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Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions.

Background Tobacco is a vital economic crop, and its cultivation is severely threatened by black shank disease, caused by Phytophthora nicotianae ( P. nicotianae ). Actin depolymerizing factor (ADF) proteins are key regulators of actin dynamics and play crucial roles in plant growth, intracellular transport, and stress responses. Although the ADF gene family has been extensively studied in various plant species, characterization in tobacco remains limited, particularly regarding its role in disease resistance. Results In this study, 24 NtADF genes were identified in the tobacco genome. Phylogenetic analysis classified them into six groups, with structural and motif compositions supporting functional conservation within groups. Evolutionary analysis revealed that whole-genome duplication (WGD) or segmental duplication (SDR) was the primary drivers of NtADFs duplication, with strong purification selection. Promoter analysis identified numerous stress- and hormone-related cis-elements, and the regulatory network predictions indicated potential interactions with transcription factors (e.g., ERF, MYB, WRKY) and microRNAs (miRNAs). Expression profiling demonstrated tissue-specific patterns and differential responses to abiotic and biotic stresses. Notably, functional characterization demonstrated that NtADF22 played a critical role in conferring resistance to black shank disease. Conclusions This study provides comprehensive insights into the NtADF gene family, highlighting its important roles in plant development and stress responses. These findings offer both theoretical and practical significance for molecular breeding and genetic improvement of tobacco. Graphical abstract

The ubiquitin–proteasome system is a highly conserved machinery that plays a crucial role in plant defense against viruses. However, the number of E3 ligases targeting viral proteins remains limited. Although RING‐between‐RING (RBR)‐type E3 ligases are evolutionarily conserved across organisms, their functions in plant responses to biotic stress remain largely unknown. Herein, it is found that the triple gene block 1 (TGB1) protein of the barley stripe mosaic virus (BSMV) undergoes ubiquitination during viral infection. Immunoprecipitation combined with mass spectrometry identified an RBR‐type E3 ligase that interacted with TGB1 in vivo and in vitro. The overexpression of Ariadne‐like protein 8 (ARI8) inhibits, whereas its knockout enhances, the local and systemic spread of BSMV. ARI8 mediated the ubiquitination of TGB1, and its Cys311 residue is required for the ARI8‐mediated degradation of TGB1 and inhibition of BSMV infection. In addition to BSMV, ARI8 negatively regulates infection by other TGB‐containing viruses, including potato virus X and beet necrotic yellow vein virus. Collectively, the findings identified a new E3 ligase that targets a plant viral protein and reveals a previously uncharacterized role for RBR‐type E3 ligases in plant responses to biotic stress, providing a potential molecular target for the development of antiviral strategies in plants. The plant RING‐between‐RING (RBR)‐type E3 ligase, Ariadne‐like protein 8 (ARI8), ubiquitinates and degrades a viral movement protein, restricting virus spread. ARI8 also inhibits other triple gene block 1 (TGB1)‐containing viruses, revealing a broad‐spectrum antiviral role. This study uncovers a previously unknown function of RBR‐type E3 ligases in plant immunity and offers a potential target for antiviral crop improvement.

BackgroundGlandular trichomes, which act as the first barrier against damage induced by insects and disease, can produce specialized metabolites that play important roles during plant development. However, the role played by microRNAs (miRNAs), which regulate many plant physiological processes, during trichome development is not unraveled in detail.ResultsIn this study, we performed RNA sequencing (RNA-Seq) and small RNA sequencing assessments of tobacco trichome, leaf minus trichome (leaf-trichome), and leaf tissue to improve our understanding of the miRNA mechanisms regulating trichome development. Totally, we identified 270 differentially expressed miRNAs (DEMs) and 10,430 differentially expressed genes (DEGs) between trichome and leaf-trichome tissues. DEM targets were mainly associated with plant hormone signal transduction, plant–pathogen interactions, and the biosynthesis of secondary metabolites. Of these, 1233 miRNA–mRNA pairs were identified with reverse expression patterns. Next, we used dual-luciferase reporter (LUC) assays to reveal that several potential targets were significantly inhibited by corresponding miRNAs, including the transcription factors (TF) NAC021, AP2, MYB36, WRKY6 and TIFY10B. Further analysis showed that miR6155-WRKY6 might perform vital roles in trichome development, and that overexpression of miR6155 resulted in decreased trichome density.ConclusionsTaken together, these findings demonstrate that miRNAs may be involved in trichome development in tobacco, and they may advance our understanding of the regulation of trichome development mediated by miRNA and can help to improve genetic engineering of trichome regulation in plants.

At present, the study on exopolysaccharid is mainly focused on lactic acid bacteria, and the research on exopolysaccharide produced by yeast, especially Sporidiobolus pararoseus, is relatively few. Therefore, the aim of this study was to explore the characterization and antioxidant activities of a novel neutral exopolysaccharide SPZ, which was isolated and purified from S. pararoseus PFY-Z1. The results showed that SPZ was mainly composed of mannose, followed by glucose, with a molecular weight was 24.98 kDa, had O-glycosidic bonds, no crystalline, and no triple helix structure. Based on fourier transform-infrared, high-performance liquid chromatography and nuclear magnetic resonance analyses, SPZ was identified to be a exopolysaccharide with some side chains, presence of α-, β-pyranose ring and nine sugar residues. Furthermore, the morphology features of SPZ have performed a relatively rough and uneven surface, covered with small pores and fissures. Moreover, SPZ had higher antioxidant activities and the maximum scavenging abilities of ⋅OH, NO2− and reducing power were 28.05 ± 0.73%, 92.76 ± 1.86% and 0.345 ± 0.024, respectively. Hence, SPZ could be used as a potential antioxidant application in the food and pharmaceutical industries.

