Genomic DNA Research Articles

Local adaptation, recombination, and the fate of neopolyploids.

Polyploidy is widely recognized as an important speciation mechanism because it isolates tetraploids from their diploid progenitors. Polyploidy also provides new genetic material that may facilitate adaptive evolution. However, new mutations are more likely to arise after a neopolyploid already has successfully invaded a population. Thus, the role of adaptive forces in establishing a polyploid remains unclear. One solution to this apparent paradox may lie in the capacity of polyploids to suppress recombination among preexisting locally adapted alleles. The local adaptation mechanism requires that spatially heterogeneous selection acts on multiple loci and that gene flow introduces maladapted alleles to the population where the polyploid forms. The mechanism requires neither strong genetic drift nor any intrinsic benefit of genome doubling and can accommodate any mode of gene action. A unique prediction of the mechanism is that adaptive alleles should predate polyploidization, a pattern consistent with observations from a few well-studied polyploids. The mechanism is also consistent with the coexistence of both diploid and tetraploid cytotypes, fitness heterogeneity among independently derived polyploids, and the prevalence of outcrossing among older polyploids. The local adaptation mechanism also makes novel predictions about circumstances favoring polyploid invasions that can be tested using molecular genetic or comparative approaches.

Cytogenetic identification and molecular mapping for the wheat-Thinopyrum ponticum introgression line with resistance to Fusarium head blight.

Xinong 511, a new wheat-Thinopyrum ponticum variety with excellent fusarium head blight resistance, the QTLs were mapped to the wheat chromosomes 5B and 7A with named QFhb.nwafu-5B and QFhb.nwafu-7A, respectively. Novel Fusarium head blight (FHB) resistance germplasms and genes are valuable for wheat improvement and breeding efforts. Thinopyrum ponticum, a wild relative of common wheat, is a valuable germplasm of disease resistance for wheat improvement and breeding. Xinong 511 (XN511) is a high-quality wheat variety widely cultivated in the Yellow and Huai Rivers Valley of China with stable FHB-resistance. Through analysis of pedigree materials of the wheat cultivar XN511, we found that the genetic material and FHB resistance from Th. ponticum were transmitted to the introgression line, indicating that the FHB resistance in XN511 likely originates from Th. ponticum. To further explore the genetic basis of FHB resistance in XN511, QTL mapping was conducted using the RILs population of XN511 and the susceptible line Aikang 58 (AK58). Survey with makers closely-linked to Fhb1, Fhb2, Fhb4, Fhb5, and Fhb7, indicated that both XN511 and the susceptible lines do not contain these QTL. Using bulked segregant analysis RNA-seq (BSR-Seq) and newly developed allele-specific PCR (AS-PCR) markers, QTLs in XN511 were successfully located on wheat chromosomes 5B and 7A. These findings are significant for further understanding and utilizing FHB resistance genes in wheat improvement.

Genome editing of mammalian cells through RNA transcript-mediated homologous recombination repair.

Double-strand break repair of eukaryotic DNA is mainly accomplished by non-homologous end joining and homologous recombination. Providing exogenous templates during homologous recombination repair can result in the editing of target genes, which is the central mechanism of the well-established CRISPR gene editing system. Currently, exogenous templates are mainly DNA molecules, which can provoke a cellular immune response within the cell. In order to verify the feasibility of RNA molecules as repair templates for homologous recombination in mammalian cell genome editing, we fused RNA template molecules to the 3´-end of sgRNA, so that the sgRNA and the homologous template RNA form a single RNA molecule. The results show this construct can be used as a repair template to achieve target gene editing in mammalian cells. In addition, the factors influencing homologous recombination mediated by RNA template molecules were investigated and it was found that increasing the length of homologous arms and inducing an R-loop near the DSB can effectively promote homologous recombination repair. Further, intracellular homologous chromosomes may compete with exogenous RNA templates. The findings in this paper provide a reference for the utilization of RNA template molecules to mediate target gene editing in eukaryotic cells, as well as a basis for the study of the mechanism by which RNA molecules mediate the repair of DSBs.

