SCN1A intronic variants impact on Nav1.1 protein expression and sodium channel function, and associated with epilepsy phenotypic severity.

High-throughput sequencing has identified numerous intronic variants in the SCN1A gene in epilepsy patients. Abnormal mRNA splicing caused by these variants can lead to significant phenotypic differences, but the mechanisms of epileptogenicity and phenotypic differences remain unknown. Two variants, c.4853-1 G>C and c.4853-25 T>A, were identified in intron 25 of SCN1A, which were associated with severe Dravet syndrome (DS) and mild focal epilepsy with febrile seizures plus (FEFS+), respectively. The impact of these variants on protein expression, electrophysiological properties of sodium channels and their correlation with epilepsy severity was investigated through plasmid construction and transfection based on the aberrant spliced mRNA. We found that the expression of truncated mutant proteins was significantly reduced on the cell membrane, and retained in the cytoplasmic endoplasmic reticulum. The mutants caused a decrease in current density, voltage sensitivity, and an increased vulnerability of channel, leading to a partial impairment of sodium channel function. Notably, the expression of DS-related mutant protein on the cell membrane was higher compared to that of FEFS+-related mutant, whereas the sodium channel function impairment caused by DS-related mutant was comparatively milder than that caused by FEFS+-related mutant. Our study suggests that differences in protein expression levels and altered electrophysiological properties of sodium channels play important roles in the manifestation of diverse epileptic phenotypes. The presence of intronic splice site variants may result in severe phenotypes due to the dominant-negative effects, whereas non-canonical splice site variants leading to haploinsufficiency could potentially cause milder phenotypes.

The O-GlcNAc database: introducing new features and tools developed from community feedback.

O-GlcNAc is a reversible post-translational modification found on serine and threonine residues of nucleocytoplasmic proteins. Four years ago, we released the O-GlcNAc Database ( oglcnac.mcw.edu ), a comprehensive catalog of O-GlcNAcylated proteins that has become one of the most cited resources in the field, with hundreds of unique users per month. We are now presenting an updated O-GlcNAc Database, which includes nearly 20,000 O-GlcNAcylated proteins and 48 species, marking substantial growth in data volume and scope. This paper presents the most noteworthy features implemented over the last year, often originating from feedback from the O-GlcNAc community. Among these features, we provide a brief overview of the database content, introduce our new protein viewer mode, and discuss the implementation of subcellular localization information and its applications in the O-GlcNAc score. We also provide an interface to use CytOVS, a tool designed to evaluate and sort O-GlcNAcome datasets derived from MS experiments. In conclusion, this new and improved O-GlcNAc Database represents a significant advancement in providing a comprehensive and expanded resource for researchers in the field of O-GlcNAc biology.

Genome-wide identification and expression analysis of the U-box gene family related to biotic and abiotic stresses in Coffea canephora L.

Plant U-box genes play an important role in the regulation of plant hormone signal transduction, stress tolerance, and pathogen resistance; however, their functions in coffee (Coffea canephora L.) remain largely unexplored. In this study, we identified 47 CcPUB genes in the C. canephora L. genome, clustering them into nine groups via phylogenetic tree. The CcPUB genes were unevenly distributed across the 11 chromosomes of C. canephora L., with the majority (11) on chromosome 2 and none on chromosome 8. The cis-acting elements analysis showed that CcPUB genes were involved in abiotic and biotic stresses, phytohormone responsive, and plant growth and development. RNA-seq data revealed diverse expression patterns of CcPUB genes across leaves, stems, and fruits tissues. qRT-PCR analyses under dehydration, low temperature, SA, and Colletotrichum stresses showed significant up-regulation of CcPUB2, CcPUB24, CcPUB34, and CcPUB40 in leaves. Furthermore, subcellular localization showed CcPUB2 and CcPUB34 were located in the plasma membrane and nucleus, and CcPUB24 and CcPUB40 were located in the nucleus. This study provides valuable insights into the roles of PUB genes in stress responses and phytohormone signaling in C. canephora L., and provided basis for functional characterization of PUB genes in C. canephora L.

