UCHL3 promotes hepatocellular carcinoma progression by stabilizing EEF1A1 through deubiquitination.

BACKGROUND: Hepatocellular carcinoma (HCC) ranks as the second leading cause of global cancer-related deaths and is characterized by a poor prognosis. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) have been proved to play important roles in various human cancers, whereas the deubiquitination of EEF1A1 was poorly understood. METHODS: The binding and regulatory relationship between Ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) and EEF1A1 was validated using clinical tissue samples, reverse transcription quantitative real-time fluorescence quantitative PCR (RT-qPCR), Western blotting, co-immunoprecipitation, and immunofluorescence, as well as ubiquitin detection and cyclohexamide tracking experiments. Finally, the impact of the UCHL3/EEF1A1 axis on HCC malignant behavior was analyzed through functional experiments and nude mouse models. RESULTS: UCHL3 was found to have a high expression level in HCC tissues. Tissue samples from 60 HCC patients were used to evaluate the correlation between UCHL3 and EEF1A1. UCHL3 binds to EEF1A1 through the lysine site, which reduces the ubiquitination level of EEF1A1. Functional experiments and nude mouse models have demonstrated that the UCHL3/EEF1A1 axis promotes the migration, stemness, and drug resistance of HCC cells. Reducing the expression of EEF1A1 can reverse the effect of UCHL3 on the malignant behavior of HCC cells. CONCLUSION: Our findings revealed that UCHL3 binds and stabilizes EEF1A1 through deubiquitination. UCHL3 and EEF1A1 formed a functional axis in facilitating the malignant progression of HCC, proving new insights for the anti-tumor targeted therapy for HCC.

EEF1B2 regulates bone marrow-derived mesenchymal stem cells bone-fat balance via Wnt/ß-catenin signaling.

The pathological advancement of osteoporosis is caused by the uneven development of bone marrow-derived mesenchymal stem cells (BMSCs) in terms of osteogenesis and adipogenesis. While the role of EEF1B2 in intellectual disability and tumorigenesis is well established, its function in the bone-fat switch of BMSCs is still largely unexplored. During the process of osteogenic differentiation, we observed an increase in the expression of EEF1B2, while a decrease in its expression was noted during adipogenesis. Suppression of EEF1B2 hindered the process of osteogenic differentiation and mineralization while promoting adipogenic differentiation. On the contrary, overexpression of EEF1B2 enhanced osteogenesis and strongly inhibited adipogenesis. Furthermore, the excessive expression of EEF1B2 in the tibias has the potential to mitigate bone loss and decrease marrow adiposity in mice with osteoporosis. In terms of mechanism, the suppression of ß-catenin activity occurred when EEF1B2 function was suppressed during osteogenesis. Our collective findings indicate that EEF1B2 functions as a regulator, influencing the differentiation of BMSCs and maintaining a balance between bone and fat. Our finding highlights its potential as a therapeutic target for diseases related to bone metabolism.

eEF1A1 regulates the expression and alternative splicing of genes associated with Parkinson's disease in U251 cells.

BACKGROUND: Eukaryotic elongation factor 1A1 (eEF1A1) is an RNA-binding protein that is associated with PARK2 activity in cells, suggesting a possible role in Parkinson's disease (PD). OBJECTIVE: To clear whether eEF1A1 plays a role in PD through transcriptional or posttranscriptional regulation. METHODS: The GSE68719 dataset was downloaded from the GEO database, and the RNA-seq data of all brain tissue autopsies were obtained from 29 PD patients and 44 neurologically normal control subjects. To inhibit eEF1A1 from being expressed in U251 cells, siRNA was transfected into those cells, and RNA-seq high-throughput sequencing was used to determine the differentially expressed genes (DEGs) and differentially alternative splicing events (ASEs) resulting from eEF1A1 knockdown. RESULTS: eEF1A1 was significantly overexpressed in PD brain tissue in the BA9 area. GO and KEGG enrichment analyses revealed that eEF1A1 knockdown significantly upregulated the expression of the genes CXCL10, NGF, PTX3, IL6, ST6GALNAC3, NUPR1, TNFRSF21, and CXCL2 and upregulated the alternative splicing of the genes ACOT7, DDX10, SHMT2, MYEF2, and NDUFAF5. These genes were enriched in pathways related to PD pathogenesis, such as apoptosis, inflammatory response, and mitochondrial dysfunction. CONCLUSION: The results suggesting that eEF1A1 involved in the development of PD by regulating the differential expression and alternative splicing of genes, providing a theoretical basis for subsequent research.

