Coat Protein Research Articles

Arachis mottle-associated virus, a new polerovirus infecting Pinto peanut.

A new polerovirus, named "arachis mottle-associated virus" (ArMoV), was identified by high-throughput sequencing in a Pinto peanut (Arachis pintoi) plant. The genome sequence was confirmed by Sanger sequencing and contains 5775 nucleotides and seven predicted open reading frames (ORFs), showing a typical polerovirus genome structure. All of the proteins encoded by ArMoV showed less than 90% amino acid sequence identity to those of other poleroviruses, the threshold to establish a new species in the genus. Phylogenetic analysis based on P1-P2 fusion protein and coat protein amino acid sequences showed that tobacco polerovirus 1 and chickpea chlorotic stunt virus, respectively, were the most closely related to ArMoV. These data suggest that ArMoV is a member of a new species of the genus Polerovirus, for which the binomial name "Polerovirus ARMOV " is proposed.

Genome-wide identification, transcript profiling and functional analyses of PCP gene family in Wucai (Brassica campestris).

Pollen coat proteins (PCPs) are cysteine-rich small-molecule proteins, which exhibit high levels of polymorphism and are expressed in gametocytes. Previous investigations have revealed that PCP genes are involved in pollen wall synthesis, pollen-stigma recognition, and pollen development and germination. However, gene expression and function of PCP family in pollen development is not well understood in Wucai (Brassica campestris L.). In this study, genome-wide identification and expression analysis of the BcPCP gene family members were conducted, including their physical and chemical properties, chromosome localization, phylogenetic relationships, gene structure, and tertiary structure. A total of 20 BcPCP genes were identified and classified into three subfamilies showing high homology to Arabidopsis thaliana. Expression pattern analysis indicated that the BcPCP gene family exhibits higher expression levels in reproductive organs, suggesting their potential involvement in the reproductive development. Notably, BraA02g002400.3C, potentially associated with male sterility, was identified through multiple transcriptomic and proteomic datasets. Subsequently, sequence analysis revealed its homology with the Arabidopsis GRP20 gene, and thus it was named BcGRP20. Functional analysis of this gene showed that overexpression of BcGRP20 gene in the Arabidopsis grp20 mutant could restore anther fertility. Overall, our findings indicate that BcGRP20 plays a critical role in pollen development and may be the causative gene for male sterility in Wucai. This study provides candidate genes for further functional identification of BcPCP genes in Wucai, which are crucial for anther development.

An improved DNA extraction method in okra for rapid PCR detection of Okra enation leaf curl virus from diverse Indian regions.

The extraction of DNA from okra (Abelmoschus esculentus) is challenging due to its high mucilage and polysaccharide content, which can hinder both the yield and quality of DNA.In this study, an improved DNA isolation method is described incorporating a key modification being the use of solution I (1 M NaCl and 2% Sarcosyl) as a pre-treatment before applying the CTAB buffer, resulting in high-purity genomic DNA in just 1h and 45min., making it suitable for handling large sample sizes due to its rapid processing capabilities. This enhanced DNA extraction method was crucial for the accurate and rapid molecular detection of Okra enation leaf curl virus (OELCuV), a monopartitebegomovirus that has spread across various regions of India. Transmitted by the whitefly (Bemisia tabaci), OELCuV causes leaf curling, enations, and stunted growth in okra, leading to significant yield losses. The surveys conducted during the 2020-21 and 2021-22 sowing seasons revealed disease incidence ranging from 14.03 to 67.57%. The extracted DNA via the improved DNA extraction method enhanced the speed of PCR based molecular identification of OELCuV, using virus-specific coat protein primers. The amplified CP genes were cloned and sequenced to study the CP gene based diversity among OELCuV isolates from different states of India. The CP gene nucleotide identity among the studied OELCuV isolates ranged from 95.57 to 99.27%, while comparison with previously reported Indian OELCuV CP sequences, the nucleotide identity ranged from 89.35 to 98.83%.The successful application of this optimized DNA extraction method sped up the detection process but also holds promise for broader use in the molecular study of okra and other mucilaginous crops, particularly in the rapid and reliable identification ofbegomoviruses. The optimized DNA extraction method significantly accelerated the detection of OELCuV, demonstrating its efficiency and reliability. This method shows strong potential for broader applications in the molecular study of okra and other mucilaginous crops, making it a valuable tool for future research and disease management.

Live Single-Cell Transcriptional Dynamics in Plant Cells.