Targeting catalase in cancer.

Healthy cells have developed a sophisticated network of antioxidant molecules to prevent the toxic accumulation of reactive oxygen species (ROS) generated by diverse environmental stresses. On the opposite, cancer cells often exhibit high levels of ROS and an altered levels of antioxidant molecules compared to normal cells. Among them, the antioxidant enzyme catalase plays an essential role in cell defense against oxidative stress through the dismutation of hydrogen peroxide into water and molecular oxygen, and its expression is often decreased in cancer cells. The elevation of ROS in cancer cells provides them proliferative advantages, and leads to metabolic reprogramming, immune escape and metastasis. In this context, catalase is of critical importance to control these cellular processes in cancer through various mechanisms. In this review, we will discuss the major progresses and challenges in understanding the role of catalase in cancer for this last decade. This review also aims to provide important updates regarding the regulation of catalase expression, subcellular localization and discuss about the potential role of microbial catalases in tumor environment. Finally, we will describe the different catalase-based therapies and address the advantages, disadvantages, and limitations associated with modulating catalase therapeutically in cancer treatment.

The endoplasmic reticulum as an active liquid network.

The peripheral endoplasmic reticulum (ER) forms a dense, interconnected, and constantly evolving network of membrane-bound tubules in eukaryotic cells. While individual structural elements and the morphogens that stabilize them have been described, a quantitative understanding of the dynamic large-scale network topology remains elusive. We develop a physical model of the ER as an active liquid network, governed by a balance of tension-driven shrinking and new tubule growth. This minimalist model gives rise to steady-state network structures with density and rearrangement timescales predicted from the junction mobility and tubule spawning rate. Several parameter-independent geometric features of the liquid network model are shown to be representative of ER architecture in live mammalian cells. The liquid network model connects the timescales of distinct dynamic features such as ring closure and new tubule growth in the ER. Furthermore, it demonstrates how the steady-state network morphology on a cellular scale arises from the balance of microscopic dynamic rearrangements.

A modified procedure for separating yeast peroxisomes from mitochondria.

Mitochondria and peroxisomes are mutually dependent organelles that share several membrane proteins that carry out the same function in both organelles. To study the unique features of these dually localized proteins in each of the two organelles, it is essential to separate mitochondria from peroxisomes. Isolating organelles from cells of Baker's yeast, Saccharomyces cerevisiae, is crucial for our understanding of the biogenesis and functions of proteins. Traditionally, subcellular fractionation and isolation of individual organelles by differential centrifugation benefit from the specific and unique density of each organelle. However, when yeast cells are grown under normal conditions, certain organelles like mitochondria and peroxisomes share strikingly similar densities. This similarity challenges the separation of these organelles from one another. In this chapter, we describe an optimized procedure to address this task. We depict growth conditions that would favor stimulation of peroxisomes to increase their number and density, and portray organellar isolation followed by gradient centrifugation, enabling an improved separation of both organelles. Additionally, we illustrate the advantage of the procedure to study the dual localization of the membrane protein Fis1.

Characterization of two phosphatase 2C domain-containing proteins PPM2A and PPM2B in Toxoplasma gondii.

Protein phosphatases Mg2+/Mn2+ dependent (PPMs), serine/threonine phosphatases, are widely distributed in apicomplexan parasites, and Toxoplasma gondii possesses the largest number of PPMs in the apicomplexan parasites. Though the function of some PPMs has been characterized in T. gondii, much less is known about two phosphatase 2C domain-containing proteins, PPM2A and PPM2B. PPM2A was identified as one of Toxoplasma Calmodulin's interacting proteins through proximity-based protein interaction BioID technology in the previous study, and PPM2B was the homolog of PPM2A in T. gondii. In this study, PPM2A was distributed in the whole tachyzoite of T. gondii, and PPM2B was mainly distributed in the cytoplasm by inserting a 10HA tag in the C-terminus of the two genes in the RH∆ku80 strain. PPM2A knockout (Δppm2a), PPM2B knockout (Δppm2b), and double knockout (ΔΔ) in RHΔhxgprt type I strain under CRISPR-Cas9 system did not result in intracellular replication defect. Besides, mouse experiments demonstrated that PPM2A, PPM2B, and double knockout did not reduce the pathogenicity of T. gondii compared with the RH∆hxgprt strain. However, the plaque size of these single knockout and double knockout strains were smaller than that in the control RH∆hxgprt strain. Our results provide new insight into the function of PPMs in the pathogenesis of T. gondii.