The elongation factor 1-alpha as storage reserve and environmental sensor in Nicotiana tabacum L. seeds.

Given their critical role in plant reproduction and survival, seeds demand meticulous regulatory mechanisms to effectively store and mobilize reserves. Within seeds, the condition of storage reserves heavily depends on environmental stimuli and hormonal activation. Unlike non-protein reserves that commonly employ dedicated regulatory proteins for signaling, proteinaceous reserves may show a unique form of 'self-regulation', amplifying efficiency and precision in this process. Proteins rely on stability to carry out their functions. However, in specific physiological contexts, particularly in seed germination, protein instability becomes essential, fulfilling roles from signaling to regulation. In this study, the elongation factor 1-alpha has been identified as a main proteinaceous reserve in Nicotiana tabacum L. seeds and showed peculiar changes in stability based on tested chemical and physical conditions. A detailed biochemical analysis followed these steps to enhance our understanding of these protein attributes. The protein varied its behavior under different conditions of pH, temperature, and salt concentration, exhibiting shifts within physiological ranges. Notably, distinct solubility transitions were observed, with the elongation factor 1-alpha becoming insoluble upon reaching specific thresholds determined by the tested chemical and physical conditions. The findings are discussed within the context of seed signaling in response to environmental conditions during the key transitions of dormancy and germination.

CRISPR Screen Identifies the RNA-Binding Protein Eef1a1 as a Key Regulator of Myogenesis.

Skeletal muscle myogenesis hinges on gene regulation, meticulously orchestrated by molecular mechanisms. While the roles of transcription factors and non-coding RNAs in myogenesis are widely known, the contribution of RNA-binding proteins (RBPs) has remained unclear until now. Therefore, to investigate the functions of post-transcriptional regulators in myogenesis and uncover new functional RBPs regulating myogenesis, we employed CRISPR high-throughput RBP-KO (RBP-wide knockout) library screening. Through this approach, we successfully identified Eef1a1 as a novel regulatory factor in myogenesis. Using CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) technologies, we successfully established cellular models for both CRISPRko and CRISPRi. Our findings demonstrated that Eef1a1 plays a crucial role in promoting proliferation in C2C12 myoblasts. Through siRNA inhibition and overexpression methods, we further elucidated the involvement of Eef1a1 in promoting proliferation and suppressing differentiation processes. RIP (RNA immunoprecipitation), miRNA pull-down, and Dual-luciferase reporter assays confirmed that miR-133a-3p targets Eef1a1. Co-transfection experiments indicated that miR-133a-3p can rescue the effect of Eef1a1 on C2C12 myoblasts. In summary, our study utilized CRISPR library high-throughput screening to unveil a novel RBP, Eef1a1, involved in regulating myogenesis. Eef1a1 promotes the proliferation of myoblasts while inhibiting the differentiation process. Additionally, it acts as an antagonist to miR-133a-3p, thus modulating the process of myogenesis.

Methionine and Leucine Promote mTOR Gene Transcription and Milk Synthesis in Mammary Epithelial Cells through the eEF1Bα-UBR5-ARID1A Signaling.

Amino acids are essential for the activation of the mechanistic target of rapamycin (mTOR), but the corresponding molecular mechanism is not yet fully understood. We previously found that Met stimulated eukaryotic elongation factor α (eEF1Bα) nuclear localization in bovine mammary epithelial cells (MECs). Herein, we explored the role and molecular mechanism of eEF1Bα in methionine (Met)- and leucine (Leu)-stimulated mTOR gene transcription and milk synthesis in MECs. eEF1Bα knockdown decreased milk protein and fat synthesis, cell proliferation, and mTOR mRNA expression and phosphorylation, whereas eEF1Bα overexpression had the opposite effects. QE-MS analysis detected that eEF1Bα was phosphorylated at Ser106 in the nucleus and Met and Leu stimulated p-eEF1Bα nuclear localization. eEF1Bα knockdown abrogated the stimulation of Met and Leu by mTOR mRNA expression and phosphorylation, and this regulatory role was dependent on its phosphorylation. Akt knockdown blocked the stimulation of Met and Leu by eEF1Bα and p-eEF1Bα expression. ChIP-PCR detected that p-eEF1Bα bound only to the -548 to -793 nt site in the mTOR promoter, and ChIP-qPCR further detected that Met and Leu stimulated this binding. eEF1Bα mediated Met and Leu' stimulation on mTOR mRNA expression and phosphorylation through inducing AT-rich interaction domain 1A (ARID1A) ubiquitination degradation, and this process depended on eEF1Bα phosphorylation. p-eEF1Bα interacted with ARID1A and ubiquitin protein ligase E3 module N-recognition 5 (UBR5), and UBR5 knockdown rescued the decrease of the ARID1A protein level by eEF1Bα overexpression. Both eEF1Bα and p-eEF1Bα were highly expressed in mouse mammary gland tissues during the lactating period. In summary, we reveal that Met and Leu stimulate mTOR transcriptional activation and milk protein and fat synthesis in MECs through eEF1Bα-UBR5-ARID1A signaling.