Unified understanding of the impact of semiflexibility, concentration, and molecular weight on macromolecular-scale ring diffusion

Conformationally fluctuating, globally compact macromolecules such as polymeric rings, single-chain nanoparticles, microgels, and many-arm stars display complex dynamic behaviors due to their rich topological structure and intermolecular organization. Synthetic rings are hybrid objects with conformations that display both ideal random walk and compact globular features, which can serve as models of genomic DNA. To date, emphasis has been placed on the effect of ring molecular weight on their unusual behaviors. Here, we combine simulations and a microscopic force-level theory to build a unified understanding for how key aspects of ring dynamics depend on different tunable molecular properties including backbone rigidity, monomer concentration, degree of traditional entanglement, and molecular weight. Our large-scale molecular dynamics simulations of ring melts with very different backbone stiffnesses reveal unanticipated behaviors which agree well with our generalized theory. This includes a universal master curve for center-of-mass diffusion constants as a function of molecular weight scaled by a chemistry and thermodynamic state-dependent critical molecular weight that generalizes the concept of an entanglement cross-over for linear chains. The key physics is how backbone rigidity and monomer concentration induced changes of the entanglement length, interring packing, degree of interpenetration, and liquid compressibility slow down space-time dynamic-force correlations on macromolecular scales. A power law decay of the center-of-mass diffusion constant with inverse molecular weight squared is the first consequence, followed by an ultraslow activated hopping transport regime. Our results set the stage to address slow dynamics and kinetic arrest in different families of compact synthetic and biological polymeric systems.

Detection of Arsenophonus in Glycaspis brimblecombei (Hemiptera: Aphalaridae) populations in Brazil.

Eucalyptus is the most intensively managed tree genus in the world. Different factors, including damage by insect pests, affect its growth and productivity. Among these pests is Glycaspis brimblecombei Moore (Hemiptera: Aphalaridae), an exotic insect of Australian origin. The evolutionary success of this insect depends on symbiotic associations with microorganisms. The influence of these microorganisms on insect pests and their natural enemies is important for integrated management tactics. Within this context, this work aimed to detect Arsenophonus in populations of G. brimblecombei in Brazil. Eucalyptus branches infested with G. brimblecombei nymphs were collected in commercial eucalyptus plantations in six Brazilian states. Specimens of this pest were sampled soon after emergence and frozen for molecular analysis. The genomic DNA of G. brimblecombei adults from each population was extracted and used to detect the endosymbiont Arsenophonus by polymerase chain reaction (PCR) employing specific primers that target its 23S rRNA gene. This endosymbiont was identified in all of the studied G. brimblecombei populations. This is the first report on the association between Arsenophonus and G. brimblecombei in Brazil.

Structure, Functions And Clinical Significance Of DNA: A Review Article

The deoxyribonucleic acid (DNA) carries hereditary codes which is translated by the cells to synthesize the ribonucleic acid (RNA) and polypeptides which can generate and perform vital function. The double helix structure is the most studied model of the DNA that was proposed by Watson and Crick. The capability of DNA to work as a genetic material can be stored and conducted during cell division to permit this information to be doubled and transmitted to the incoming generation. Any damage in the structure of DNA is an essential direct cause for the progression of cancer and other disorders. The factors for DNA damage can be classified as exogenous and endogenous factors. In this review article, we highlight the evidence-supported information about the structure, functions and clinical significance of DNA.

Low evolutionary potential for blue-green egg colouration in a wild bird population.

Exploring the evolutionary architecture of female sexual traits and their potential evolvability is important to understand their possible role as post-mating sexual signals. Egg colouration has been proposed to be one of these post-mating sexual signals, honestly advertising female quality in birds, especially in blue-green laying species. In this study, we used an animal model in a Bayesian framework to estimate the evolvability of multiple descriptors of blue-green egg colouration and egg size in a wild long-term monitored population of spotless starlings (Sturnus unicolor). Our results show low to moderate heritability (h2 = 0.31 - 0.44) for three egg colour descriptors (blue-green chroma, chroma and lightness) and egg size. Using the coefficient of additive genetic variance (CVA) and the evolvability (IA) as proxies of evolutionary potential of all components of this trait, we found low values of CVA for all these variables, suggesting a small evolutionary potential of these phenotypic traits, contrasting to previous results reported in another blue-green egg laying species. Our results indicate a modest raw genetic material of this trait on which sexual selection can act upon and, therefore, a small probability for these traits to respond easily to selection.

