The H9N2 subtype avian influenza virus (AIV) hemagglutinin (HA) protein is a major immunogen in which HA1 is a genetic variant and HA2 is relatively conserved.  Identifying broad-spectrum antigen epitopes targeting HA1 is crucial for vaccine design and detection.  Based on the phylogenetic and serological analyses, we identified 2 antigenic groups and 3 representative viruses: A/chicken/Jiangsu/JY040218C/2019, A/pigeon/Jiangsu/JY020616/2019, and A/chicken/Jiangsu/WX090312/2018.  An overlapping peptide library was synthesized using HA1 amino acid sequences of the viruses as templates.  Through peptide scanning of the sera against different strains of H9N2 subtype AIV, we identified peptides from 4 regions (H9-2/3, H9-20/21, H9-26, and H9-29/30/31) that demonstrated broad-spectrum reactivity.  Immunological assay results demonstrated that H9-21 (²¹⁹RIFKPLIGPRPLVNGLMGRI²³⁹), H9-26 (²⁶⁹SGESHGRILKTDLKMGSCTV²⁸⁹), and H9-30 (³⁰⁹YAFGNCPKYI GVKSLKLAVG³²⁹) effectively induced antibody generation and conferred partial protective efficacy against the parent virus JY040218C.  The results of lymphocyte proliferation and ELISpot assays indicated that peptides H9-15 (¹⁵⁹MRWLTQKNNAYPTQDAQYTN¹⁷⁹), H9-22 (²²⁹PLVNGLMGRINYYWSVLKP G²⁴⁹), and H9-23 (²³⁹NYYWSVLKPGQTLRIKSDGN²⁵⁹) could effectively stimulate the expression of interferon-gamma in peripheral blood lymphocytes of chickens immunized against different strains of H9N2 AIV.  Collectively, 5 novel cell epitopes H9-15, H9-22, H9-23, H9-26, and H9-30, including the best B cell epitope H9-26 and the best T cells epitope H9-22, were identified that could be targeted for vaccine design or detection approaches against H9N2 AIVs.

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, is widely spread worldwide and causes huge economic losses.  The effective measure to control the viral infection is to develop ideal vaccines.  Here, the collagenase equivalent domain (COE) of PEDV was displayed on the surface of nanoparticles (NPs) in order to develop a newer, safer and more effective subunit vaccine against PEDV.  The monomeric COE was displayed on the mi3 protein, which self-assembles into nanoparticles composed of 60 subunits, using the SpyTag/SpyCatcher system.  The size, zeta potential, microstructure of the COE-mi3 virus-like particles (VLPs) were investigated.  The COE-mi3 VLPs that possessed good security, stability and better retention can be more efficiently taken up by antigen-presenting cells (APCs) and help promote dendritic cells (DCs) maturation.  Moreover, COE-mi3 VLPs could prominently improve specific antibody levels including neutralizing antibodies (NAbs), and serum IgG, mucosal IgA.  Moreover, COE-mi3 VLPs elicited more activation of CD4⁺ and CD8⁺ T cells and production of IFN-γ and IL-4 cytokines.  In particular, COE-mi3 VLPs is an effectual antigen-delivery platform to enhance germinal center (GC) B cell responses.  This structure-based self-assembly of NP gives great potential to be developed as a new subunit vaccines attractive platform, and may also provide new ideas for the development of other enteric coronavirus vaccines.

Quantitative inversion is a major topic in remote sensing science.  The development of visible light-based hyperspectral reconstruction techniques has opened novel prospects for low-cost, high-precision remote sensing inversion in agriculture.  The aim of this study was to assess the effectiveness of hyperspectral reconstruction technology in agricultural remote sensing applications.  Hyperspectral images were reconstructed using the MST++ hyperspectral reconstruction model and compared with the original visible light images in terms of their correlations with physiological parameters, the accuracy of single-feature modeling, and the accuracy of combined feature modeling.  The results showed that compared to the visible light image, the reconstructed data exhibited a stronger correlation with the measured physiological parameters, and the accuracy was improved for both the single feature and combined feature inversion modes.  However, compared to multispectral sensors, hyperspectral reconstruction provided limited improvement of the inversion model accuracy.  The results suggest that for physiological parameters that are not easy to observe directly, deep mining of features in visible light data through hyperspectral reconstruction technology can improve the accuracy of the inversion model.  However, appropriate feature selection and simple models are more suitable for the remote sensing inversion task of traditional agronomic plot experiments.  To strengthen the application of hyperspectral reconstruction technology in agricultural remote sensing, further development is necessary with broader wavelength ranges and more diverse agricultural scenarios.

Starch serves as a critical storage component, significantly influencing the grain yield and quality of maize (Zea mays L.).  Understanding the genetic basis of natural variation in kernel starch content (SC) is essential for maize breeding to meet future demands.  A genome-wide association study (GWAS) identified 84 and 96 loci associated with kernel SC across two years, overlapping with 185 candidate genes.  The candidate gene ZmMYB71, encoding a MYB-related transcription factor, demonstrated the highest co-expression frequency with starch synthesis genes.  Analysis revealed that ZmMYB71 functions as a nuclear located transcription repressor, and mutants exhibited increased kernel SC by over 2.32%, with minimal impact on amylose content or 100-grain weight.  Sh1, Sh2, and GBSSI exhibited up-regulation in mutants by 1.56-, 1.45- and 1.32-fold, respectively, aligning with RNA sequencing results; their promoter activities appear directly repressed by ZmMYB71 through the GATATC and TTAGGG motifs.  Additionally, the ZmMYB71 elite haplotype Hap1 occurred in over 55% of the high-starch maize sub-populations Iowa Stiff Stalk Synthetic (BSSS) and Partner B (PB), but only in 7.14% of the low-starch sub-population Partner A (PA).  Analysis of Hap1 haplotype frequencies across breeding stages revealed a significant increase to 40.28% in inbred groups released after 2010, compared to 28.57 and 27.94% in 1980 and 1990, and 2000, respectively.  These findings enhance understanding of natural variation in maize kernel SC and establish ZmMYB71 as a negative regulator with potential applications in SC improvement.