Phylogenetic Assessment of Understudied Families in Hymenochaetales (Basidiomycota, Fungi)-Reporting Uncovered Species and Reflecting the Recent Taxonomic Updates in the Republic of Korea.

Hymenochaetales Oberw. is an order classified in Basidiomycota of Fungi, and species in this order display notable diversity. They exhibit various fruiting body shapes, including clavarioid, effused-reflexed, and resupinate basidiomes. Few mycorrhizal species have been reported in Hymenochaetales, but wood-decaying species dominate the order. Hymenochaetaceae Imazeki & Toki and Schizoporaceae Jülich are the most species-rich families within Hymenochaetales, and most species in the Republic of Korea belong to these two families. As such, current taxonomic classification and nomenclature are not reflected upon species in the remaining Hymenochaetales families. For this study, a multifaceted morphological and multigenetic marker-based phylogenetic investigation was conducted to, firstly, comprehensively identify understudied Hymenochaetales specimens in Korea and, secondly, reflect the updates on the species classification. Five genetic markers were assessed for the phylogenetic analysis: nuclear small subunit ribosomal DNA (nSSU), internal transcribed spacer (ITS), nuclear large subunit ribosomal DNA (nLSU), RNA polymerase II subunit 2 gene (RPB2), and translation elongation factor 1 gene (TEF1). The results from phylogenetic analysis supported 18 species classified under eight families (excluding Hymenochaetaceae and Schizoporaceae) in Korea. Species formerly placed in Rickenellaceae and Trichaptum sensu lato have been systematically revised based on recent taxonomic reconstructions. In addition, our findings revealed one new species, Rickenella umbelliformis, and identified five formerly nationally unreported species classified under five understudied families. Our findings contribute to a better understanding of Hymenochaetales diversity and highlight the need for continued research.

Agaricus subgenus Pseudochitonia in Malakand, Pakistan: An updated phylogeny and description of three new species.

Agaricus is a genus with more than 500 species. Most of the new species reported since 2000 are tropical or subtropical. The study area, the Malakand region, located in the north of Pakistan, has a subtropical climate. In this study, nine species, including three new species, of Agaricus subgenus Pseudochitonia, are reported from this region. Description of the new species are based on morphological characteristics and phylogenetic analyses using three DNA regions: nuc ribosomal DNA internal transcribed spacers (ITS), fragments of the large subunit of nuc ribosomal DNA (28S), and the translation elongation factor 1-alpha gene (TEF1). One new species, Agaricus lanosus, with wooly squamules on its cap, forms a lineage within Agaricus sect. Bivelares and cannot be classified with certainty in one of the two subsections (Cupressorum and Hortenses) of this section. Agaricus rhizoideus with rhizoid-like structure at the base of the stipe forms a basal clade in Agaricus sect. Hondenses. Specimens of the third new species, Agaricus malakandensis, form a species-level clade within Agaricus sect. Catenulati and exhibits the morphological characteristics of this section. Due to their similar ITS sequences, two previously unnamed specimens from Thailand (A. sp. LD2012162 and CA799) are considered conspecific with A. malakandensis.

The eEF1A protein in cancer: Clinical significance, oncogenic mechanisms, and targeted therapeutic strategies.