Inclusion of TAT and NLS sequences in lipopeptide molecules generates homogenous nanoparticles for gene delivery applications

PurposesThe objective of this study is to develop a versatile gene carrier based on lipopeptides capable of delivering genetic material into target cells with minimal cytotoxicity. MethodsTwo lipopeptide molecules, palmitoyl-CKKHH and palmitoyl-CKKHH-YGRKKRRQRRR-PKKKRKV, were synthesized using solid phase peptide synthesis and evaluated as transfection agents. Physicochemical characterization of the lipopeptides included a DNA shift mobility assay, particle size measurement, and transmission electron microscopy (TEM) analysis. Cytotoxicity was assessed in CHO-K1 and HepG2 cells using the MTT assay, while transfection efficiency was determined by evaluating the expression of the green fluorescent protein-encoding gene. ResultsOur findings demonstrate that the lipopeptides can bind, condense, and shield DNA from DNase degradation. The inclusion of the YGRKKRRQRRR sequence, a transcription trans activator, and the PKKKRKV sequence, a nuclear localization signal, imparts desirable properties. Lipopeptide-based TAT-NLS/DNA nanoparticles exhibited stability for up to 20 days when stored at 6–8 °C, displaying uniformity with a compact size of approximately 120 nm. Furthermore, the lipopeptides exhibited lower cytotoxicity compared to the poly-L-lysine. Transfection experiments revealed that protein expression mediated by the lipopeptide occurred at a charge ratio ranging from 4.0 to 8.0. ConclusionThese results indicate that the lipopeptide, composed of a palmitoyl alkyl chain and TAT and NLS sequences, can efficiently condense and protect DNA, form stable and uniform nanoparticles, and exhibit promising characteristics as a potential gene carrier with minimal cytotoxicity.

Do tunneling nanotubes drive chemoresistance in solid tumors and other malignancies?

Intercellular communication within the tumor microenvironment (TME) is essential for establishing, mediating, and synchronizing cancer cell invasion and metastasis. Cancer cells, individually and collectively, react at the cellular and molecular levels to insults from standard-of-care treatments used to treat patients with cancer. One form of cell communication that serves as a prime example of cellular phenotypic stress response is a type of cellular protrusion called tunneling nanotubes (TNTs). TNTs are ultrafine, actin-enriched contact-dependent forms of membrane protrusions that facilitate long distance cell communication through transfer of various cargo, including genetic materials, mitochondria, proteins, ions, and various other molecules. In the past 5-10 years, there has been a growing body of evidence that implicates TNTs as a novel mechanism of cell-cell communication in cancer that facilitates and propagates factors that drive or enhance chemotherapeutic resistance in a variety of cancer cell types. Notably, recent literature has highlighted the potential of TNTs to serve as cellular conduits and mediators of drug and nanoparticle delivery. Given that TNTs have also been shown to form in vivo in a variety of tumor types, disrupting TNT communication within the TME provides a novel strategy for enhancing the cytotoxic effect of existing chemotherapies while suppressing this form of cellular stress response. In this review, we examine current understanding of interplay between cancer cells occurring via TNTs, and even further, the implications of TNT-mediated tumor-stromal cross-talk and the potential to enhance chemoresistance. We then examine tumor microtubes, an analogous cell protrusion heavily implicated in mediating treatment resistance in glioblastoma multiforme, and end with a brief discussion of the effects of radiation and other emerging treatment modalities on TNT formation.

Can self-amplifying RNA vaccines and viruses exchange genetic material?

Can self-amplifying RNA vaccines and viruses exchange genetic material?