Increased artemisinin production in Artemisia annua L. by co-overexpression of six key biosynthetic enzymes.

Malaria remains a global health issue, especially in resource-limited regions. Artemisinin, a key antimalarial compound from Artemisia annua, is crucial for treatment, but low natural yields hinder large-scale production. In this study, we employed advanced transgenic technology to co-overexpress six key biosynthetic enzymes-Isopentenyl Diphosphate Isomerase (IDI), Farnesyl Pyrophosphate Synthase (FPS), Amorpha 4,11-diene Synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), cytochrome P450 oxidoreductase (AACPR) and artemisinic aldehyde D11 reductase (DBR2)-in A. annua to significantly enhance artemisinin production. Our innovative approach utilized a co-expression strategy to optimize the artemisinin biosynthetic pathway, leading to a remarkable up to 200 % increase in artemisinin content in T1 transgenic plants compared to non-transgenic controls. The stability and efficacy of this transformation were confirmed in subsequent generations (T2), achieving a potential 232 % increase in artemisinin levels. Additionally, we optimized transgene expression to maintain plant growth and development, and performed untargeted metabolite analysis using GC-MS, which revealed significant changes in metabolite composition among T2 lines, indicating effective diversion of farnesyl diphosphate into the artemisinin pathway. This metabolic engineering breakthrough offers a promising and scalable solution for enhancing artemisinin production, representing a major advancement in the field of plant biotechnology and a potential strategy for more cost-effective malaria treatment.

Bioaccumulation and toxicological effects of dietborne arsenic exposure on the apple snail (Pomacea canaliculata).

An eight-compartment physiologically based pharmacokinetic (PBPK) model was used to simulate the bioaccumulation and distribution of arsenic (As) within the apple snail (Pomacea canaliculata) following the ingestion of As-contaminated lettuce. The bioaccumulation results revealed that the shell contained the majority (67.21 %) of the total As content, with the liver and the head-foot containing approximately 11.14 % and 10.45 % of the total As content in the snail, respectively. Modeling quantified the process of intestine-stomach absorption of dietborne As and revealed its crucial role in the subsequent distribution of As within the body. The liver is the primary metabolic site, whereas the shell is the primary storage site. Exposure to dietborne As leads to pronounced physiological and biochemical alterations in apple snails. Total protein levels decreased by 24.06 %, superoxide dismutase (SOD) activity decreased by 24.43 %, malondialdehyde (MDA) content increased by 47.51 %, glutathione (GSH) content decreased by 46.99 %, and glutathione S-transferase (GST) activity decreased by 42.22 %. Furthermore, the subcellular-level results indicated that dietborne As exposure altered subcellular distribution in the liver. Additionally, dietborne As exposure significantly reduced the abundance of gut microbiota in apple snails.

Sequence characteristics, expression and subcellular localization of PtCYP721A57 gene from cytochrome P450 family in Polygala tenuifolia willd.