Eukaryotic elongation factor 1A (eEF1A) is among the most abundant proteins in eukaryotic cells. Evolutionarily conserved across species, eEF1A is in charge of translation elongation for protein biosynthesis as well as a plethora of non-translational moonlighting functions for cellular homeostasis. In malignant cells, however, eEF1A becomes a pleiotropic driver of cancer progression via a broad diversity of pathways, which are not limited to hyperactive translational output. In the past decades, mounting studies have demonstrated the causal link between eEF1A and carcinogenesis, gaining deeper insights into its multifaceted mechanisms and corroborating its value as a prognostic marker in various cancers. On the other hand, an increasing number of natural and synthetic compounds were discovered as anticancer eEF1A-targeting inhibitors. Among them, plitidepsin was approved for the treatment of multiple myeloma whereas metarrestin was currently under clinical development. Despite significant achievements in these two interrelated fields, hitherto there lacks a systematic examination of the eEF1A protein in the context of cancer research. Therefore, the present work aims to delineate its clinical implications, molecular oncogenic mechanisms, and targeted therapeutic strategies as reflected in the ever expanding body of literature, so as to deepen mechanistic understanding of eEF1A-involved tumorigenesis and inspire the development of eEF1A-targeted chemotherapeutics and biologics.

Translation elongation factor-1α is pivotal for plant heat tolerance despite its pronounced heat-induced aggregation.

The translation elongation factor 1α (EF1α) protein is a highly conserved G protein that is crucial for protein translation in all eukaryotic organisms. EF1α quickly became insoluble at temperatures 42 °C treatment for 2h in vitro, but generally remained soluble in vivo even after being exposed to temperatures as high as 45 °C for an extended period, which suggests that protective mechanisms exist for keeping EF1α soluble in plant cells under heat stress. EF1α had fast in vivo insolubilization when exposed to 45 °C, resulting in about 40% of the protein aggregating after 9 h. Given its established role in protein translation, heat-induced aggregation is most likely to impact the function of the elongation factor. Overexpression of constitutive mutants in both GTP-bound and GDP-bound forms of EF1α resulted in significantly decreased heat tolerance. These findings provide evidence to support the critical role of EF1α, a thermosensitive protein, in the heat tolerance of plants.

MILIP Binding to tRNAs Promotes Protein Synthesis to Drive Triple-Negative Breast Cancer.

Patients with triple-negative breast cancer (TNBC) have a poor prognosis due to the lack of effective molecular targets for therapeutic intervention. Here we found that the long noncoding RNA (lncRNA) MILIP supports TNBC cell survival, proliferation, and tumorigenicity by complexing with transfer RNAs (tRNA) to promote protein production, thus representing a potential therapeutic target in TNBC. MILIP was expressed at high levels in TNBC cells that commonly harbor loss-of-function mutations of the tumor suppressor p53, and MILIP silencing suppressed TNBC cell viability and xenograft growth, indicating that MILIP functions distinctively in TNBC beyond its established role in repressing p53 in other types of cancers. Mechanistic investigations revealed that MILIP interacted with eukaryotic translation elongation factor 1 alpha 1 (eEF1α1) and formed an RNA-RNA duplex with the type II tRNAs tRNALeu and tRNASer through their variable loops, which facilitated the binding of eEF1α1 to these tRNAs. Disrupting the interaction between MILIP and eEF1α1 or tRNAs diminished protein synthesis and cell viability. Targeting MILIP inhibited TNBC growth and cooperated with the clinically available protein synthesis inhibitor omacetaxine mepesuccinate in vivo. Collectively, these results identify MILIP as an RNA translation elongation factor that promotes protein production in TNBC cells and reveal the therapeutic potential of targeting MILIP, alone and in combination with other types of protein synthesis inhibitors, for TNBC treatment. SIGNIFICANCE: LncRNA MILIP plays a key role in supporting protein production in TNBC by forming complexes with tRNAs and eEF1α1, which confers sensitivity to combined MILIP targeting and protein synthesis inhibitors.

DNA methylation profiling identifies epigenetic signatures of early gastric cancer.

Research on the DNA methylation status of gastric cancer (GC) has primarily focused on identifying invasive GC to develop biomarkers for diagnostic. However, DNA methylation in noninvasive GC remains unclear. We conducted a comprehensive DNA methylation profiling study of differentiated-type intramucosal GCs (IMCs). Illumina 850K microarrays were utilized to assess the DNA methylation profiles of formalin-fixed paraffin-embedded tissues from eight patients who were Epstein-Barr virus-negative and DNA mismatch repair proficient, including IMCs and paired adjacent nontumor mucosa. Gene expression profiling microarray data from the GEO database were analyzed via bioinformatics to identify candidate methylation genes. The final validation was conducted using quantitative real-time PCR, the TCGA methylation database, and single-sample gene set enrichment analysis (GSEA). Genome-wide DNA methylation profiling revealed a global decrease in methylation in IMCs compared with nontumor tissues. Differential methylation analysis between IMCs and nontumor tissues identified 449 differentially methylated probes, with a majority of sites showing hypomethylation in IMCs compared with nontumor tissues (66.1% vs 33.9%). Integrating two RNA-seq microarray datasets, we found one hypomethylation-upregulated gene: eEF1A2, overlapped with our DNA methylation data. The mRNA expression of eEF1A2 was higher in twenty-four IMC tissues than in their paired adjacent nontumor tissues. GSEA indicated that the functions of eEF1A2 were associated with the development of IMCs. Furthermore, TCGA data indicated that eEF1A2 is hypomethylated in advanced GC. Our study illustrates the implications of DNA methylation alterations in IMCs and suggests that aberrant hypomethylation and high mRNA expression of eEF1A2 might play a role in IMCs development.