Geographical distribution of MTHFR C677T gene polymorphisms among the reproductive-age women in Chinese Han populations: based on migration

BackgroundMethylenetetrahydrofolate reductase (MTHFR) is essential for the metabolism of folic acid and homocysteine. The MTHFR C677T polymorphism is associated with several disorders. Our study aims to explore the geographical distributions of the MTHFR C677T polymorphism of women in China and how migration affected the polymorphism in Suzhou.MethodsA total of 7188 women of reproductive age were recruited in Suzhou of the study. Subjects were classified according to their native places after data extraction. MTHFR C677T gene polymorphisms were detected by quantitative PCR with genomic DNA isolated from blood samples.ResultsThe frequencies of the 677T allele and 677TT genotype were higher in northern China than that in southern China and decreased in geographical gradients from north to south. The frequencies were considerably higher in the migrant population than that in the indigenous population of Suzhou. The migrant population have gradually changed the prevalence in Suzhou.ConclusionsOur study suggested that the prevalence of MTHFR C677T polymorphisms among women varied across different geographical regions in Chinese Han populations. The 677T allele frequencies of the northern populations were significantly higher than those of the southern populations. The migrant population gradually changed the prevalence of the MTHFR C677T polymorphism in Suzhou.

The interplay of DNA damage and repair, gene expression, and mutagenesis in mammalian cells during oxidative stress.

We investigated the interplay among oxidative DNA damage and repair, expression of genes encoding major base excision repair (BER) enzymes and bypass DNA polymerases, and mutagenesis in mammalian cells. Primary mouse embryonic fibroblasts were challenged with oxidative stress induced by methylene blue plus visible light, and formation and repair of DNA damage, changes in gene expression, and mutagenesis were determined at increasing intervals post-treatment (0 - 192 hours). Significant formation of oxidative DNA damage together with upregulation of Ogg1, Polβ, and Polκ, and no changes in Mutyh and Nudt1 expression were found in treated cells. There was a distinct interconnection between Ogg1 and Polβ expression and DNA damage formation and repair whereby changes in expression of these two genes were proportionate to the levels of oxidative DNA damage, once a 3-plus hour lag time passed (P < 0.05). Equally notable was the matching pattern of Polκ expression and kinetics of oxidative DNA damage and repair (P < 0.05). The DNA damage and gene expression data were remarkably consistent with mutagenicity data in the treated cells; the induced mutation spectrum is indicative of erroneous bypass of oxidized DNA bases and incorporation of oxidized deoxynucleoside triphosphates during replication of the genomic DNA. Our findings support follow-up functional studies to elucidate how oxidation of DNA bases and the nucleotide pool, overexpression of Polκ, delayed upregulation of Ogg1 and Polβ, and inadequate expression of Nudt1 and Mutyh collectively affect mutagenesis consequent to oxidative stress.

First report of Colletotrichum orbiculare causing anthracnose of pointed gourd in India.

