Recently, CRISPR/Cas13a-based biosensors have been combined with non-traditional lateral flow assays (NTLFAs) successfully used to clinical detection of target RNA due to their simplicity, portability, and costeffectiveness. However, N-TLFAs used in such biosensors have several disadvantages, such as inversion of test and control zones, in which color intensity depends on reporter probe cleavage. Herein, we propose a novel biosensor in which CRISPR/Cas13a is integrated with traditional lateral flow assays (TLFAs) to facilitate the sequential direct location of test/control zones by dual hairpin probes, namely DNA hairpin reporter probe B-HPs (5 ' Biotin-hairpins, B-HPs, loop containing 5 rU) and F-HPs (5 ' FAM-hairpins, F-HPs). Cas13a activated by crRNAtargeted RNA duplexes can indiscriminately cleave B-HPs reporter probes to release short sequences to open hairpin F-HPs reporter probes to form Biotin-dsDNA-FAM reporter probes. The advantage of this platform is that detection results only rely on the formed Biotin-dsDNA-FAM reporter probes, which can be used for SARS-CoV-2 RNA detection after preliminary detection. Under optimized conditions, the SARS-CoV-2 RNA detection range was 10 aM to 1 mu M and the limit of detection was as low as 5.34 aM. This proof-of-concept study demonstrates that dual-hairpin reporter-probe binding to TLFAs opens up new opportunities for CRISPR/Cas13a activity detection and bioanalytical applications.

Plants are constantly challenged by a diversity of abiotic stressors, and growth arrest is a common plant response aimed at enhancing stress tolerance. Because of this growth/stress tolerance antagonism, plants must finely modulate their growth and responses to environmental stimuli. Here, we demonstrate that HSFB1, a heat shock transcription factor, plays a critical role in the coordination of plant growth and drought stress responses in Arabidopsis thaliana. First, we found that HSFB1 negatively regulates plant growth and development under normal conditions and that HSFB1 expression is enhanced under drought stress. Conversely, the loss-of-function mutant hsfb1 exhibited increased plant growth and reduced drought stress tolerance compared with the wild-type. Consistently, overexpression of HSFB1 suppressed plant growth and enhanced drought stress tolerance. Subsequently, via chromatin immunoprecipitation sequencing, RNA sequencing, and transient expression assays, we screened and identified the heat shock protein 101 (HSP101) gene as a direct transcriptional target of HSFB1. Genetic analysis suggested that HSP101 functions downstream of HSFB1 to positively regulate drought tolerance in plants. Furthermore, we found that HSFB1 physically interacts with the eukaryotic translation initiation factor eIF3G1, and this interaction appears to be further enhanced under drought stress. Notably, the mutation of eif3g1 increased the severity of drought-induced growth inhibition in the hsfb1 mutant, and eIF3G1 enhanced the transcriptional activation of HSFB1 on the HSP101 promoter under drought stress. Altogether, our findings highlight HSFB1 as a key regulator coordinating plant growth and drought stress responses in Arabidopsis.

In this study, we characterized the holin-like protein ORF70 from the cyanophage MaMV-DC, offering valuable insights into its role in phage-mediated host cell lysis. ORF70 shares key features with class III holins, such as a hydrophobic transmembrane domain and membrane-associated localization, which are crucial for its bacteriolytic activity. Subcellular localization studies suggested its association with the membrane, supporting its classification as a holin-like protein. Overexpression of ORF70 in E. coli resulted in significant growth inhibition, increased β-galactosidase leakage, and visual confirmation of cell death through live/dead staining. Additionally, ORF70’s sensitivity to the energy toxin 2,4-dinitrophenol (DNP) further indicated its holin-like activity by promoting membrane depolarization. Transmission electron microscopy and Gram staining revealed characteristic morphological changes in E. coli cells, including membrane disruption, consistent with damage caused by holins. These results suggest that ORF70 acts as a holin-like protein that disrupts the host membrane, leading to bacterial cell death. Our study provides evidence supporting the holin-like activity of ORF70 from cyanophage MaMV-DC. This research significantly enhances our understanding of phage-host interactions and opens new avenues for developing phage-based therapies, offering promising alternatives to traditional antibiotics amidst the growing challenge of antibiotic resistance.

