Search Results (32)

Wheat (Triticum aestivum L.) is a primary crop globally. Among the numerous pathogens affecting wheat production, Puccinia striiformis f. sp. tritici (Pst) is a significant biotic stress agent and poses a major threat to world food security by causing stripe rust or yellow rust disease. Understanding the molecular basis of plant–pathogen interactions is crucial for developing new means of disease management. It is well established that the effector proteins play a pivotal role in pathogenesis. Therefore, studying effector proteins has become an important area of research in plant biology. Our previous work identified differentially expressed candidate secretory effector proteins of stripe rust based on transcriptome sequencing data from susceptible wheat (Avocet S) and resistant wheat (Avocet YR10) infected with Pst. Among the secreted effector proteins, PSTG_14090 contained an ancient double-psi beta-barrel (DPBB) fold, which is conserved in the rare lipoprotein A (RlpA) superfamily. This study investigated the role of PSTG_14090 in plant immune responses, which encodes a protein, here referred to as Pst-DPBB, having 131 amino acids with a predicted signal peptide (SP) of 19 amino acids at the N-terminal end, and the DNA sequence of this effector is highly conserved among different stripe rust races. qRT-PCR analysis indicated that expression levels are upregulated during the early stages of infection. Subcellular localization studies in Nicotiana benthamiana leaves and wheat protoplasts revealed that it is distributed in the cytoplasm, nucleus, and apoplast. We demonstrated that Pst-DPBB negatively regulates the immune response by functioning in various compartments of the plant cells. Based on Co-IP and structural predictions and putative interaction analyses by AlphaFold 3, we propose the probable biological function(s). Pst-DPBB behaves as a papain inhibitor of wheat cysteine protease; Pst-DPBB has high structural homology to kiwellin, which is known to interact with chorismate mutase, suggesting that Pst-DPBB inhibits the native function of the host chorismate mutase involved in salicylic acid synthesis. The DPBB fold is also known to interact with DNA and RNA, which may suggest its possible role in regulating the host gene expression. Full article

Caliciviruses including noro- and sapoviruses of family Caliciviridae are important enteric human and swine pathogens, while others, like valoviruses, are less known. In this study, we developed a detection and typing pipeline for the most prevalent swine enteric caliciviruses—sapovirus GIII (Sw-SaV), norovirus GII (Sw-NoV), and valovirus GI (Sw-VaV). The pipeline integrates triplex RT-qPCR, 3′RACE semi-nested PCR, and next-generation sequencing (NovaSeq, Illumina) techniques. A small-scale epidemiological investigation was conducted on archived enteric and, for the first time, on oral fluid/saliva samples of diarrheic and asymptomatic swine of varying ages from Hungary and Slovakia. In enteric samples, Sw-SaV was the most prevalent, detected in 26.26% of samples, primarily in diarrheic pigs with low Cq values, followed by Sw-NoV (2.53%) in nursery pigs. In oral fluid samples, Sw-NoV predominated (7.46%), followed by Sw-SaV (4.39%). Sw-VaVs were sporadically found in both sample types. A natural, asymptomatic Sw-SaV outbreak was retrospectively detected where the transient shedding of the virus was <2 weeks. Complete capsid sequences (n = 59; 43 Sw-SaV, 13 Sw-NoV, and 3 Sw-VaV) including multiple (up to five) co-infecting variants were identified. Sw-SaV sequences belong to seven genotypes, while Sw-NoV and Sw-VaV strains clustered into distinct sub-clades, highlighting the complex diversity of these enteric caliciviruses in swine. Full article

