targets for the control of TZSV. Full article

Brassica juncea is an important leafy vegetable, and flowering time is a key determinant of its yield and quality. In this study, one significantly up-regulated gene, BjuAGL9-2, was identified from RNA-Seq data. qRT-PCR analysis confirmed that BjuAGL9-2 expression was significantly elevated in reproductive organs and reproductive stages. Further five BjuAGL9-2 over-expression (OE) lines were subsequently generated, which showed an early-flowering and pale-yellow leaf phenotype compared to the wild type. qRT-PCR assays found that the mRNA of core floral integrator genes was changed in Arabidopsis OE lines. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated that BjuAGL9-2 interacted with BjuTUA5, BjuZFP7, BjuGSTU5, and BjuMAPK16 in vivo. Sub-cellular localization assays showed that BjuAGL9-2 localizes in the nucleus, whereas its interacting partners localize in the cytoplasm. qRT-PCR assays further revealed that BjuTUA5 and BjuGSTU5 were up-regulated in flower buds, while BjuZFP7 and BjuMAPK16 were down-regulated. During vegetative stages, all four genes were up-regulated in B. juncea. As for BjuAGL9-2 interaction protein-encoding homolog genes, except AtGSTU5, the other three genes were up-regulated in Arabidopsis OE lines. Additionally, qRT-PCR analysis of chlorophyll biosynthesis-related genes showed that 19 of 27 genes were up-regulated, while 8 genes were down-regulated, in Arabidopsis OE lines. Collectively, these findings suggest that BjuAGL9-2 promotes flowering and contributes to the pale-yellow phenotype by regulating its interacting protein-coding genes, floral integrators, and chlorophyll biosynthesis genes. Full article

Calcium signaling is essential for coordinating plant responses to diverse stimuli and regulating growth and development. Among calcium sensors, calmodulin (CaM) and CaM-like proteins (CMLs) represent a class that, despite increasing research, remains incompletely characterized in wheat, with many interacting partners and biological functions remaining largely elusive. This study conducted bioinformatics analyses of subgroup II CaM/CMLs, characterizing their phylogenetic relationships, conserved motifs, sequence features, and cis-elements. Expression analysis revealed that TaCML49-B was significantly upregulated in roots under salt stress. Moreover, TaCML49-B was localized to nucleus, cytoplasm, and membrane. Function characterization demonstrated that overexpression of TaCML49-B in Arabidopsis enhanced salt tolerance, whereas the BSMV-VIGS silencing of TaCML49-B reduced salt resistance in wheat. Furthermore, STRING database prediction analysis and bimolecular fluorescence complementation (BiFC) assay confirmed that TaCML49-B can physically interact with TaIQD23, which encodes an IQ67 domain protein, suggesting its potential involvement in the salt stress signaling pathway. Collectively, our findings indicate that TaCML49-B functions as a positive role in wheat salt stress response, thereby providing novel insights into the functions of TaCML genes and calcium signaling in wheat. Full article

The early flowering and less lateral branches in watermelon hold significant agricultural value. The synergistic effects of these traits provide an ideal template for watermelon plant architecture improvement. However, the molecular regulatory networks underlying the development of lateral organs (including branches and flowers) in watermelon remain unclear. In this study, we found ClTFL1 knockout lines significantly promote flowering time and inhibit lateral branching and tendril formation, while also leading to a mild apical flower phenotype. These findings indicate that the function of ClTFL1 in watermelon is more extensive than that of its homologous genes in Arabidopsis, rice, and tomato. Through yeast two-hybrid screening, we identified the interacting proteins of ClTFL1, including members of the 14-3-3 family ClGRF8, ClGRF9, and ClGRF12. Bimolecular fluorescence complementation (BiFC) assays further demonstrated ClTFL1 could directly interact with the ClGRF8, ClGRF9, and ClGRF12 protein. The knockout of ClGRF8 and ClGRF12 leads to reduced lateral branches and early flowering. These phenotypes are highly consistent with those of ClTFL1 knockout mutants. Our findings demonstrate the important role of the ClTFL1-ClGRFs module in regulating lateral organ development and flowering time in watermelon, offering important targets for watermelon plant architectural modification and molecular breeding. Full article