Bovine herpesvirus type 1 (BoHV-1) is a widespread respiratory infection that significantly impacts cattle health worldwide. To address this issue in China, we previously developed a novel double gene-deleted vaccine targeting gG and tk. In this study, we further evaluated the efficacy of this vaccine by challenging vaccinated cattle with a prevalent wild-type BoHV-1 strain and comparing its effectiveness against a commercially available inactivated BoHV-1 vaccine. Post-immunization, all cattle maintained normal rectal temperatures and exhibited no respiratory symptoms. Cattle receiving the gene-deleted vaccine showed a significant increase in the expression of immune markers IFN-gamma and TNF-alpha. Following exposure to wild-type BoHV-1, all immunized groups produced high levels of neutralizing antibodies and specific gB antibodies. Notably, virus shedding was significantly lower in the vaccinated groups compared to the non-immune challenge group. Histological analysis of lung tissues revealed that vaccinated calves had more intact lung structure than their unimmunized counterparts after the challenge. Additionally, the gG-/tk- gene-deleted vaccine demonstrated a higher protective rate based on the average scores of clinical symptoms and lung lesions. Overall, the BoHV-1 gG-/tk- gene-deleted vaccine outperformed the other vaccines tested. This study confirms that the gene-deleted vaccine provides robust protection and superior immunogenicity compared to existing inactivated vaccines, underscoring its potential for future market application.

Heterotrimeric G-proteins are multifunctional modulators that participate in a wide range of growth and developmental processes in eukaryotic species, including yeast, plants, and animals. In this study, we characterized a maize mutant, ct2, that showed a compact architecture and reproductive-organ-related phenotypic variation. Heredity analysis indicated that the mutant phenotypes resulted from monogenic inheritance. The target gene, CT2, was cloned using bulked segregant analysis and map-based cloning. Sequence alignment showed that the ct2 mutation was the result of a 185-bp sequence insertion at the 3′ terminal of CT2. Protein structure prediction and phylogenetic analysis indicated that CT2 is a canonical Gα of monocotyledonous plants. Through phenotypic identification, we found that CT2 was involved in yield-related traits in maize. Furthermore, our findings indicated that CT2 promotes cell proliferation in stem internodes, which may result from the upregulation of zeatin biosynthesis by CT2. This research provides direction for further studies in the biological function of CT2 in cell proliferation and in yield-related traits, which will be beneficial for maize breeding through screening and application of beneficial alleles.

Our study explores the genetic mechanisms underlying the spotted leaf phenotype in rice, focusing on the rpt5a mutant characterized by persistent reddish-brown leaf spots from seedling stage to maturity, leading to extensive leaf necrosis. Through map-based cloning, we localized the responsible locus to a 330Kb region on chromosome 2 and identified LOC_Os02g56000 (named OsRPT5A) as the causative gene. A point mutation in OsRPT5A, substituting valine for glutamic acid, was identified as the critical factor for the phenotype. Functional complementation and generation of knockout lines in the IR64 background confirmed the central role of OsRPT5A in controlling this trait. Our study also revealed that OsRPT5A is constitutively expressed across various tissues, with its subcellular localization unaffected by mutations. Notably, we observed an abnormal accumulation of reactive oxygen species (ROS) in rpt5a mutants, suggesting a disruption in the ROS system. Complementation studies indicated OsRPT5A's involvement in ROS homeostasis and catalase activity regulation. Moreover, the rpt5a mutant exhibited enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo), highlighting OsRPT5A's role in rice's pathogen resistance mechanisms. These findings elucidate the genetic basis of the spotted leaf phenotype and emphasize the intricate interplay between plant defense responses and developmental pathways.

