Search Results (34)

Phytophthora cinnamomi is a major plant pathogen that affects economically important crops and natural ecosystems, posing a threat to global biodiversity. While gene editing has emerged as a powerful tool for functional genomics in various Phytophthora species, its application in P. cinnamomi remains underexplored. To address this gap, our study investigated the challenges of implementing CRISPR/Cas9-mediated gene editing in P. cinnamomi, with the insights gained applicable to other gene editing platforms. We designed guide RNAs (gRNAs) targeting β-cinnamomin, a highly basic elicitin expressed by the pathogen during early infection stages, known for its role in sterol recruitment. Using an “all-in-one” plasmid containing the gRNA, Cas9, and an antibiotic resistance gene as a selectable marker, we transformed P. cinnamomi protoplasts via PEG/CaCl₂-mediated methods. The successful integration of the nptII gene, which confers geneticin (G418) resistance, was confirmed in heterokaryotic regenerants. To isolate pure mutants and eliminate wild-type dominance, we derived homokaryotic colonies from nptII-positive transformants. Mutation screening was performed using mismatch detection assays, T7 endonuclease 1 (T7E1), and restriction fragment length polymorphism (RFLP), followed by Sanger sequencing. Despite the integration of the nptII gene, the β-cinnamomin sequence in the transformants remained identical to the wild-type sequence, indicating challenges in detecting and achieving targeted gene disruption. This study identifies critical steps for optimising mutagenesis in P. cinnamomi, highlighting the importance of thorough preliminary screening, effective separation of heterokaryotic populations, and the isolation of homokaryotic colonies. Full article

Onychostoma macrolepis is not only a protected Cyprinid species in the wild but also an emerging commercial aquaculture fish in China. The objective of this research was to genetically modify the genes associated with commercial traits by CRISPR/Cas9 for the protection and utilization of the germplasm resources of O. macrolepis. To that end, one-cell stage embryos were obtained via hormone-induced ovulation and artificial insemination in O. macrolepis. Eight genes related to body color, growth, intermuscular bone, and sex differentiation were mutated in O. macrolepis using the CRISPR/Cas9 system by microinjection of gRNA/Cas9 mRNA. The optimal dose of gRNA/Cas9 mRNA was determined by injection of different concentrations of tyr (tyrosinase)-gRNA/Cas9 and examination of the mutation rate and hatching rate of embryos. Indels were detected by T7 endonuclease I digestion and Sanger sequencing. F0 mutants with high mutation rates were selected for phenotype analyses. Disruption of body color gene tyr, mpv17 (mitochondrial inner membrane protein MPV17), and csf1ra (colony-stimulating factor 1 receptor, a) resulted in obvious phenotype with decreased or even absence of melanophores, iridophores, and xanthophores, respectively. Mutation of mstnb (myostatin b) led to improved growth performance. Mutation of mc4r (melanocortin 4 receptor) led to no obvious phenotype. Mutation of runx2b (RUNX family transcription factor 2b) and bmp6 (bone morphogenetic protein 6) resulted in decreased or absence of intermuscular bones, as revealed by alizarin red S staining. Mutation of cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a) resulted in ovarian degeneration as revealed by gonadal histological examination. Therefore, this study successfully obtained mutants with obvious phenotypes of genes associated with body color, growth, intermuscular bone, and sex differentiation by CRISPR/Cas9 in O. macrolepis. Full article

Trichoderma is a widely studied ascomycete fungal genus, including more than 400 species. However, genetic information on Trichoderma is limited, with most species reporting only DNA barcodes. Mitochondria possess their own distinct DNA that plays a pivotal role in molecular function and evolution. Here, we report 42 novel mitochondrial genomes (mitogenomes) combined with 18 published mitogenomes of Trichoderma. These circular mitogenomes exhibit sizes of 26,276–94,608 bp, typically comprising 15 core protein-coding genes (PCGs), 2 rRNAs, and 16–30 tRNAs; however, the number of endonucleases and hypothetical proteins encoded in the introns of PCGs increases with genome size enlargement. According to the result of phylogenetic analysis of the whole mitogenome, these strains diverged into six distinct evolutionary branches, supported by the phylogeny based on 2830 single-copy nuclear genes. Comparative analysis revealed that dynamic Trichoderma mitogenomes exhibited variations in genome size, gene number, GC content, tRNA copy, and intron across different branches. We identified three mutation hotspots near the regions encoding nad3, cox2, and nad5 that caused major changes in the mitogenomes. Evolutionary analysis revealed that atp9, cob, nad4L, nad5, and rps3 have been influenced by positive selection during evolution. This study provides a valuable resource for exploring the important roles of the genetic and evolutionary dynamics of Trichoderma mitogenome in the adaptive evolution of biocontrol fungi. Full article