N-Cinnamoylpyrrole-derived alkaloids from the genus Piper as promising agents for ischemic stroke by targeting eEF1A1.

BACKGROUND: Ischemic stroke (IS) is a serious cerebrovascular disease characterized by significantly elevated mortality and disability rates, and the treatments available for this disease are limited. Neuroinflammation and oxidative stress are deemed the major causes of cerebral ischemic injury. N-Cinnamoylpyrrole alkaloids form a small group of natural products from the genus Piper and have not been extensively analyzed pharmacologically. Thus, identifying the effect and mechanism of N-cinnamoylpyrrole-derived alkaloids on IS is worthwhile. PURPOSE: The present research aimed to explore the antineuroinflammatory and antioxidative stress effects of N-cinnamoylpyrrole-derived alkaloids isolated from the genus Piper and to explain the effects and mechanism on IS. METHODS: N-cinnamoylpyrrole-derived alkaloids were isolated from Piper boehmeriaefolium var. tonkinense and Piper sarmentosum and identified by various chromatographic methods. Lipopolysaccharide (LPS)-induced BV-2 microglia and a mouse model intracerebroventricularly injected with LPS were used to evaluate the antineuroinflammatory and antioxidative stress effects. Oxygen‒glucose deprivation/reperfusion (OGD/R) and transient middle cerebral artery occlusion (tMCAO) models were used to evaluate the effect of PB-1 on IS. To elucidate the fundamental mechanism, the functional target of PB-1 was identified by affinity-based protein profiling (ABPP) strategy and verified by cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), and circular dichroism (CD) analyses. The effect of PB-1 on the NF-κB and NRF2 signaling pathways was subsequently evaluated via western blotting and immunofluorescence staining. RESULTS: The results showed that N-cinnamoylpyrrole-derived alkaloids significantly affected neuroinflammation and oxidative stress. The representative compound, PB-1 not only inhibited neuroinflammation and oxidative stress induced by LPS or OGD/R insult, but also alleviated cerebral ischemic injury induced by tMCAO. Further molecular mechanism research found that PB-1 promoted antineuroinflammatory and antioxidative stress activities via the NF-κB and NRF2 signaling pathways by targeting eEF1A1. CONCLUSION: Our research initially unveiled that the therapeutic impact of PB-1 on cerebral ischemic injury might rely on its ability to target eEF1A1, leading to antineuroinflammatory and antioxidative stress effects. The novel discovery highlights eEF1A1 as a potential target for IS treatment and shows that PB-1, as a lead compound that targets eEF1A1, may be a promising therapeutic agent for IS.

Arcyria similaris: A new myxomycete species from China.

A new myxomycete species, Arcyria similaris, was reported herein. The specimens were found and collected in the field on dead bark from Jingangtai National Geopark in Henan Province of China. This species has distinct and unique morphological characteristics, including dark grayish olive sporothecae that fade to smoke gray with age, shallow saucer-shaped cups with marked reticulations and thick papillae on the inner surface, a netted capillitium with many bulges, uniformly marked with low, dense, and irregular reticulations, and spores (8.0-)9.3-10.1(-10.9) µm in diameter, marked with sparse small warts and grouped prominent warts. Apart from a comprehensive morphological study, partial sequences of the nuclear 18S rDNA and elongation factor-1 alpha (EF-1α) genes were also provided in this study. This new species was described and illustrated morphologically. The specimens are deposited in the Herbarium of Fungi of Nanjing Normal University (HFNNU).

eEF1A2 promotes PTEN-GSK3ß-SCF complex-dependent degradation of Aurora kinase A and is inactivated in breast cancer.

The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3ß, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.