Heterotrimeric G-proteins are multifunctional modulators that participate in a wide range of growth and developmental processes in eukaryotic species, from yeast to plants and animals. Component detection and the study of G protein signaling in most plants, including maize, are in the initial stages. In this study, we characterized a maize mutant, ct2 , that showed a compact architecture and reproductive organ-related phenotypic variation. The target gene CT2 was cloned using bulked segregant analysis and map-based cloning. Gene structure prediction and phylogenetic analysis indicated that CT2 is a canonical Gα protein belonging to the monocotyledonous group. Promoter analysis of CT2 and RNA sequencing revealed cis -acting regulatory elements and differentially expressed genes involved in JA signaling and stress tolerance. The transcription of CT2 was repressed by NaCl and PEG treatments, and ct2 mutation in the ct2 line compromised stress tolerance in maize. On the basis of our results, we proposed a schema diagram of CT2 -regulated biological process and their feedback on CT2 transcription. This research provides clues for further studies of CT2 function in hormone signaling and stress tolerance, which is beneficial for maize breeding through the screening and application of beneficial alleles. ### Competing Interest Statement The authors have declared no competing interest. Datasets generated during the current study are available from the corresponding author on reasonable request.

The combination of magnetic nanomaterials with the immobilized metal affinity (IMA) technique has emerged as a powerful method to specifically separate histidine (His)-rich proteins. However, obtaining a high-performance and sustainable magnetic adsorbent with good dispersibility and minimal copper leakage remains a major challenge. Herein, we propose a simple ligand-free method to acquire Cu-doped magnetic nanorings (Fe3O4-Cu NR) for the highly selective enrichment of His-rich proteins. Importantly, the Fe3O4-Cu NR with unique ring-like structural features offers numerous exposed Cu2+ binding sites for His-rich proteins while maintaining a high magnetic behavior (61.7 emu g(-1)). Thus, the Fe3O4-Cu NR exhibits a remarkable adsorption capacity (932.2 mg g(-1)) and high selectivity (SF < 0.1) for bovine hemoglobin (BHb), surpassing most of the reported IMA magnetic adsorbents. Moreover, Fe3O4-Cu NR can be easily regenerated with negligible copper ion leakage (<0.1 mu mol L-1) and without requiring a reincubation step, demonstrating reusability for up to six cycles. In addition, the removal efficiency and recovery of Fe3O4-Cu NR for BHb in the diluted bovine blood were found to be 89.79 and 70.25%, respectively, further indicating the specificity of Fe3O4-Cu NR to BHb in complex biological samples.

Blumeria graminis f. sp. tritici ( Bgt ) is a significant wheat fungal pathogen, posing threats to both yield and quality. Antimicrobial peptides, with their broad-spectrum activities, hold promise in combating Bgt -induced wheat fungal diseases.In this study, we identified TaW662 , an antifungal peptide gene sourced from the wheat- Thinopyrum intermedium disomic alien addition line SN6306. Through third-generation transcriptome sequencing, we obtained the full-length transcript of TaW662 . Notably, TaW662 exhibited upregulated expression in response to powdery mildew infection in SN6306. Subcellular localization analysis revealed TaW662’s extracellular secretion, suggesting its role in defense mechanisms. Additionally, the TaW662 protein was expressed in Escherichia coli , and the purified protein could inhibit the growth of Bgt in vitro . Utilizing the online alphafold2 server, we predicted the three-dimensional structure of TaW662, aiding in understanding its fungicidal mechanisms. Analysis of TaW662’s physicochemical properties further supported its potential efficacy as a fungicide against Bgt . In conclusion, TaW662 emerges as a promising candidate for combating Bgt -induced wheat fungal diseases, warranting further exploration for agricultural disease management strategies. Highlights TaW662 , a secreted protein homologous to TaWIR1 , is induced in wheat by Blumeria graminis f. sp. tritici ( Bgt ). The expression pattern of TaW662 in wheat under induced by Bgt was analysed using RNA-Seq technology. The three-dimensional structure of TaW662 was predicted using AlphaFold2. The growth of Bgt is inhibited by recombinant TaW662. ### Competing Interest Statement The authors have declared no competing interest. * AMP : antimicrobial peptide SSH : suppression subtractive hybridization GFP : green fluorescent protein FPKM : Fragments Per Kilo bases per Million fragments SN6306 : Wheat varieties Shan Nong 6306 YN15 : Wheat varieties Yan Nong 15 WIR : wheat-induced resistance

Schematic diagram of the preparation of the enzyme-responsive nanoplatform HA-CP@Fe3O4 and its application in the study of antibacterial infection in vivo.