Cervical cancer is the fourth most common malignancy in women globally. Chemotherapies, targeted therapies, and immunotherapies in the treatment of cervical cancer are usually accompanied by effective and adverse effects. Therefore, finding other efficient and accurate molecular targets remains essential to improve the treatment benefits of cervical cancer patients. MCPIP1 (monocyte chemoattractant protein-induced protein 1) is a kind of endonuclease with a CCCH zinc finger domain and a PilT-N-terminal (PIN) domain, and its function in cervical cancer is unknown. We found that MCPIP1 inhibits cell proliferation and promotes cell apoptosis of cervical cancer. Additionally, MCPIP1 suppresses mRNA and protein expression of the apoptotic inhibitor XIAP by decreasing its mRNA stability. Mechanically, MCPIP1 binds to the XIAP mRNA via its CCCH zinc finger domain and degrades the XIAP mRNA via the endonuclease activity coming from its PIN domain. Our study clarifies that MCPIP1 promotes cervical cancer cell apoptosis by suppressing the expression of XIAP, thereby impeding cervical cancer progression. Moreover, targeted delivery of MCPIP1 with engineered Salmonella typhimurium leads to tumor growth retardation in the HeLa xenograft tumor model in mice. Therefore, our study may provide a theoretical basis for formulating clinical treatment strategies for cervical cancer. Full article

Environmental stressors can induce paternal epigenetic modifications that are a key determinant of the intergenerational inheritance of acquired phenotypes in mammals. Some of them can affect phenotypic expression through inducing changes in tRNA-derived small RNAs (tsRNAs), which modify paternal epigenetic regulation in sperm. However, it is unclear how these stressors can affect changes in the expression levels of tsRNAs and their related endonucleases in the male reproductive organs. We found that Ribonuclease inhibitor 1 (RNH1), an oxidation responder, interacts with ANG to regulate sperm tsRNA generation in the mouse caput epididymis. On the other hand, inflammation and oxidative stress induced by either lipopolysaccharide (LPS) or palmitate (PA) treatments weakened the RNH1-ANG interaction in the epididymal epithelial cells (EEC). Accordingly, ANG translocation increased from the nucleus to the cytoplasm, which led to ANG upregulation and increases in cytoplasmic tsRNA expression levels. In conclusion, as an antioxidant, RNH1 regulates tsRNA generation through targeting ANG in the mouse caput epididymis. Moreover, the tsRNA is an epigenetic factor in sperm that modulates paternal inheritance in offspring via the fertilization process. Full article

Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs’ eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek’s disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs. Full article

CAR-T cell therapy involves genetically engineering T cells to recognize and attack tumour cells by adding a chimeric antigen receptor (CAR) to their surface. In this study, we have used dual transduction with AAV serotype 6 (AAV6) to integrate an anti-CD19 CAR into human T cells at a known genomic location. The first viral vector expresses the Cas9 endonuclease and a guide RNA (gRNA) targeting the T cell receptor alpha constant locus, while the second vector carries the DNA template for homology-mediated CAR insertion. We evaluated three gRNA candidates and determined their efficiency in generating indels. The AAV6 successfully delivered the CRISPR/Cas9 machinery in vitro, and molecular analysis of the dual transduction showed the integration of the CAR transgene into the desired location. In contrast to the random integration methods typically used to generate CAR-T cells, targeted integration into a known genomic locus can potentially lower the risk of insertional mutagenesis and provide more stable levels of CAR expression. Critically, this method also results in the knockout of the endogenous T cell receptor, allowing target cells to be derived from allogeneic donors. This raises the exciting possibility of “off-the-shelf” universal immunotherapies that would greatly simplify the production and administration of CAR-T cells. Full article

Ageritin from poplar mushrooms is a specific endonuclease that hydrolyzes a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA, thereby blocking protein synthesis. Considering the possible biotechnological use of this enzyme, here we report its antifungal activity against virulent fungi affecting crops of economic interest. Our results show that ageritin (200 µg/plug; ~13.5 nmole) inhibits the growth of Botrytis cinerea (57%), Colletotrichum truncatum (42%), and Alternaria alternata (57%), when tested on potato dextrose agar plates. At the same time, no effect was observed against Trichoderma harzianum (a fungus promoting beneficial effects in plants). To verify whether the antifungal action of ageritin against B. cinerea and T. harzianum was due to ribosome damage, we tested ageritin in vitro on partially isolated B. cinerea and T. harzianum ribosomes. Interestingly, ageritin was able to release the Endo’s fragment from both tested fungal ribosomes. We therefore decided to test the antifungal effect of ageritin on B. cinerea and T. harzianum using a different growth condition (liquid medium). Differently from the result in solid medium, ageritin can inhibit both B. cinerea and T. harzianum fungal growth in liquid medium in a concentration-dependent manner up to 35.7% and 38.7%, respectively, at the highest concentration tested (~200 µg/mL; 12 µM), and the analysis of RNA isolated from ageritin-treated cells revealed the presence of Endo’s fragment, highlighting its ability to cross the fungal cell wall and reach the ribosomes. Overall, these data highlight that the efficacy of antifungal treatment to prevent or treat a potential fungal disease may depend not only on the fungal species but also on the conditions of toxin application. Full article