Ogataea nonmethanolica f.a, sp. nov., a novel yeast species isolated from rotting wood in Brazil and Colombia.

Three yeast isolate candidates for a novel species were obtained from rotting wood samples collected in Brazil and Colombia. The Brazilian isolate differs from the Colombian isolates by one nucleotide substitution in each of the D1/D2 and small subunit (SSU) sequences. The internal transcribed spacer (ITS) and translation elongation factor 1-α gene sequences of the three isolates were identical. A phylogenetic analysis showed that this novel species belongs to the genus Ogataea. This novel species is phylogenetically related to Candida nanaspora and Candida nitratophila. The novel species differs from C. nanaspora by seven nucleotides and two indels, and by 17 nucleotides and four indels from C. nitratophila in the D1/D2 sequences. The ITS sequences of these three species differ by more than 30 nucleotides. Analyses of the sequences of the SSU and translation elongation factor 1-α gene also showed that these isolates represent a novel species of the genus Ogataea. Different from most Ogataea species, these isolates did not assimilate methanol as the sole carbon source. The name Ogataea nonmethanolica sp. nov. is proposed to accommodate these isolates. The holotype of Ogataea nonmethanolica is CBS 13485T. The MycoBank number is MB 851195.

Genetic Diversity and Pathogenicity of the Fusarium Species Complex on Soybean in Serbia.

Using morphological and cultural characteristics for identification, 36 Fusarium isolates were recovered from diseased roots, stems, and seeds of soybean from several localities throughout Vojvodina Province, Serbia. Based on molecular characterization, 12 Fusarium species were identified: F. acuminatum, F. avenaceum, F. commune, F. equiseti, F. graminearum, F. incarnatum, F. oxysporum, F. proliferatum, F. solani, F. sporotrichioides, F. subglutinans, and F. tricinctum. The elongation factor 1-α-based phylogeny grouped the isolates into 12 well-supported clades, but polymorphisms among sequences in some clades suggested the use of the species complex concept: (i) F. incarnatum-equiseti species complex (FIESC)-F. incarnatum and F. equiseti; (ii) F. oxysporum species complex (FOSC)-F. oxysporum; (iii) F. solani species complex (FSSC)-F. solani; and (iv) F. acuminatum/F. avenaceum/F. tricinctum species complex (FAATSC)-F. acuminatum, F. avenaceum, and F. tricinctum. Pathogenicity tests showed that the most aggressive species causing soybean seed rot were F. sporotrichioides, F. graminearum, FIESC, and F. avenaceum. Furthermore, F. subglutinans, FSSC, and F. proliferatum showed a high percentage of pathogenicity on soybean seeds (80 to 100%), whereas variability in pathogenicity occurred within isolates of F. tricinctum. FOSC, F. commune, and F. acuminatum had the lowest pathogenicity. To our knowledge, this is the first study of the characterization of Fusarium species on soybean in Serbia. This study provides valuable information about the composition of Fusarium species and pathogenicity that will be used in further research on soybean resistance to Fusarium-based diseases.

Evaluation of Suitable Reference Genes for Quantitative Real-Time PCR in Various Tissues of Apocynum venetum.

Apocynum venetum L. is an economically valuable plant with tolerance to drought and salinity. Its leaves are utilized in tea production and pharmaceuticals, while the stem bark serves as a high-quality fiber material. To gain insights into the gene expression patterns of A. venetum using quantitative real-time PCR (qRT-PCR), it is crucial to identify appropriate reference genes. This study selected nine candidate genes, including α-tubulin (TUA), ß-tubulin (TUB), actin (ACT), cyclophilin (CYP), elongation factor-1α (EF-1α), the B family of regulatory subunits of protein phosphatase (PPP2R2, PPP2R3, and PPP2R5), and phosphoglycerate kinase (PGK), to determine the most appropriate reference genes in the leaf, stem, and root tissues of A. venetum. A comprehensive ranking by geNorm, NormFinder, BestKeeper, and RefFinder software and Venn diagrams was used to screen more stable reference genes in different tissues. The two most stable reference genes were CYP and TUA in leaves, PGK and PPP2R3 in stems, and TUA and EF-1α in roots, respectively. The relative expression values of the four genes involved in proline metabolism under polyethylene glycol treatment were used to validate the screened reference genes, and they exhibited highly stable expression levels. These findings represent the first set of stable reference genes for future gene expression studies in A. venetum. They significantly contribute to enhancing the accuracy and reliability of gene expression analyses in this economically important plant species.