The sequence of Aquaporin 12 (AQP12) cDNA was amplified from spiny dogfish (Squalus acanthias) cDNAs using degenerate PCR, followed by 5′ and 3′ RACE PCR. The AQP12 nucleotide sequence had an open reading frame of 300 amino acids, which included one or more N-glycosylation sites. Degenerate and tissue PCRs revealed that AQP12 is expressed at the highest levels in the liver, followed by the pyloric stomach and the esophagus/cardiac stomach, with a small amount potentially present in the eye. A polyclonal antibody was made using a peptide from the derived amino acid sequence. Western blotting with the antibody showed faint banding around the size expected (33 kDa) by the 300 amino acid protein. A few more intense bands were seen at around 40 kDa and larger sizes. Immunohistochemistry in cardiac stomach tissue sections showed staining in a few sporadic paneth-like secretory cells along the surface of the epithelium. High-magnification imaging showed that the AQP12 staining was located in the membrane of secretory granules in the apical pole of the cells. This localization is reminiscent of the AQP12 localization in pancreatic acinar cells, where it is found in the membrane of zymogen granules containing digestive enzymes. Full article

Targeted treatment of advanced or metastatic urothelial carcinomas (UCs) requires the identification of druggable mutations. This study describes the development of a 3′ Rapid Amplification of cDNA Ends (3′ RACE)-based targeted RNA sequencing panel which accounts for the status of all genes relevant to UC treatment, namely, FGFR1-4, KRAS, NRAS, BRAF, ERBB2 (HER2), CD274 (PD-L1) and PIK3CA. FGFR2/3-activating point mutations or fusions were found in 54/233 (23.2%) tumors. FGFR3 rearrangements were identified in 11 patients, with eight of them being undetectable by commonly used PCR kits. In addition, one tumor contained a high-copy FGFR2 gene amplification accompanied by strong overexpression of the gene. Mutations in RAS/RAF genes were present in 30/233 (12.9%) UCs and were mutually exclusive with alterations affecting FGFR2/3 genes. On the contrary, activating events in the HER2 oncogene (point mutations and overexpression), as well as PIK3CA mutations, which were relatively common, occurred with similar frequencies in RAS/RAF- or FGFR2/3-positive vs. negative samples. High PD-L1 mRNA expression was associated with advanced disease stage and was not observed in tumors with increased HER2 mRNA expression or in UCs with evidence for FGFR2/3 activation. Three of the studied carcinomas had high-level microsatellite instability (MSI). Overall, more than half of the UCs had potentially druggable genetic alterations. The proposed NGS panel permits comprehensive and cost-efficient analysis of UC-specific molecular targets and may be considered in clinical routine. Full article

Wheat leaf rust fungus is an obligate parasitic fungus that can absorb nutrients from its host plant through haustoria and secrete effector proteins into host cells. The effector proteins are crucial factors for pathogenesis as well as targets for host disease resistance protein recognition. Exploring the role of effector proteins in the pathogenic process of Puccinia triticina Eriks. (Pt) is of great significance for unraveling its pathogenic mechanisms. We previously found that a cysteine-rich effector protein, Pt1641, is highly expressed during the interaction between wheat and Pt, but its specific role in pathogenesis remains unclear. Therefore, this study employed techniques such as heterologous expression, qRT-PCR analysis, and host-induced gene silencing (HIGS) to investigate the role of Pt1641 in the pathogenic process of Pt. The results indicate that Pt1641 is an effector protein with a secretory function and can inhibit BAX-induced programmed cell death in Nicotiana benthamiana. qRT-PCR analyses showed that expression levels of Pt1641 were different during the interaction between the high-virulence strain THTT and low-virulence strains FGD and Thatcher, respectively. The highest expression level in the low-virulence strain FGD was four times that of the high-virulence strain THTT. The overexpression of Pt1641 in wheat near-isogenic line TcLr1 induced callose deposition and H₂O₂ production on TcLr1. After silencing Pt1641 in the Pt low-virulence strain FGD on wheat near-isogenic line TcLr1, the pathogenic phenotype of Pt physiological race FGD on TcLr1 changed from “;” to “3”, indicating that Pt1641 plays a non-toxic function in the pathogenicity of FGD to TcLr1. This study helps to reveal the pathogenic mechanism of wheat leaf rust and provides important guidance for the mining and application of Pt avirulent genes. Full article