The Cytochrome P450 (CYP450) family is the largest enzyme protein family in plants, distributed across various organs and involved in significant catalytic activities in primary and secondary metabolic processes. In this study, we cloned the PtCYP721A57 gene, characterized its open reading frame (ORF), and conducted comprehensive analyses including physicochemical properties, evolutionary relationships, subcellular localization, prokaryotic expression, and correlation between the relative expression of different parts and the content of tenuifolin, hormones, and abiotic stress response associated with the encoded protein. The ORF of PtCYP721A57 was 1,521 bp, with a secondary structure predominantly composed of α-helices and random coils. Subcellular localization experiments confirmed the presence of PtCYP721A57 in the endoplasmic reticulum. For prokaryotic expression, we constructed the recombinant plasmid pET28a-PtCYP721A57 using pET28a as the vector, which was then transformed into BL21(DE3). Induction with Isopropyl ß-D-1-thiogalactopyranoside (IPTG) at temperatures of 16 and 25 °C and varying concentrations (0.1, 0.2, 0.5, 1, 2 mM) resulted in the formation of inclusion bodies, with higher expression observed at 25 °C. Our qPCR analyses revealed that PtCYP721A57 exhibited the highest expression in the cortex of Polygala tenuifolia, followed by roots and xylem, correlating with the observed tenuifolin content distribution. Induction with abscisic acid (ABA) and chitosan (CHT) initially decreased PtCYP721A57 expression followed by a subsequent increase, peaking at 48 h. Similarly, drought stress induced a gradual increase in PtCYP721A57 expression, also peaking at 48 h. NaCl treatment for 6 h significantly upregulated PtCYP721A57 expression. In conclusion, our study provides foundational insights into the PtCYP721A57 gene in Polygala tenuifolia, laying the groundwork for further exploration of its role in the biosynthesis pathway of triterpenoid saponins.

Quantitative dynamics of intracellular NMN by genetically encoded biosensor.

Nicotinamide mononucleotide (NMN) is the direct precursor and a major booster of NAD+ with increasing applications in NAD+- and aging-related pathologies. However, measuring live cell NMN dynamics was not possible, leaving key questions in NMN uptake and intracellular regulation unanswered. Here we developed genetically encoded bioluminescent and fluorescent sensors to quantify subcellular NMN in live cells by engineering specific NMN-responsive protein scaffolds fused to luciferase and fluorescent proteins. The sensor dissected the multimechanistic uptake of exogenous NMN and nicotinamide riboside (NR) in live cells and further measured the NMN levels across different subcellular compartments, as well as the perturbed NMN/NAD+ ratios by external supplements. Moreover, we measured the NMN regulation by NAD(H) hydrolase Nudts and peroxisomal carrier Pxmp2 and identified Slc25a45 as a potential mitochondrial NMN regulator for its unique fingerprint on the local NMN/NAD+ ratio. Collectively, the genetically encoded sensors provide a useful tool for visualizing NMN metabolism.

Transgenic sensors reveal compartment-specific effects of aggregation-prone proteins on subcellular proteostasis during aging.

Loss of proteostasis is a hallmark of aging that underlies many age-related diseases. Different cell compartments experience distinctive challenges in maintaining protein quality control, but how aging regulates subcellular proteostasis remains underexplored. Here, by targeting the misfolding-prone FlucDM luciferase to the cytoplasm, mitochondria, and nucleus, we established transgenic sensors to examine subcellular proteostasis in Drosophila. Analysis of detergent-insoluble and -soluble levels of compartment-targeted FlucDM variants indicates that thermal stress, cold shock, and pro-longevity inter-organ signaling differentially affect subcellular proteostasis during aging. Moreover, aggregation-prone proteins that cause different neurodegenerative diseases induce a diverse range of outcomes on FlucDM insolubility, suggesting that subcellular proteostasis is impaired in a disease-specific manner. Further analyses with FlucDM and mass spectrometry indicate that pathogenic tauV337M produces an unexpectedly complex regulation of solubility for different FlucDM variants and protein subsets. Altogether, compartment-targeted FlucDM sensors pinpoint a diverse modulation of subcellular proteostasis by aging regulators.

The effect of biochar amendment on Cd accumulation in Bidens pilosa L: Changing Cd subcellular distribution, cell wall polysaccharide Cd-binding capacity and composition.