Numerous observations have supported the idea that various types of noncoding RNAs, including tRNA fragments (tRFs), are involved in communications between the host and its microbial community. The possibility of using their signaling function has stimulated the study of secreted RNAs, potentially involved in the interspecies interaction of bacteria. This work aimed at identifying such RNAs and characterizing their maturation during transport. We applied an approach that allowed us to detect oligoribonucleotides secreted by Prevotella copri (Segatella copri) or Rhodospirillum rubrum inside Escherichia coli cells. Four tRFs imported by E. coli cells co-cultured with these bacteria were obtained via chemical synthesis, and all of them affected the growth of E. coli. Their successive modifications in the culture medium and recipient cells were studied by high-throughput cDNA sequencing. Instead of the expected accidental exonucleolysis, in the milieu, we observed nonrandom cleavage by endonucleases continued in recipient cells. We also found intramolecular rearrangements of synthetic oligonucleotides, which may be considered traces of intermediate RNA circular isomerization. Using custom software, we estimated the frequency of such events in transcriptomes and secretomes of E. coli and observed surprising reproducibility in positions of such rare events, assuming the functionality of ring isoforms or their permuted derivatives in bacteria. Full article

Biosensors based on endonuclease Cas12 provide high specificity in pathogen detection. Sensitive detection using Cas12-based assays can be achieved using trans-cleaved DNA probes attached to simply separated carriers, such as magnetic particles (MPs). The aim of this work was to compare polyA, polyC, and polyT single-stranded (ss) DNA with different lengths (from 10 to 145 nt) as trans-target probes were immobilized on streptavidin-covered MPs. Each ssDNA probe was labeled using fluorescein (5′) and biotin (3′). To compare the probes, we used guide RNAs that were programmed for the recognition of two bacterial pathogens: Dickeya solani (causing blackleg and soft rot) and Erwinia amylovora (causing fire blight). The Cas12 was activated by targeting double-stranded DNA fragments of D. solani or E. amylovora and cleaved the MP–ssDNA conjugates. The considered probes demonstrated basically different dependencies in terms of cleavage efficiency. PolyC was the most effective probe when compared to polyA or polyT probes of the same length. The minimal acceptable length for the cleavage follows the row: polyC < polyT < polyA. The efficiencies of polyC and polyT probes with optimal length were proven for the DNA targets’ detection of D. solani and E. amylovora. The regularities found can be used in Cas12a-based detection of viruses, bacteria, and other DNA/RNA-containing analytes. Full article

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox regulation. The redox activity of APE1/Ref-1 is involved in inflammatory responses and regulation of DNA binding of transcription factors related to cell survival pathways. However, the effect of APE1/Ref-1 on adipogenic transcription factor regulation remains unknown. In this study, we investigated the effect of APE1/Ref-1 on the regulation of adipocyte differentiation in 3T3-L1 cells. During adipocyte differentiation, APE1/Ref-1 expression significantly decreased with the increased expression of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBP)-α and peroxisome proliferator-activated receptor (PPAR)-γ, and the adipocyte differentiation marker adipocyte protein 2 (aP2) in a time-dependent manner. However, APE1/Ref-1 overexpression inhibited C/EBP-α, PPAR-γ, and aP2 expression, which was upregulated during adipocyte differentiation. In contrast, silencing APE1/Ref-1 or redox inhibition of APE1/Ref-1 using E3330 increased the mRNA and protein levels of C/EBP-α, PPAR-γ, and aP2 during adipocyte differentiation. These results suggest that APE1/Ref-1 inhibits adipocyte differentiation by regulating adipogenic transcription factors, suggesting that APE1/Ref-1 is a potential therapeutic target for regulating adipocyte differentiation. Full article