The cultivation of pineapple (Ananas comosus) is threatened worldwide by mealybug wilt disease of pineapple (MWP), whose etiology is not yet fully elucidated. In this study, we characterized pineapple mealybug wilt-associated ampeloviruses (PMWaVs, family Closteroviridae) from a diseased pineapple plant collected from Reunion Island, using a high-throughput sequencing approach combining Illumina short reads and Nanopore long reads. Reads co-assembly resulted in complete or near-complete genomes for six distinct ampeloviruses, including the first complete genome of pineapple mealybug wilt-associated virus 5 (PMWaV5) and that of a new species tentatively named pineapple mealybug wilt-associated virus 7 (PMWaV7). Short reads data provided high genome coverage and sequencing depths for all six viral genomes, contrary to long reads data. The 5′ and 3′ ends of the genome for most of the six ampeloviruses could be recovered from long reads, providing an alternative to RACE-PCRs. Phylogenetic analyses did not unveil any geographic structuring of the diversity of PMWaV1, PMWaV2 and PMWaV3 isolates, supporting the current hypothesis that PMWaVs were mainly spread by human activity and vegetative propagation. Full article

Ophibolin A, a fungal sesterterpene, exerts a pivotal influence in a diverse array of biological processes, encompassing herbicidal, bactericidal, fungicidal, and cytotoxic activities. Sixty genes associated with sesterterpene compound biosynthesis were obtained from Bipolaris eleusines via transcriptome sequencing, and those closely linked to ophiobolin A biosynthesis were subsequently filtered. A gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) that catalyzes the first committed step of ophiobolin biosynthesis in the mevalonic acid (MVA) pathway was isolated and characterized using RACE (Rapid Amplification of cDNA Ends) technology from ophiobolin A-producing fungus, B. eleusines. The full-length cDNA of the B. eleusines HMGR gene (BeHMGR) was 3906 bp and contained a 3474 bp open reading frame (ORF) encoding 1157 amino acids. Sequence analysis revealed that deduced BeHMGR had high homology to the known HMGRs from Pyrenophora tritici-repentis and Leptosphaeria maculans. It had a calculated molecular mass of about 124.65 kDa and an isoelectric point (pI) of 6.90. It contained two putative HMG-CoA-binding motifs and two NADP(H)-binding motifs. Induced expression analysis of the BeHMGR gene by methyl jasmonate treatment using quantitative fluorescence PCR showed that it significantly elevated after 3 h of methyl jasmonate treatment, peaked at 6 h, and then gradually decreased. This demonstrates that BeHMGR gene expression is induced by methyl jasmonate. Full article

COVID-19 breakthrough infection (BTI) can occur despite vaccination. Using a multi-centre, prospective, observational Canadian cohort of people with HIV (PWH) receiving ≥2 COVID-19 vaccines, we compared the SARS-CoV-2 spike (S) and receptor-binding domain (RBD)-specific IgG levels 3 and 6 months post second dose, as well as 1 month post third dose, in PWH with and without BTI. BTI was defined as positivity based on self-report measures (data up to last study visit) or IgG data (up to 1 month post dose 3). The self-report measures were based on their symptoms and either a positive PCR or rapid antigen test. The analysis was restricted to persons without previous COVID-19 infection. Persons without BTI remained COVID-19-naïve until ≥3 months following the third dose. Of 289 participants, 92 developed BTI (31.5 infections per 100 person-years). The median days between last vaccination and BTI was 128 (IQR 67, 176), with the most cases occurring between the third and fourth dose (n = 59), corresponding to the Omicron wave. In analyses adjusted for age, sex, race, multimorbidity, hypertension, chronic kidney disease, diabetes and obesity, a lower IgG S/RBD (log10 BAU/mL) at 1 month post dose 3 was significantly associated with BTI, suggesting that a lower IgG level at this time point may predict BTI in this cohort of PWH. Full article