Biochar can reduce Cd uptake by plants, thereby reducing its biotoxicity, but the mechanisms involved at the subcellular level have not been thoroughly elucidated. In this work, we explored the effect of maize straw biochar on Cd accumulation by Bidens pilosa L. and its mechanism at subcellular levels. After 90 days of potting experiment, the subcellular fractions were extracted by differential centrifugation, and the polysaccharide fractions of root cell walls were extracted by leaching centrifugation, and then the Cd content of each fraction was determined. Results showed that Cd was preferentially distributed in cell walls of three organs. Additionally, biochar addition resulted in a greater distribution of Cd from cell wall to soluble fractions and organelles in stems. These results suggested that cell wall immobilization and intracellular compartmentalization were critical detoxification mechanisms tackling Cd stress with biochar addition of Bidens pilosa L. Pectin was the main sink where Cd was stored. And galacturonic acid content in pectin occupied the highest ratio among the three polysaccharide fractions. After biochar addition, in hemicellulose the change of Cd content was consistent with the change of galacturonic acid content. These results suggested that the galacturonic acid in hemicellulose played an important role in Cd binding. Biochar addition reduced the bioavailability of soil Cd and improved the growth environment, thus inducing Bidens pilosa L. to change the composition of root cell wall in response to Cd stress.

The highly conserved region within exonuclease III-like in PML-I regulates the cytoplasmic localization of PML-NBs.

The sub-nuclear protein structure PML-NB regulates a wide range of important cellular functions, while its abnormal cytoplasmic localization may have pathological consequences. However, the nature of this aberrant localization remains poorly understood. In this study, we unveil that PML-I, the most conserved and abundant structural protein of PML-NB, possesses potent cytoplasmic targeting ability within the N-terminal half of the exonuclease III-like domain encoded by its unique exon 9, independent of the known nuclear localization signal. Fusion of this region to PML-VI can relocate PML-VI from the nucleus to the cytosol. Structural and deletion analysis revealed that the cytoplasmic targeting ability of this domain was restrained by the sequences encoded by exon 8a and the 3' portion of exon 9 in PML-I. Deletion of either of these regions relocates PML-I to the cytosol. Furthermore, we observed a potential interaction between the ER-localized TREX1 and the cytoplasmic-located PML-I mutants. Our results suggest that perturbation of the EXO-like domain of PML-I may represent an important mode to translocate PMLs from the nucleus to the cytosol, thereby interfering with the normal nuclear functions of PML-NBs.

Genome-wide identification and analysis of anthocyanin synthesis-related R2R3-MYB genes in Fragaria pentaphylla.

BACKGROUND: MYB transcription factors regulate anthocyanin biosynthesis across numerous plant species. However, comprehensive genome-wide investigations regarding the R2R3-MYB gene family and its involvement in regulating anthocyanin biosynthesis in the red and white fruit color morphs of Fragaria pentaphylla remain scarce. RESULTS: A total of 101 FpR2R3-MYB genes were identified from the F. pentaphylla genome and were divided into 34 subgroups based on phylogenetic analysis. Gene structure (exon/intron) and protein motifs were particularly conserved among the FpR2R3-MYB genes, especially members within the same subgroup. The FpR2R3-MYB genes were distributed over seven F. pentaphylla chromosomes. Analysis of gene duplication events revealed five pairs of tandem duplication genes and 16 pairs of segmental duplication genes, suggesting that segmental duplications are the major pattern for expansion of the FpR2R3-MYB gene family expansion in F. pentaphylla. Cis-regulatory elements of the FpR2R3-MYB promoters were involved in cellular development, phytohormones, environmental stress and photoresponse. Based on the analysis of the FpR2R3-MYB gene family and transcriptome sequencing (RNA-seq) data, FpMYB9 was identified as a key transcription factor involved in the regulation of anthocyanin synthesis in F. pentaphylla fruits. The expression of FpMYB9 increases significantly during the ripening stage of red fruits, as confirmed by reverse transcription quantitative real-time PCR. In addition, subcellular localization experiments further confirmed the nuclear presence of FpMYB9, supporting its role as a transcription factor involved in anthocyanin biosynthesis. CONCLUSION: Our results showed that the FpR2R3-MYB genes are highly conserved and play important roles in the anthocyanin biosynthesis in F. pentaphylla fruits. Our results also provide a compelling basis for further understanding of the regulatory mechanism underlying the role of FpMYB9 in anthocyanin formation in F. pentaphylla fruits.