Peanut (Arachis hypogaea L.) is an important cash and oil seed crop, mostly distributed in arid and semi-arid areas. In recent years, due to the influence of atmospheric circulation anomalies and other factors, drought has become frequent and increasingly serious in China. This has posed serious challenges to peanut production. The objective of this study was to investigate the potential of the endophytic fungus Piriformospora indica to form a symbiotic relationship with peanut plants and to evaluate the drought tolerance of P. indica-colonized peanut plants subjected to a simulated drought stress treatment using 20% polyethylene glycol 6000 (PEG6000). The endophytic fungus P. indica affected the physiological characteristics of the host plant by colonizing the plant roots, thereby conferring greater resistance to drought stress. This fungus strongly colonized the roots of peanuts and was found to enhance root activity after 24 h of P. indica colonization under PEG6000. Catalase (CAT) and peroxidase (POD) activities were increased at 24 h in peanut leaves colonized with P. indica. Expression of drought-related genes, such as AhNCED1, AhP5CS, and DREB2A was upregulated at 24 h of P. indica colonization. In addition, after PEG6000 treatment, proline, soluble protein, and abscisic acid (ABA) concentrations in plants were increased, while the accumulation of malondialdehyde (MDA), and hydrogen peroxide (H2O2) was decreased in P. indica colonized peanut. In conclusion, P. indica mediated peanut plant protection against the detrimental effects of drought resulted from enhanced antioxidant enzyme activities, and the upregulated expression of drought-related genes for lower membrane damage.

Members of the ADP-ribosylation factor family, which are GTP-binding proteins, are involved in metabolite transport, cell division, and expansion. Although there has been a significant amount of research on small GTP-binding proteins, their roles and functions in regulating maize kernel size remain elusive. Here, we identified ZmArf2 as a maize ADP-ribosylation factor-like family member that is highly conserved during evolution. Maize zmarf2 mutants showed a characteristic smaller kernel size. Conversely, ZmArf2 overexpression increased maize kernel size. Furthermore, heterologous expression of ZmArf2 dramatically elevated Arabidopsis and yeast growth by promoting cell division. Using expression quantitative trait loci (eQTL) analysis, we determined that ZmArf2 expression levels in various lines were mainly associated with variation at the gene locus. The promoters of ZmArf2 genes could be divided into two types, pS and pL, that were significantly associated with both ZmArf2 expression levels and kernel size. In yeast-one-hybrid screening, maize Auxin Response Factor 24 (ARF24) is directly bound to the ZmArf2 promoter region and negatively regulated ZmArf2 expression. Notably, the pS and pL promoter types each contained an ARF24 binding element: an auxin response element (AuxRE) in pS and an auxin response region (AuxRR) in pL, respectively. ARF24 binding affinity to AuxRR was much higher compared with AuxRE. Overall, our results establish that the small G-protein ZmArf2 positively regulates maize kernel size and reveals the mechanism of its expression regulation.

Rhizosphere microbial communities have a relationship with plant growth and soil fertility and play an important role in the ecosystem. Here, we investigated the effects of different fertilization on the microbial community structure in the peanut rhizosphere soil using Illumina MiSeq high throughput sequencing. Yubao No. 4 cultivar was used in this study as the experimental material. Compound fertilizer (CF), compound fertilizer + microbial agent (CF+MA), compound fertilizer + microbial fertilizer (CF+MF) and compound fertilizer + microbial agent + microbial fertilizer (CF+MM) was applied to the soil. Results showed that CF+MA, CF+MF and CF+MM treatments significantly increased peanut yield. The activities of catalase, urease and acid phosphatase in the soil were significantly raised in CF+MA and CF+MM treatment compared to CF during the blossom stage and pod-setting stage. Sequencing result indicated that the bacteria richness in rhizosphere soil of peanut was increased after CF+MA was applied. The fungal diversity was decreased after CF+MA, CF+MF and CF+MM was applied. The dominant bacterial phyla found in all samples were Proteobacteria, Actinobacteria and Acidobacteria. Members of the Ascomycota and Basidiomycota phyla dominated the fungal component of the rhizosphere soil microbiome across all treatments. Our results indicate that microbial agent and microbial fertilizer alter the microbial community structure of peanut rhizosphere soil. The abundance of potentially beneficial bacteria ( Bradyrhizobium , Rhizobium and Burkholderia ) and fungi ( Trichoderma and Cladophialophora ) increased, while that of potentially pathogenic fungi ( Penicillium and Fusarium ) decreased, thereby significantly promoting plant growth and yield of peanut.