SPL transcription factors affect plant growth and development, including blooming and photoperiod control. The investigation began with transcriptome data screening of 28 JrSPL genes in walnut (Junglans regia L.) ‘Wen185’. These genes were discovered on all chromosomes except 6 and 15. Phylogenetic study divides the 28 JrSPL genes into five groupings. The biggest cluster, cluster IV, has 12 JrSPL genes. The expression of JrSPL genes in different tissues was investigated by qRT-PCR. JrSPL02 gene expression was greater in walnut female and male flower tissues than other genes. Subcellular localization has shown the JrSPL02 gene resides in the nucleus. Jre-miR156 may target JrSPL02’s 3′-UTR region, according to miRNA sequencing, RACE, and BiFC studies. Arabidopsis plants expressing the JrSPL02 gene flowered 3 days faster than the wild type, according to phenotypic observation. Transgenic lines had more stem branches and siliques than the control group but fewer rosette leaves. In summary, this study functionally analyses the metamorphosis of the miR156-SPL module during the blooming stage and the underlying mechanisms that govern early fruiting in early-fruiting walnuts in Xinjiang. Full article

Various attempts to amplify an AQP11 cDNA from tissues of the spiny dogfish (Squalus acanthias) were made. Two pairs of deoxy-inosine-containing degenerate primers were designed based on conserved amino acid sequences from an AQP11 alignment. These primers yielded some faint bands from gill cDNA that were sequenced. Blast searches with the sequences showed they were not AQP11. An elasmobranch AQP11 nucleotide sequence alignment was produced to identify conserved regions to make further degenerate primers. One primer pair produced a short 148 bp fragment showing particularly strong amplification in gill and intestine. It was sequenced and represented a piece of the AQP11 gene. However, as the fragment may have resulted from contaminating genomic DNA (in total RNA used to make cDNA), 5′ and 3′ RACE were performed to amplify the two ends of the putative cDNA. Furthermore, 5′ and 3′ RACE amplifications depend on the presence of a 5′ cap nucleotide and a poly A tail, respectively on the putative AQP11 mRNA. Hence, successful amplification was only possible from cDNA and not genomic DNA. Nested RACE amplifications were performed using gill and intestinal RACE cDNA, but none of the DNA fragments sequenced were AQP11. Consequently, the spiny dogfish AQP11 gene may represent a pseudogene. Full article

This study describes the cloning, expression and functional characterization of α-humulene synthase, responsible for the formation of the key aromatic compound α-humulene in agarwood originating from Aquilaria malaccensis. The partial sesquiterpene synthase gene from the transcriptome data of A. malaccensis was utilized for full-length gene isolation via a 3′ RACE PCR. The complete gene, denoted as AmDG2, has an open reading frame (ORF) of 1671 bp and encodes for a polypeptide of 556 amino acids. In silico analysis of the protein highlighted several conserved motifs typically found in terpene synthases such as Asp-rich substrate binding (DDxxD), metal-binding residues (NSE/DTE), and cytoplasmic ER retention (RxR) motifs at their respective sites. The AmDG2 was successfully expressed in the E. coli:pET-28a(+) expression vector whereby an expected band of about 64 kDa in size was detected in the SDS-PAGE gel. In vitro enzyme assay using substrate farnesyl pyrophosphate (FPP) revealed that AmDG2 gave rise to two sesquiterpenes: α-humulene (major) and β-caryophyllene (minor), affirming its identity as α-humulene synthase. On the other hand, protein modeling performed using AlphaFold2 suggested that AmDG2 consists entirely of α-helices with short connecting loops and turns. Meanwhile, molecular docking via AutoDock Vina (Version 1.5.7) predicted that Asp307 and Asp311 act as catalytic residues in the α-humulene synthase. To our knowledge, this is the first comprehensive report on the cloning, expression and functional characterization of α-humulene synthase from agarwood originating from A. malaccensis species. These findings reveal a deeper understanding of the structure and functional properties of the α-humulene synthase and could be utilized for metabolic engineering work in the future. Full article

Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance to Fusarium oxysporum f. sp. Cubense (Foc), the causal agent of this disease, is genetically dissected in this study using two Musa acuminata ssp. Malaccensis segregating populations, segregating for Foc Tropical (TR4) and Subtropical (STR4) race 4 resistance. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on chromosome 3 of ‘DH-Pahang’ reference assembly v4. Within this region, there was a cluster of pattern recognition receptors, namely leucine-rich repeat ectodomain containing receptor-like protein kinases, cysteine-rich cell-wall-associated protein kinases, and leaf rust 10 disease-resistance locus receptor-like proteins, positioned in an interspersed arrangement. Their transcript levels were rapidly upregulated in the resistant progenies but not in the susceptible F₂ progenies at the onset of infection. This suggests that one or several of these genes may control resistance at this locus. To confirm the segregation of single-gene resistance, we generated an inter-cross between the resistant parent ‘Ma850’ and a susceptible line ‘Ma848’, to show that the STR4 resistance co-segregated with marker ‘28820’ at this locus. Finally, an informative SNP marker 29730 allowed the locus-specific resistance to be assessed in a collection of diploid and polyploid banana plants. Of the 60 lines screened, 22 lines were predicted to carry resistance at this locus, including lines known to be TR4-resistant, such as ‘Pahang’, ‘SH-3362’, ‘SH-3217’, ‘Ma-ITC0250’, and ‘DH-Pahang/CIRAD 930’. Additional screening in the International Institute for Tropical Agriculture’s collection suggests that the dominant allele is common among the elite ‘Matooke’ NARITA hybrids, as well as in other triploid or tetraploid hybrids derived from East African highland bananas. Fine mapping and candidate gene identification will allow characterization of molecular mechanisms underlying the TR4 resistance. The markers developed in this study can now aid the marker-assisted selection of TR4 resistance in breeding programs around the world. Full article

The Clostridium perfringens (C. perfringen) beta2 (CPB2) toxin produced by C. perfringens type C (CpC) can cause necrotizing enteritis in piglets. Immune system activation in response to inflammation and pathogen infection is aided by long non-coding RNAs (lncRNAs). In our previous work, we revealed the differential expression of the novel lncRNA LNC_001186 in CpC-infected ileum versus healthy piglets. This implied that LNC_001186 may be a regulatory factor essential for CpC infection in piglets. Herein, we analyzed the coding ability, chromosomal location and subcellular localization of LNC_001186 and explored its regulatory role in CPB2 toxin-induced apoptosis of porcine small intestinal epithelial (IPEC-J2) cells. RT-qPCR results indicated that LNC_001186 expression was highly enriched in the intestines of healthy piglets and significantly increased in CpC-infected piglets’ ileum tissue and CPB2 toxin-treated IPEC-J2 cells. The total sequence length of LNC_001186 was 1323 bp through RACE assay. CPC and CPAT, two online databases, both confirmed that LNC_001186 had a low coding ability. It was present on pig chromosome 3. Cytoplasmic and nuclear RNA isolation and RNA-FISH assays showed that LNC_001186 was present in the nucleus and cytoplasm of IPEC-J2 cells. Furthermore, six target genes of LNC_001186 were predicted using cis and trans approaches. Meanwhile, we constructed ceRNA regulatory networks with LNC_001186 as the center. Finally, LNC_001186 overexpression inhibited IPEC-J2 cells’ apoptosis caused by CPB2 toxin and promoted cell viability. In summary, we determined the role of LNC_001186 in IPEC-J2 cells’ apoptosis caused by CPB2 toxin, which assisted us in exploring the molecular mechanism of LNC_001186 in CpC-induced diarrhea in piglets. Full article