MSlocPRED: deep transfer learning-based identification of multi-label mRNA subcellular localization.

Subcellular localization of messenger ribonucleic acid (mRNA) is a universal mechanism for precise and efficient control of the translation process. Although many computational methods have been constructed by researchers for predicting mRNA subcellular localization, very few of these computational methods have been designed to predict subcellular localization with multiple localization annotations, and their generalization performance could be improved. In this study, the prediction model MSlocPRED was constructed to identify multi-label mRNA subcellular localization. First, the preprocessed Dataset 1 and Dataset 2 are transformed into the form of images. The proposed MDNDO-SMDU resampling technique is then used to balance the number of samples in each category in the training dataset. Finally, deep transfer learning was used to construct the predictive model MSlocPRED to identify subcellular localization for 16 classes (Dataset 1) and 18 classes (Dataset 2). The results of comparative tests of different resampling techniques show that the resampling technique proposed in this study is more effective in preprocessing for subcellular localization. The prediction results of the datasets constructed by intercepting different NC end (Both the 5' and 3' untranslated regions that flank the protein-coding sequence and influence mRNA function without encoding proteins themselves.) lengths show that for Dataset 1 and Dataset 2, the prediction performance is best when the NC end is intercepted by 35 nucleotides, respectively. The results of both independent testing and five-fold cross-validation comparisons with established prediction tools show that MSlocPRED is significantly better than established tools for identifying multi-label mRNA subcellular localization. Additionally, to understand how the MSlocPRED model works during the prediction process, SHapley Additive exPlanations was used to explain it. The predictive model and associated datasets are available on the following github: https://github.com/ZBYnb1/MSlocPRED/tree/main.

Endogenous Magnetic Lipid Droplet-Mediated Cascade-Targeted Sonodynamic Therapy as an Approach to Reversing Breast Cancer Multidrug Resistance.

Multidrug resistance (MDR) has emerged as a major barrier to effective breast cancer treatment, contributing to high rates of chemotherapy failure and disease recurrence. There is thus a pressing need to overcome MDR and to facilitate the efficient and precise treatment of breast cancer in a targeted manner. In this study, endogenous functional lipid droplets (IR780@LDs-Fe3O4/OA) were developed and used to effectively overcome the limited diffusion distance of reactive oxygen species owing to their amenability to cascade-targeted delivery, thereby facilitating precise and effective sonodynamic therapy (SDT) for MDR breast cancer. Initially, IR780@LDs-Fe3O4/OA was efficiently enriched within tumor sites in a static magnetic field, achieving the visualization of tumor treatment. Subsequently, the cascade-targeted SDT combined with the Fenton effect induced lysosome membrane permeabilization and relieved lysosomal sequestration, thus elevating drug concentration at the target site. This treatment approach also suppressed ATP production, thereby inhibiting P-glycoprotein-mediated chemotherapeutic drug efflux. This cascade-targeted SDT strategy significantly increased the sensitivity of MDR cells to doxorubicin, increasing the IC50 value of doxorubicin by approximately 10-fold. Moreover, the cascade-targeted SDT also altered the gene expression profiles of MDR cells and suppressed the expression of MDR-related genes. In light of these promising results, the combination of cascade-targeted SDT and conventional chemotherapy holds great clinical promise as an effective treatment modality with excellent biocompatibility that can improve MDR breast cancer patient outcomes.

Members of WRKY Group III Transcription Factors Are Important in Mite Infestation in Strawberry (Fragaria × ananassa Duch.).