Heat stress (HS) seriously restricts the growth and development of plants. When plants are exposed to extreme high temperature, the heat stress response (HSR) is activated to enable plants to survive. Sessile plants have evolved multiple strategies to sense and cope with HS. Previous studies have established that PHYTOCHROME INTERACTING FACTOR 4 (PIF4) acts as a key component in thermomorphogenesis; however, whether PIF4 regulates plant thermotolerance and the molecular mechanism linking this light transcriptional factor and HSR remain unclear. Here, we show that the overexpression of PIF4 indeed provides plants with a stronger basal thermotolerance and greatly improves the survival ability of Arabidopsis under severe HS. Via phylogenetic analysis, we identified two sets (six) of PIF4 homologs in wheat, and the expression patterns of the PIF4 homologs were conservatively induced by heat treatment in both wheat and Arabidopsis. Furthermore, the PIF4 protein was accumulated under heat stress and had an identical expression level. Additionally, we found that the core regulator of HSR, HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), was highly responsive to light and heat. Followed by promoter analysis and ChIP-qPCR, we further found that PIF4 can bind directly to the G-box motifs of the HSFA2 promoter. Via effector–reporter assays, we found that PIF4 binding could activate HSFA2 gene expression, thereby resulting in the activation of other HS-inducible genes, such as heat shock proteins. Finally, the overexpression of PIF4 led to a stronger basal thermotolerance under non-heat-treatment conditions, thereby resulting in an enhanced tolerance to severe heat stress. Taken together, our findings propose that PIF4 is linked to heat stress signaling by directly binding to the HSFA2 promoter and triggering the HSR at normal temperature conditions to promote the basal thermotolerance. These functions of PIF4 provide a candidate direction for breeding heat-resistant crop cultivars.

Heat shock factors (Hsfs) play pivotal roles in plant stress responses and confer stress tolerance. However, the functions of several Hsfs in rice (Oryza sativa L.) are not yet known. In this study, genome-wide analysis of the Hsf gene family in rice was performed. A total of 25 OsHsf genes were identified, which could be clearly clustered into three major groups, A, B, and C, based on the characteristics of the sequences. Bioinformatics analysis showed that tandem duplication and fragment replication were two important driving forces in the process of evolution and expansion of the OsHsf family genes. Both OsHsfB4b and OsHsfB4d showed strong responses to the stress treatment. The results of subcellular localization showed that the OsHsfB4b protein was in the nucleus whereas the OsHsfB4d protein was located in both the nucleus and cytoplasm. Over-expression of the OsHsfB4b gene in Arabidopsis and rice can increase the resistance to drought stress. This study provides a basis for understanding the function and evolutionary history of the OsHsf gene family, enriching our knowledge of understanding the biological functions of OsHsfB4b and OsHsfB4d genes involved in the stress response in rice, and also reveals the potential value of OsHsfB4b in rice environmental adaptation improvement.

Traditional antibiotics have made great contributions to human health and animal husbandry since the discovery of penicillin in 1928, but bacterial resistance and drug residues are growing threats to global public health due to the long-term uncontrolled application of antibiotics. There is a critical need to develop new antimicrobial drugs to replace antibiotics. Antimicrobial peptides (AMPs) are distributed in all kingdoms of life, presenting activity against pathogens as well as anticancer, anti-inflammatory, and immunomodulatory activities; consequently, they have prospects as new potential alternatives to antibiotics. Porcine myeloid antimicrobial peptides (PMAPs), the porcine cathelicidin family of AMPs, have been reported in the literature in recent years. PMAPs have become an important research topic due to their strong antibacterial activity. This review focuses on the universal trends in the biochemical parameters, structural characteristics and biological activities of PMAPs.