Summary RNA silencing plays a crucial role in defending against viral infections in diverse eukaryotic hosts. Despite extensive studies on core components of the antiviral RNAi pathway such as DCLs, AGOs and RDRs proteins, host factors involved in antiviral RNAi remain incompletely understood. In this study, we employed the proximity labelling approach to identify the host factors required for antiviral RNAi in Nicotiana benthamiana. Using the barley stripe mosaic virus (BSMV)‐encoded γb, a viral suppressor of RNA silencing (VSR), as the bait protein, we identified the DEAD‐box RNA helicase RH20, a broadly conserved protein in plants and animals with a homologous human protein known as DDX5. We demonstrated the interaction between RH20 and BSMV γb. Knockdown or knockout of RH20 attenuates the accumulation of viral small interfering RNAs, leading to increased susceptibility to BSMV, while overexpression of RH20 enhances resistance to BSMV, a process requiring the cytoplasmic localization and RNA‐binding activity of RH20. In addition to BSMV, RH20 also negatively regulates the infection of several other positive‐sense RNA viruses, suggesting the broad‐spectrum antiviral activity of RH20. Mechanistic analysis revealed the colocalization and interaction of RH20 with SGS3/RDR6, and disruption of either SGS3 or RDR6 undermines the antiviral function of RH20, suggesting RH20 as a new component of the SGS3/RDR6 bodies. As a counter‐defence, BSMV γb VSR subverts the RH20‐mediated antiviral defence by interfering with the RH20–SGS3 interaction. Our results uncover RH20 as a new positive regulator of antiviral RNAi and provide new potential targets for controlling plant viral diseases.

[Objective] This study aimed to investigate the protective effects of proteins, salts, sugars and trace elements on soybean rhizobium strain HW-05 by response surface methodology. [Method] Different types, combinations, ratios and concentrations of protective agents were designed. Under simulated conditions, the optimal protective agent formulation was screened for improving the survival rate and survival time of soybean rhizobium strain HW-05. The optimal combination of significant factors was determined by Box-Behnken central composite design. [Result] Three significant factors affecting effective number of viable cells were screened, including peptone, xanthan gum and NaCl. The final concentration of each compound was optimized 0. 13% peptone, 0.011% xanthan gum, 0.30% NaCl. [Conclusion] After addition of protective agent and preservation at room temperature for six months, effective number of viable cells of soybean rhizobium strain HW-05 reached 3.185 ×10^8 CFU/ml. The survival rate of HW-05 cells was improved by more than 25% compared with the control group （2.458×10^8 CFU /ml）.

We have previously identified 1 241 regions of somatic copy number alterations （CNAs） in hepatocellular carcinoma （HCC）. In the present study, we found that a novel recurrent focal amplicon, lq24.1-24.2, targets the MPZL1 gene in HCC. Notably, there is a positive correlation between the expression levels of MPZL1 and intrahepatic metastasis of the HCC specimens. MPZL1 can significantly enhance the migratory and metastatic potential of the HCC cells. Moreover, we found that one of the mechanisms by which MPZL1 promotes HCC cell migration is by inducing the phosphorylation and activation of the pro-metastatic protein, cortactin. Additionally, we found that Src kinase mediates the phosphorylation and activation of cortactin induced by MPZL1 overexpression. Taken together, these findings suggest that MPZL1 is a novel pro-metastatic gene targeted by a recurrent region of copy number amplification at lq24.1-24.2 in HCC.

With the continuous growth of the inkjet printing market, it has become particulary important to endow dye thermal transfer paper with fast-drying performance and fine transfer effect. In order to achieve this goal, the nanohybrid materials of HNTs/MSN (halloysite nanotubes/mesoporous silica) were prepared by Stöber method and in situ growth method, and then, the thermal sublimation transfer papers coated with CMC/MSN, CMC/HNTs, and CMC/HNTs/MSN were prepared, respectively. Characterizations of MSN, HNTs, and HNTs/MSN were conducted using TEM, FTIR, XRD, XPS, and BET techniques, followed by testing the properties of the coated papers. The results showed that the HNTs/MSN nanohybrid materials were formed by deposited MSN on HNTs with the Stöber method and in situ growth method. Among the three types of papers, CMC/HNTs/MSN paper exhibited the best comprehensive performance with fast-drying performance and fine transfer effect. Therefore, CMC/HNTs/MSN can be used as a novel type of thermal transfer paper coating with fast ink drying speed and fine transfer effect.

Longitudinal vibration of non-uniform rod has been of great significance in various engineering occasions. The existing works are usually limited to the certain area variation and/or classical boundary condition. Motivated by this limitation, an efficient accurate solution is developed for the longitudinal vibration of a general variable cross-section rod with arbitrary boundary condition. Displacement function is invariantly expressed as the summation of standard Fourier series and supplementary polynomials, with an aim to make the calculation of all derivatives more straightforwardly in the whole solving region [0, L]. Energy principle is employed for system dynamics formulation, with the elastic boundaries considered as potential energy stored in the restraining spring. Arbitrary cross-section area variation is uniformly expanded into Fourier series. Numerical examples are presented for the natural frequency and mode shapes of non-uniform rod of free and clamped boundary conditions and compared with literature data. Results show good agreement with the previous analytical solutions. Influence of cross section area variation on vibration characteristics of non-uniform rods is then studied and discussed. One of the most advantages of the proposed model is that there is no need to reformulate the problem or rewrite the codes when the cross-section area distribution and/or boundary conditions change arbitrarily.