The intramuscular fat (or marbling fat) content is an essential economic trait of beef cattle and improves the flavor and palatability of meat. Several studies have highlighted the correlation between long non-coding RNAs (lncRNAs) and intramuscular fat development; however, the precise molecular mechanism remains unknown. Previously, through a high-throughput sequencing analysis, we found a lncRNA and named it a long non-coding RNA BNIP3 (lncBNIP3). The 5′ RACE and 3′ RACE explored 1945 bp total length of lncBNIP3, including 1621 bp of 5′RACE, and 464 bp of 3′RACE. The nucleoplasmic separation and FISH results explored the nuclear localization of lncBNIP3. Moreover, the tissue expression of lncBNIP3 was higher in the longissimus dorsi muscle, followed by intramuscular fat. Furthermore, down-regulation of lncBNIP3 increased the 5-Ethynyl-2′- deoxyuridine (EdU)-EdU-positive cells. The flow cytometry results showed that the number of cells in the S phase was significantly higher in preadipocytes transfected with si-lncBNIP3 than in the control group (si-NC). Similarly, CCK8 results showed that the number of cells after transfection of si-lncBNIP3 was significantly higher than in the control group. In addition, the mRNA expressions of proliferative marker genes CyclinB1 (CCNB1) and Proliferating Cell Nuclear Antigen (PCNA) in the si-lncBNIP3 group were significantly higher than in the control group. The Western Blot (WB) results also showed that the protein expression level of PCNA transfection of si-lncBNIP3 was significantly higher than in the control group. Similarly, the enrichment of lncBNIP3 significantly decreased the EdU-positive cells in the bovine preadipocytes. The results of flow cytometry and CCK8 assay also showed that overexpression of lncBNIP3 inhibited the proliferation of bovine preadipocytes. In addition, the overexpression of lncBNIP3 significantly inhibited the mRNA expressions of CCNB1 and PCNA. The WB results showed that the overexpression of lncBNIP3 significantly inhibited the expression of the CCNB1 protein level. To further explore the mechanism of lncBNIP3 on the proliferation of intramuscular preadipocytes, RNA-seq was performed after interference with si-lncBNIP3, and 660 differentially expressed genes (DEGs) were found, including 417 up-regulated DEGs and 243 down-regulated DEGs. The KEGG pathway analysis showed that the cell cycle was the most significant pathway for the functional enrichment of DEGs, followed by the DNA replication pathway. The RT-qPCR quantified the expression of twenty DEGs in the cell cycle. Therefore, we speculated that lncBNIP3 regulated intramuscular preadipocyte proliferation through the cell cycle and DNA replication pathways. To further confirm this hypothesis, the cell cycle inhibitor Ara-C was used to inhibit DNA replication of the S phase in intramuscular preadipocytes. Herein, Ara-C and si-lncBNIP3 were simultaneously added to the preadipocytes, and the CCK8, flow cytometry, and EdU assays were performed. The results showed that the si-lncBNIP3 could rescue the inhibitory effect of Ara-C in the bovine preadipocyte proliferation. In addition, lncBNIP3 could bind to the promoter of cell division control protein 6 (CDC6), and down-regulation of lncBNIP3 promoted the transcription activity and the expression of CDC6. Therefore, the inhibitory effect of lncBNIP3 on cell proliferation might be understood through the cell cycle pathway and CDC6 expression. This study provided a valuable lncRNA with functional roles in intramuscular fat accumulation and revealed new strategies for improving beef quality. Full article

Adenovirus has been detected in a wide range of hosts like dogs, foxes, horses, bats, avian animals, and raccoon dogs. Canine adenoviruses with two serotypes host mammals and are members of the mastadenovirus family. Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis and infectious bronchial disease, respectively. In this study, we investigated the prevalence of CAdV-1 and 2 in wild Nyctereutes procyonoides in Korea in 2017–2020 from 414 tissue samples, including the liver, kidney, lung, and intestine, collected from 105 raccoon dog carcasses. Only CAdV-2 was detected in two raccoon dogs, whereas CAdV-1 was not detected. Tissue samples from raccoon dogs were screened for CAdV-1 and CAdV-2 using conventional PCR. Adenovirus was successfully isolated from PCR positive samples using the Vero cell line, and the full-length gene sequence of the isolated viruses was obtained through 5’ and 3’ rapid amplification of cDNA ends (RACE). The major genes of the isolated CAdV-2/18Ra54 and CAdV-2/18Ra-65 strains showed the closest relationship with that of the CAdV-2 Toronto A26/61 strain isolated from Canada in 1976. There is no large mutation between CAdV-2, which is prevalent worldwide, and CAdV-2, which is prevalent in wild animals in Korea. In addition, it is still spreading and causing infections. The Toronto A26/61 strain, which showed the most similarity to CAdV-2/18Ra-54, was likely transmitted to wild animals through vaccinated companion animals, suggesting that further research is needed on safety measures surrounding animal vaccination. This study provides information on the genetic characteristics and prevalence of canine adenovirus in domestic wild animals and provides a better understanding of canine adenovirus. Full article