Pointed gourd (Trichosanthes dioica Roxb.), of the Cucurbitaceae family, is widely cultivated as a vegetable in many countries such as Bangladesh, India, Pakistan, Myanmar, Nepal and Sri Lanka. Over 800,000 metric tons of pointed gourds are produced annually in India, where cultivation is estimated to occupy over 33,000 hectares of land (MoA & FW, Government of India). In summer 2018, significant losses (approximately 15-20%) occurred in the sub-Himalayan region in West Bengal state of India (21.14-21.30° N, 78.82-79.02°E) due to a disease with typical anthracnose-like symptoms on the fruits. Light yellowish, small sized round to irregular spots were also apparent on the leaves. These spots gradually increased in size and turned into light brown and were surrounded by yellow halo. The lesions on the fruits were circular, yellow-brown, necrotic and sunken. A survey of four fields (1.5 ha) was conducted and a disease incidence of 30-40% was observed. Necrotic tissues from fruit as well as leaves were cut into approximately 5 mm2, surface sterilized with 0.1% HgCl2, plated in potato dextrose agar and incubated at 28ºC for 7 days in the dark. A total of 50 morphologically similar colonies were obtained from 20 sampled fruits and 10 sampled leaves. Fungal colonies were initially white, becoming gray as the cultures aged on PDA. The cultures developed black acervuli around the center of the colony. Setae were brown in colour, 1-5 septate, 40-100 µm long. Conidia were also observed through light and scanning electron microscopy and exhibit as (4-6 ×13-19 µm) hyaline, aseptate, cylindrical to oblong, with one end round and other truncate. The morphological characteristics were found similar to Colletotrichum orbiculare Damm, P.F. Cannon & Crous as reported by Damm et al. (2013). Ten isolates were obtained by transferring hyphal tips to new PDA plates and incubating under the same conditions. To confirm the identity of the pathogen, genomic DNA was extracted from five pure isolates (PG-Pha, PG-Pha-2, PG-Pha-3, PG-Pha-4, PG-Pha-5) with the cetyltrimethylammonium bromide (CTAB). Further, the ITS1-5.8S-ITS2 region, D1/D2 region of the 28S rRNA large subunit (LSU), Actin (ACT) gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified using specific primers, ITS1/ITS4 (White et al. 1990), NL1/NL4, ACT1/ACT2 and GDF1/GDR1 respectively and PCR conditions described in Damm et al. (2012). A GenBank BLAST search showed 99-100% identity to the Colletotrichum orbiculare (Acc. Nos. KP898988 for ITS1-5.8S-ITS2, Z18997 for 28S rRNA, AB778553 for ACT gene, and KF178482 for GAPDH). All obtained sequences were submitted to the GenBank (Acc. Nos. MN006616, OP811046-OP811049, [ITS1-5.8-ITS2], MN006684, PP391616-PP391619 [28S rRNA], MN168524, PP400822-PP400825 [ACT gene], OP627091, PP400826-PP400829 [GAPDH]). For phylogenetic analysis, MEGA version 11 (Tamura et al. 2021) was used to construct a maximum likelihood tree with 1000 bootstrap replicates, based on a concatenation alignment of three gene sequences (ITS, Actin and GAPDH) of the all the five C. orbiculare isolates as well as sequences of other Colletotrichum species obtained from GenBank. The cluster analysis revealed that, isolate PG-Pha form a cluster with other C. orbiculare isolates. Pathogenicity tests were conducted to confirm Koch's postulates. Pathogenicity tests were performed in mature fruits by inoculating them (n=8) with 10 µl of a 1×106 conidia/ml suspension at needle puncture wound sites. In control set up sterile distilled water was pipetted on fruits. Fruits were placed on sterile trays covered with glasses and incubated at humid chambers at 28±2ºC with 12 h of light. Healthy one-month old potted pointed gourd plants (n=15) were sprayed with conidial suspension until run-off. A set of 15 plants were sprayed with sterile distilled water and maintained as control. The plants were kept in a greenhouse at 25ºC, >75% relative humidity, and a 16/8 h day/night cycle for 15 days. Sterile distilled water was sprayed on the plants at one day interval to maintain the humidity. Inoculated fruits started showing yellowing symptoms one day post inoculation and gradually yellow-brown sunken spots became visible at the place of puncture, whereas control fruits remain symptomless even after 7 days of inoculation. Inoculated leaves showed disease symptoms similar to those observed in the field whereas leaves of control sets were symptomless even after 15 days. The pathogenicity test was repeated thrice under the same conditions mentioned before. C. orbiculare was successfully re-isolated from all the symptomatic tissues of leaves as well as fruits, completing Koch's postulates. Previously, the pathogen has been reported as an important anthracnose pathogen of Cucurbitaceae, especially of cucumber (Cucumis sativus), melons (Cucumis melo), watermelon (Citrullus lanatus), pumpkin (Cucurbita pepo) and squash (Cucurbita maxima) (Farr and Rossman 2020). To our knowledge, this is the first report of C. orbiculare causing anthracnose of pointed gourd. This disease represents a threat to producers in India and central Asia. Further research may contribute to the development of management strategies for this disease.