Pseudorabies (PR) is an acute infectious disease of pigs caused by the PR virus (PRV) and results in great economic losses to the pig industry worldwide.  PRV glycoprotein E (gE)-based enzyme-linked immunosorbent assay (ELISA) has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries.  Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent.  However, there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies.  In the present study, the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel.  Then, two nanobodies against PRV-gE were screened from the immunized camel by phage display technique.  Subsequently, two nanobody-HRP fusion proteins were expressed with HEK293T cells.  The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA (bELISA) to detect anti-PRV-gE antibodies.  Through optimizing the conditions of bELISA, the amount of coated antigen was 200 ng per well, and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5.  The cut-off value of bELISA was 24.20%, and the sensitivity and specificity were 96.43 and 92.63%, respectively.  By detecting 233 clinical pig sera with the developed bELISA and a commercial kit, the results showed that the coincidence rate of two assays was 93.99%.  Additionallly, epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains.  Simple, great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed.  The bELISA could be used for monitoring and eradicating PR.

Rapeseed (Brassica napus L.) is one of the most important oilseed crops worldwide.  Development of rapeseed varieties with high-quality oil is a long-term breeding goal.  Reducing the contents of palmitic acid, the main saturated fatty acid in rapeseed oil, could greatly improve oil quality.  Here, we performed genome-wide association study (GWAS) and transcriptome-wide association study (TWAS) of seed palmitic acid content (SPAC) using 393 diverse B. napus accessions.  Four genes (BnaA08.DAP, BnaA08.PAA1, BnaA08. DUF106, and BnaC03.DAP) were identified by both GWAS and TWAS.  The transcripts per million (TPM) values of these candidate genes at 20 and 40 days after flowering (DAF) were significantly correlated with SPAC in this association panel.  Based on genetic variation in the candidate genes, we identified four low-SPAC haplotypes by combining candidate gene association analysis and haplotype analysis.  Brassica napus accessions carrying low-SPAC haplotypes had lower SPAC than those carrying high-SPAC haplotypes without affecting seed oil content, seed protein content, or seed yield.  Based on the functional single-nucleotide polymorphism (SNP) chrA08_9529850 (C/A) in the promoter of BnaA08.DUF106, we developed a molecular marker (Bn_A8_SPAC_Marker) that could be used to facilitate breeding for low SPAC in B. napus.  Our findings provide valuable information for studying the genetic control of SPAC in B. napus.  Moreover, the candidate genes, favorable haplotypes, and molecular marker identified in this study will be useful for breeding low-SPAC B. napus varieties.

Cucumber (Cucumis sativus L.) is a major vegetable crop worldwide, and its yield and quality are closely linked to flower development. AGAMOUS-LIKE 6 (AGL6), a member of the ancient MADS-box transcription factor family, plays a crucial role in flower development. However, the specific functions of its homolog in cucumber remain poorly understood. In this study, we demonstrate that CsAGL6 is predominantly expressed in flowers, with high expression levels observed in all floral organ primordia during the early stages of floral development. The petals of Csagl6 mutants exhibit a greener color compared to wild-type plants, along with a significant increase in total chlorophyll content. Additionally, the mutants show abnormal petal morphology, including changes in size and shape, as well as enlarged sepals resembling leaves occasionally. Molecular analysis reveals that the A-class gene CAULIFLOWER (CAL) and the E-class gene SEPALLATA 4 (SEP4) are significantly downregulated in the mutants, while the chlorophyll synthesis gene Early Light-Induced Protein 1 (ELIP1) and several stress-related genes in the chloroplasts are dramatically upregulated. Our findings provide novel insights into the functional role of CsAGL6 in regulating sepal and petal development, and offer a potential avenue for understanding the genetic control of flower pigmentation and organ morphology in Cucumis species.

The CONSTANS/CONSTANS-LIKE (CO/COL) gene family plays important roles in plants flowering and stress response. In this study, two variants of the MiCOL14B gene were identified from two different mango cultivars; they were designated as MiCOL14B-GQ and MiCOL14B-JH, which exhibited significant differences in sequence and B-box domain. Both genes are expressed in various tissues of mango, localized in the nucleus, and responsive to drought and salt stress. In transgenic Arabidopsis thaliana, MiCOL14B-GQ delayed flowering, while MiCOL14B-JH promoted flowering. This phenotypic divergence stemmed from their molecular regulatory specificity. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays demonstrated that both variants directly bind to the promoters of florigen genes (MiFTs), with MiCOL14B-GQ repressing their transcription and MiCOL14B-JH enhancing it. Altered expression levels of MiFTs in the roots of transgenic mango further validated this mechanism. Moreover, both MiCOL14B-GQ and MiCOL14B-JH improved stress tolerance under drought and salt conditions in transgenic A. thaliana as well as in transgenic mango roots. These variants significantly increased stress tolerance by increasing proline (Pro) content and superoxide dismutase (SOD) activity, while reducing malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) accumulation. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays revealed that MiCOL14B-GQ and MiCOL14B-JH interact with several stress-related proteins. This study demonstrates for the first time the potential function of MiCOL14B gene sequence variation in regulating flowering and stress responses, providing valuable genetic resources for mango molecular breeding.