Strawberry is frequently attacked by mites, which directly affects the yield and quality of this fruit species. The WRKY Group III transcription factors (TFs) play an important role in plant tolerance to biotic sources of stress, such as pathogens and insect pests. In this study, six Group III WRKY TFs (FaWRKY25, FaWRKY31, FaWRKY32, FaWRKY43, FaWRKY44, and FaWRKY45) were identified in strawberry. A phylogenetic analysis showed that the six WRKY III TFs were divided into two clades and all had a conserved WRKYGQK domain and the C-X7-C-X23-H-T-C zinc finger motif. An interaction network analysis revealed that FaWRKY44 was co-expressing with FaWRKY25 and FaWRKY45. The expression patterns showed that the WRKY Group III genes responded to plant hormones and mite infestation in strawberry. To further verify the role of FaWRKY25 in plant resistance to mites, we cloned the FaWRKY25 gene and overexpressed it in transgenic plants. An in vivo subcellular localization analysis indicated that the FaWRKY25 protein was localized in the nucleus. Fewer mites were also detected on the wild-type plants than on FaWRKY25-overexpressing transgenic plants, suggesting that FaWRKY25 negatively regulates the resistance of strawberry to mites. The present study advances our understanding on a potential target that mites use to manipulate host plant defenses.

Protein P5 of pear chlorotic leaf spot-associated virus is a pathogenic factor that suppresses RNA silencing and enhances virus movement.

Pear chlorotic leaf spot-associated virus (PCLSaV) is a newly described emaravirus that infects pear trees. The virus genome consists of at least five single-stranded, negative-sense RNAs. The P5 encoded by RNA5 is unique to PCLSaV. In this study, the RNA silencing suppression (RSS) activity of P5 and its subcellular localization were determined in Nicotiana benthamiana plants by Agrobacterium tumefaciens-mediated expression assays and green fluorescent protein RNA silencing induction. Protein P5 partially suppressed local RNA silencing, strongly suppressed systemic RNA silencing and triggered reactive oxygen species accumulation. The P5 self-interacted and showed subcellular locations in plasmodesmata, endoplasmic reticulum and nucleus. Furthermore, P5 rescued the cell-to-cell movement of a movement defective mutant PVXΔP25 of potato virus X (PVX) and enhanced the pathogenicity of PVX. The N-terminal 1-89 amino acids of the P5 were responsible for the self-interaction ability and RSS activity, for which the signal peptide at positions 1-19 was indispensable. This study demonstrated the function of an emaravirus protein as a pathogenic factor suppressing plant RNA silencing to enhance virus infection and as an enhancer of virus movement.

Expression analysis of the apple HSP70 gene family in abiotic stress and phytohormones and expression validation of candidate MdHSP70 genes.

Heat shock protein 70 (HSP70) is one kind of molecular chaperones which are widely found in organisms, and its members are highly conserved among each other, with important roles in plant growth and development. In this study, 56 HSP70 genes were identified from the apple genome database. Analysis of gene duplication events showed that tandem and segmental duplication events play an important role in promoting the amplification of the MdHSP70 gene family. Collinearity analysis showed that HSP70 family members of apple were more closely related to HSP70 family members of Arabidopsis, tomato and soybean. The promoter region of the apple HSP70 genes contains a large number of cis-acting elements in response to hormones and stress. Tissue-specific expression analysis showed that some of the genes were associated with various stages of the apple growth process. Codon preference analysis showed small differences between codon bases 1 and 3 in the apple HSP70 genome, and the codon base composition had a small effect on codon usage preference. The multiple expression patterns of the MdHSP70 gene suggested that MdHSP70 gene members play important roles in growth and development and in response to hormonal and abiotic stresses. The yeast two-hybrid (Y2H) demonstrated that MdHSP70-53 interacts with MdDVH24_032563. The qRT-PCR analysis showed that most MdHSP70 members' hormonal and abiotic stresses (MdHSP70-6, MdHSP70-26 and MdHSP70-45) appeared to be highly expressed. To further elucidate the function of MdHSP70 (6, 26, 45), we introduced them into tobacco to confirm subcellular locations and noted that these genes are located in the cytoplasm and cell membrane. This study serves as a theoretical basis for further studies of the MdHSP70 gene and helps to further investigate the functional characterization of MdHSP70 gene.