A novel SSR4 variant associated with congenital disorder of glycosylation: a case report and related analysis

IntroductionCongenital disorders of glycosylation (CDG) refer to monogenetic diseases characterized by defective glycosylation of proteins or lipids causing multi-organ disorders. Here, we investigate the clinical features and genetic variants of SSR4-CDG and conduct a preliminary investigation of its pathogenesis.MethodsWe retrospectively report the clinical data of a male infant with early life respiratory distress, congenital diaphragmatic eventration, cosmetic deformities, and moderate growth retardation. Peripheral blood was collected from the case and parents, genomic DNA was extracted and whole-exome sequencing was performed. The mRNA expression of SSR4 gene was quantified by Real-time Quantitative PCR. RNA sequencing analysis was subsequently performed on the case and a healthy child.ResultsWhole-exome sequencing of the case and his parents’ genomic DNA identified a hemizygous c.80_96del in SSR4, combined with the case’s clinical features, the diagnosis of CDG was finally considered. In this case, the expression of SSR4 was downregulated. The case were present with 1,078 genes downregulated and 536 genes upregulated. SSR4 gene expression was significantly downregulated in the case. Meanwhile, gene set enrichment analysis (GSEA) revealed that SSR4-CDG may affect hemostasis, coagulation, catabolism, erythrocyte development and homeostatic regulation, and muscle contraction and regulation, etc. Improvement of growth retardation in case after high calorie formula feeding and rehabilitation training.ConclusionOur study expanded the SSR4-CDG variant spectrum and clinical phenotype and analyzed pathways potentially affected by SSR4-CDG, which may provide further insights into the function of SSR4 and help clinicians better understand this disorder.

Regulatory Considerations for Genome-Edited T-cell Therapies.

Methods to engineer the genomes of human cells for therapeutic intervention continue to advance at a remarkable pace. Chimeric antigen receptor-engineered T lymphocytes have pioneered the way for these therapies, initially beginning with insertions of chimeric antigen receptor transgenes into T-cell genomes using classical gene therapy vectors. The broad use of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies to edit endogenous genes has now opened the door to a new era of precision medicine. To add complexity, many engineered cellular therapies under development integrate gene therapy with genome editing to introduce novel biological functions and enhance therapeutic efficacy. Here, we review the current state of scientific, translational, and regulatory oversight of gene-edited cell products.

Symbolic recording of signalling and cis-regulatory element activity to DNA.

Measurements of gene expression or signal transduction activity are conventionally performed using methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm in which such biological activities are stably recorded to the genome. Enhancer-driven genomic recording of transcriptional activity in multiplex (ENGRAM) is based on the signal-dependent production of prime editing guide RNAs that mediate the insertion of signal-specific barcodes (symbols) into a genomically encoded recording unit. We show how this strategy can be used for multiplex recording of the cell-type-specific activities of dozens to hundreds of cis-regulatory elements with high fidelity, sensitivity and reproducibility. Leveraging signal transduction pathway-responsive cis-regulatory elements, we also demonstrate time- and concentration-dependent genomic recording of WNT, NF-κB and Tet-On activities. By coupling ENGRAM to sequential genome editing via DNA Typewriter1, we stably record information about the temporal dynamics of two orthogonal signalling pathways to genomic DNA. Finally we apply ENGRAM to integratively record the transient activity of nearly 100 transcription factor consensus motifs across daily windows spanning the differentiation of mouse embryonic stem cells into gastruloids, an in vitro model of early mammalian development. Although these are proof-of-concept experiments and much work remains to fully realize the possibilities, the symbolic recording of biological signals or states within cells, to the genome and over time, has broad potential to complement contemporary paradigms for how we make measurements in biological systems.

First report of two Pantoea species causing bacterial leaf blight on wheat in the United States.