The protection of current influenza vaccines is limited due to the viral antigenic shifts and antigenic drifts. The universal influenza vaccine is a new hotspot in vaccine research that aims to overcome these problems. Polydopamine (PDA), a versatile biomaterial, has the advantages of an excellent biocompatibility, controllable particle size, and distinctive drug loading approach in drug delivery systems. To enhance the immunogenicities and delivery efficiencies of H9N2 avian influenza virus (AIV) epitope peptide vaccines, PDA nanoparticles conjugated with the BPP-V and BP-IV epitope peptides were used to prepare the nano BPP-V and BP-IV epitope peptide vaccines, respectively. The characteristics of the newly developed epitope peptide vaccines were then evaluated, revealing particle sizes ranging from approximately 240 to 290 nm (PDI<0.3), indicating that the synthesized nanoparticles were stable. Simultaneously, the immunoprotective effects of nano BPP-V and BP-IV epitope peptide vaccines were assessed. The nano BPP-V and BP-IV epitope vaccines, especially nano BP-IV epitope vaccine, quickly induced anti-hemagglutinin (HA) antibody production and a sustained immune response, significantly promoted humoral and cellular immune responses, reduced viral lung damage and provided effective protection against AIV viral infection. Together, these results reveal that PDA, as a delivery carrier, can improve the immunogenicities and delivery efficiencies of H9N2 AIV nano epitope vaccines, thereby providing a theoretical basis for the design and development of PDA as a carrier of new universal influenza vaccines.

Short peptide antigens covering conserved T or B cell epitopes have been investigated in influenza vaccines. Bursal pentapeptide V (BPP-V) and bursal peptide IV (BP-IV) are small molecular peptides that were isolated and identified from the bursa of Fabricius (BF) and induce a strong immune response at both the humoural and cellular levels. To explore the molecular adjuvant potential of BPP-V and BP-IV with an epitope vaccine, an epitope peptide (HA284-298, GNCVVQCQTERGGLN) rich in T and B cell epitopes for the H9N2 avian influenza virus (AIV) haemagglutinin (HA) protein was selected. BPP-V and BP-IV were coupled with the epitope peptide sequence to form BPP-V and BP-IV-epitope vaccines, respectively. The immunoefficacy of BPP-V and BP-IV-epitope peptide vaccines was evaluated. The results showed that the epitope peptide had weak immunogenicity. The BPP-V-epitope peptide vaccine promoted only the secretion of anti-HA IgG and IgG1 antibodies. The BP-IV-epitope peptide vaccine not only promoted the production of anti-HA IgG and IgG1 antibodies but also significantly induced the production of the IgG2a antibody. The BP-IV-epitope peptide vaccine significantly promoted the production of interleukin (IL-4) and interferon-γ (IFN-γ) (the BPP-V epitope peptide vaccine promoted only the production of IL-4), enhanced the cytotoxic T lymphocyte (CTL) response, and increased the proportion of CD3+ T lymphocytes. Moreover, the BP-IV-epitope peptide vaccine promoted a cell-mediated immune response similar to that of the AIV vaccine group. Furthermore, BPP-V and BP-IV-epitope peptide vaccines could also accelerate the clearance of pulmonary virus and reduce pathological damage after the challenge with H9N2 AIV. This study demonstrates the potential of BP-IV as an effective adjuvant for the epitope peptide vaccine for the H9N2 AIV.

抗生素的耐药性和动物源性食品中的药物残留问题严重威胁全球公共卫生系统.因此,开发出不易产生耐药性、抗菌活性高的新型抗菌药物迫在眉睫.抗菌肽因其分子量小、抗菌谱广、不易产生耐药性等优点受到科学家们的广泛关注,但天然抗菌肽具有抗菌活性低、溶血活性和细胞毒性等缺陷.随着抗菌肽序列和结构的不断优化,多种具有显著体内外抗菌活性且安全高效的新型抗菌药物被研发出来.猪源抗菌肽PMAP-36是从猪骨髓细胞中分离出来的一种具有典型两亲性a-螺旋结构的高阳离子抗菌肽.本文就国内外关于猪源抗菌肽PMAP-36的序列设计及其结构优化等方面的研究进展进行综述.