Transcription of mitochondrial DNA generates long polycistronic precursors whose nucleolytic cleavage yields the individual mtDNA-encoded transcripts. In most cases, this cleavage occurs at the 5′- and 3′-ends of tRNA sequences by the concerted action of RNAseP and RNaseZ/ELAC2 endonucleases, respectively. Variants in the ELAC2 gene have been predominantly linked to severe to mild cardiomyopathy that, in its milder forms, is accompanied by variably severe neurological presentations. Here, we report five patients from three unrelated families. Four of the patients presented mild to moderate cardiomyopathy and one died at 1 year of age, one patient had no evidence of cardiomyopathy. The patients had variable neurological presentations that included intellectual disability, ataxia, refractory epilepsy, neuropathy and deafness. All patients carried previously unreported missense and nonsense variants. Enzymatic analyses showed multiple OXPHOS deficiencies in biopsies from two patients, whereas immunoblot analyses revealed a decreased abundance of ELAC2 in fibroblasts from three patients. Northern blot analysis revealed an accumulation of unprocessed mt-tRNA^Val-precursor consistent with the role of ELAC2 in transcript processing. Our study expands the genetic spectrum of ELAC2-linked disease and suggests that cardiomyopathy is not an invariably present clinical hallmark of this pathology. Full article

Baloxavir marboxil (BXM) is an antiviral drug that targets the endonuclease of the influenza polymerase acidic (PA) protein. Antiviral resistance, mainly mediated by the I38T PA substitution, readily occurs in both A(H1N1) and A(H3N2) viruses following a single dose of BXM. Influenza B resistance to BXM remains poorly documented. We aimed to generate baloxavir-resistant contemporary influenza B/Yamagata/16/1988- and B/Victoria/2/1987-like viruses by in vitro passages under baloxavir acid (BXA) pressure to identify resistance mutations and to characterize the fitness of drug-resistant variants. Influenza B/Phuket/3073/2013 recombinant virus (rg-PKT13, a B/Yamagata/16/1988-like virus) and B/Quebec/MCV-11/2019 (MCV19, a B/Victoria/2/1987-like isolate) were passaged in ST6GalI-MDCK cells in the presence of increasing concentrations of BXA. At defined passages, viral RNA was extracted for sequencing the PA gene. The I38T PA substitution was selected in MCV19 after six passages in presence of BXA whereas no PA change was detected in rg-PKT13. The I38T substitution increased the BXA IC₅₀ value by 13.7-fold in the MCV19 background and resulted in reduced viral titers compared to the wild type (WT) at early time points in ST6GalI-MDCK and at all time-points in human epithelial cells. By contrast, the I38T substitution had no impact on MCV19 polymerase activity, and this mutation was genetically stable over four passages. In conclusion, our results show a similar pathway of resistance to BXA in influenza B viruses highlighting the major role of the I38T PA substitution and suggest that I38T may differently impact the fitness of influenza variants depending on the viral type, subtype, or lineage. Full article

Fusarium musae has recently been described as a cross-kingdom pathogen causing post-harvest disease in bananas and systemic and superficial infection in humans. The taxonomic identity of fungal cross-kingdom pathogens is essential for confirming the identification of the species on distant infected hosts. Understanding the level of variability within the species is essential to decipher the population homogeneity infecting human and plant hosts. In order to verify that F. musae strains isolated from fruits and patients are part of a common population and to estimate their overall diversity, we assembled, annotated and explored the diversity of the mitogenomes of 18 F. musae strains obtained from banana fruits and human patients. The mitogenomes showed a high level of similarity among strains with different hosts’ origins, with sizes ranging from 56,493 to 59,256 bp. All contained 27 tRNA genes and 14 protein-coding genes, rps3 protein, and small and large ribosomal subunits (rns and rnl). Variations in the number of endonucleases were detected. A comparison of mitochondrial endonucleases distribution with a diverse set of Fusarium mitogenomes allowed us to specifically discriminate F. musae from its sister species F. verticillioides and the other Fusarium species. Despite the diversity in F. musae mitochondria, strains from bananas and strains from human patients group together, indirectly confirming F. musae as a cross-kingdom pathogen. Full article

Plant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene to study the molecular requirements for virus replication and encapsidation. Sequence comparisons among RRV isolates and structural modeling of the RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) revealed three natural mutations of the type species isolate that we reverted to the common species sequences: (a) twenty-one amino acid truncations near the endonuclease domain (named delA), (b) five amino acid substitutions near the putative viral RNA binding loop (subT), and (c) four amino acid substitutions in N (NISE). The delA and subT in the RdRp influenced the levels of GFP, gRNA, and agRNA at 3 but not 5 days post inoculation (dpi), suggesting these sequences are essential for initiating RNA synthesis and replication. The NISE mutation led to sustained GFP, gRNA, and agRNA at 3 and 5 dpi indicating that the N supports continuous replication and GFP expression. Next, we showed that the cucumber mosaic virus (CMV strain FNY) 2b singularly enhanced GFP expression and RRV replication. Including agRNA2 with the RRV replicon produced observable virions. In this study we developed a robust reverse genetic system for investigations into RRV replication and virion assembly that could be a model for other emaravirus species. Full article