Wheat (Triticum aestivum) is an important crop worldwide, contributing to about one third of the global caloric intake. In June 2021, leaves with bacterial blight symptoms, including yellow and necrotic lesions running parallel to veins, were found in several fields across five counties in eastern Colorado (Weld, Morgan, Sedgwick, Baca, and Kit Carson). Plants exhibiting these symptoms were scattered throughout fields, but symptoms appeared consistent across counties. To determine the causal agent and complete Koch's postulates, a 1 cm2 symptomatic leaf area was excised and macerated in 0.5 mL of sterilized water from four field samples. The lysate was spread on yeast extract dextrose calcium carbonate medium (YDC agar, 1% yeast extract, 2% dextrose, 2% calcium carbonate, 1.5% agar) to isolate bacteria. Single colonies of yellow, mucoid morphology were selected and streaked on new YDC plates. Isolate genomic DNA was extracted (Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit, #D6005), and ~30 ng of gDNA was used to amplify the 16S rRNA, gyrB, and rpoB genes of all four isolates (Barret et al., 2015; Delétoile et al., 2009; Krawczyk et al., 2020; Ogier et al., 2019). Amplified PCR products were cleaned (Zymo DNA Clean & Concentrator kit, #D4033) and Sanger sequenced, and all sequences have been deposited in NCBI (16S rRNA: OR707336, OR707337, OR707338, OR707339), (gyrB: PP407951, PP407952, PP407953, PP407954), (rpoB: PP407955, PP407956, PP407957, PP407958). A BLAST search against whole genomes identified one isolate from Kit Carson county (CO314) and two isolates from Baca county (CO316 and CO317) as Pantoea agglomerans with 100% identity for the 16S rRNA, gyrB, and rpoB genes, and one isolate from Weld county (CO315) was 100% identical to Pantoea allii for all three genes. To complete Koch's postulates and confirm Pantoea sp. as the causal disease agents, isolates were grown as lawns on DifcoTM Nutrient Agar (NA) medium (48h, 28℃), suspended in 10 mM MgCl2 using a final optical density of 0.1 (~109 colony forming units per milliliter (CFU/mL)), and syringe-infiltrated into the entire leaf area of 10-day-old wheat seedling leaves (var. Hatcher). Treatments of 10mM MgCl2 and a field isolate that does not cause symptoms, identified as Pseudomonas synxantha by 16S rRNA and gyrB sequencing, were negative controls. Inoculated wheat plants were transferred to a growth chamber (22℃, 90% relative humidity). Symptoms developed 14 days post inoculation (dpi), with the most severe appearing 21 dpi. Each of the four Pantoea isolates were re-isolated from symptomatic leaves by grinding them in a Tissue Lyser II (Qiagen) with two metal beads and diluting with 0.4 mL of sterile water. A 20 µL sample of each isolate was plated on NA (24h, 28℃). The colonies appeared phenotypically identical to the original isolates, and Sanger sequencing confirmed the identities of the isolates. To our knowledge, this is the first report of P. agglomerans causing disease in wheat in the United States, and the first report of P. allii as a wheat disease-causing agent. This report is consistent with previous communications showing P. agglomerans causing wheat disease in China (Gao et al., 2023), and P. ananatis in Poland (Krawczyk et al., 2020). The growing numbers of reports of Pantoea spp. as pathogens in recent years suggests increasing novel disease emergence on cereals worldwide.

First Report of Polygonatum kingianum Leaf Spot Caused by Alternaria alternata in Kunming, China.

Polygonatum kingianum Coll. et Hemsl., a Polygonatum species in the Asparagaceae family, plays an important role in Chinese herbal medicine (Zhao et al. 2018). P. kingianum is widely planted in the Southwestern China. In September 2023, we observed a leaf spot of P. kingianum with disease incidence of 100%, and disease index reached 60 in commercial plantings in Kunming, Yunnan province, China (24.3610°N, 102.3740°E). In the initial stage of infection, symptoms manifested as a small circular brown spot. As the spots gradually expanded, they formed oval to irregular shaped lesions with grayish-white or dark-brown borders. Progressively the entire leaf withered and died. For identification of the causal agent of the leaf spot, leaf sections (5×5 mm2) were cut from the margin of the lesion and soaked in 75% ethanol for 10 s, 1% sodium hypochlorite for 3 min, washed with sterile distilled water, dried on sterilized tissue paper and placed on potato dextrose agar (PDA). The Petri dishes were then incubated at 28℃ for 3 days with a 12-h photoperiod. A predominant fungus was isolated from 95% of the samples. Three monosporic isolates were screened using a single-spore isolation method. After 4 days of incubation the colonies were white, after 7 days turned yellow-white. Conidia were black-brown, oblong or fusiform, with 3-7 transverse septa and 0-3 longitudinal septa, with dimensions of 19.5 to 49.5 × 8.7 to 17.6 µm (n = 30). Total genomic DNA of these three isolates was extracted from mycelia by the cetyltrimethylammonium bromide (CTAB) protocol. The nucleotide sequences of the elongation factor 1-alpha (EF1α), nuclear ribosomal internal transcribed spacer (ITS), 28S nuclear ribosomal large subunit rRNA gene (LSU), 18S nuclear ribosomal small subunit rRNA gene (SSU), and the second largest subunit of nuclear DNA-directed RNA polymerase II (RPB2) gene regions were amplified using the primer pairs EF1-728F/EF1-986R (Carbone and Kohn 1999), ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Schoch et al. 2012), NS1/NS4 (Schoch et al. 2012), and fRPB2-5F/fRPB2-7Cr (Liu et al. 1999), respectively. Amplicons were cloned in a pMDTM19-T vector (code no. 6013, Takara, Kusatsu, Japan) and bidirectionally sequenced. All three isolates had identical nucleotide sequences. Sequences from one isolate (PkF03) were deposited in GenBank. BLASTn analyses showed that sequences of EF1α (GenBank accession no. PP695240), ITS (PP694046), LSU (PP683406), SSU (PP683407), and RPB2 (PP695241) of isolate PkF03 were 99.6 (KP125134), 100 (KP124358), 100 (KP124510), 99.9 (KP124980), and 100% (KP124826), respectively, identical with Alternaria alternata (Fr.) Keissl. strain CBS 118815. Based on the nucleotide sequences of EF1α, ITS, LSU, SSU, and RPB2, a maximum likelihood phylogenetic tree was constructed using MEGAX with Tamura-Nei model. Isolate PkF03 was grouped in the same clade as A. alternata. According to the morphology and sequence analyses isolate PkF03 was identified as A. alternata (Woudenberg et al. 2013). To determine pathogenicity of isolate PkF03, a spore suspension (106 spores/mL) was sprayed on 1-year-old healthy leaves of P. kingianum. The control leaves were sprayed with sterile water. All plants were incubated at 28℃, 70% relative humidity, and a 12-h photoperiod. The pathogenicity tests were repeated three times with six plants in each treatment. Fifteen days post-inoculation, the inoculated leaves showed brown-yellow lesions, whereas the control leaves remained symptomless. A. alternata was reisolated from infected leaves. To our knowledge, this is the first report of A. alternata causing leaf spot on P. kingianum in Kunming, China. The results provide a scientific basis for prevention and control of the disease.

In vitro higher-order oligomeric assembly of the respiratory syncytial virus M2-1 protein with longer RNAs.

The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs in vitro using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments in vivo. Reconstructing the system in vitro uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment.

MRNA-based HIV-1 vaccines.

SUMMARYThe success of the Severe Acute Respiratory Syndrome Coronavirus 2 mRNA vaccines to lessen/prevent severe COVID-19 opened new opportunities to develop RNA vaccines to fight other infectious agents. HIV-1 is a lentivirus that integrates into the host cell genome and persists for the lifetime of infected cells. Multiple mechanisms of immune evasion have posed significant obstacles to the development of an effective HIV-1 vaccine over the last four decades since the identification of HIV-1. Recently, attempts to address some of these challenges have led to multiple studies that manufactured, optimized, and tested, in different animal models, mRNA-based HIV-1 vaccines. Several clinical trials have also been initiated or are planned to start soon. Here, we review the current strategies applied to HIV-1 mRNA vaccines, discuss different targeting approaches, summarize the latest findings, and offer insights into the challenges and future of HIV-1 mRNA vaccines.