Transcriptional reprogramming plays a key role in a variety of biological processes. Recent advances in RNA imaging techniques have allowed to visualize, in vivo, transcription-related mechanisms in different organisms. The MS2 system constitutes a robust method that has been used for over two decades to image multiple steps of a transcript's life cycle from "birth to death" with high spatiotemporal resolution in the animal field. It is based on the high affinity binding of the MS2 bacteriophage coat protein to its RNA hairpin ligands. Despite its broad applicability, a limited number of studies have implemented the system in plants, but without exploiting its full potential. Here, we describe the transposition of the MS2 technique to Arabidopsis. Combined with microfluidics, it allows to visualize the transcriptional repression of a phosphate starvation induced gene (SPX1) upon phosphate refeeding in vivo. The system provided access to the transcriptional response kinetics of individual cells, gene expression heterogeneity, and revealed bursting phenomena in plantae. The described methods provide new insights for multiple applications.

A Mycovirus Representing a Novel Lineage and a Mitovirus of Botrytis cinerea Co-Infect a Basidiomycetous Fungus, Schizophyllum commune

Strain IBc-114 was isolated from a gray mold lesion and was identified as the fungus Schizophyllum commune. In this strain, two mycoviruses, Schizophyllum commune RNA virus 1 (ScRV1, C_AA053475.1) and Botrytis cinerea mitovirus 9 strain IBc-114 (BcMV9/IBc-114, C_AA053476.1), were isolated and characterized. ScRV1 has flexuous filamentous particles about 20 ± 2.1 nm in diameter and 1000 ± 94.2 nm in length. The genome of ScRV1 is 7370 nt in length and contains two open reading frames (ORFs) which encode a polyprotein and a coat protein, respectively. The polyprotein has 1967 aa, including a helicase domain and an RdRp domain which has the highest identity of 28.21% with that of Entomophthora benyvirus E (EbVE). The coat protein has 241 aa which is mostly phylogenetically close to the coat proteins of Alphatetraviridae. Based on the phylogenetic analysis of ScRV1 and viruses selected, ScRV1 might represent a new family (temporarily named Mycobenyviridae) of the order Hepelivirales. The genome of BcMV9/IBc-114 that infects S. commune is 2729 nt in length and has only one ORF encoding an RdRp protein with 719 aa. BcMV9/IBc-114 has the highest identity of 98.61% with Botrytis cinerea mitovirus 9 (BcMV9) (MT089704). ScRV1, but not BcMV9/IBc-114, has certain effects on the host growth of S. commune. Furthermore, BcMV9/IBc-114 has been demonstrated to replicate in the ascomycetous fungi Botrytis cinerea and Sclerotinia sclerotiorum, and it negatively affects the growth and pathogenicity of B. cinerea, but it does not affect S. sclerotiorum. This is the first report of mycoviruses in S. commune and cross-phyla transmission of mitovirus in nature.

Molecular detection and sequencing of beet necrotic yellow vein virus and beet cryptic virus 2 in sugar beet from Kazakhstan

IntroductionBeet necrotic yellow vein virus (BNYVV) is a common viral pathogen that causes considerable economic loss globally. In the present study, a commercial realtime PCR test system and custom loop mediated amplification primers were used to detect the virus in asymptomatic sugar beet samples.MethodsA total of 107 of 124 samples tested positive for the presence of the A type BNYVV coat protein gene. Near complete sequences of RNA-3 and RNA-4 were obtained using reverse transcription, followed by nanopore sequencing of 14 samples.Results and discussionA comparison with available sequences, including previously published isolates Kas2 and Kas3 from Kazakhstan, identified RNA-3 as similar to such of the P-type isolates Puthiviers and Kas3. RNA-5 was not detected using real-time PCR or cDNA amplification. Unique variable sites were identified in the p25 protein sequence translated from RNA-3. Another virus, beet cryptic virus 2 (BCV2), was identified and sequenced in samples infected with BNYVV. With 85.28% genome coverage, the identified BCV2 samples were very similar to the previously reported isolates from Hungary and Germany

First Report of Pepper Chlorosis-Associated Virus in Burdock (Arctium lappa) in Korea.

Burdock (Arctium lappa) is a fiber-rich root vegetable widely used in Asian cuisine with many health benefits. In July 2023, two burdock leaf samples showing viral symptoms, such as yellowing and interveinal chlorosis, were collected from a field in Daegu, South Korea, with an incidence rate of about 10%. The samples were pooled, ground with liquid nitrogen, and total RNA was extracted using a Maxwell® 16 LEV Plant RNA Kit (Promega, Madison, USA). A cDNA library was constructed using TruSeq Standard Total RNA with Ribo-Zero (Illumina, San Diego, USA) and sequenced on an Illumina NovaSeq 6000 (BIOTO, Daejeon, Korea) with 100 bp paired-end reads. Of the approximately 84 million reads generated, about 61 million clean reads were identified using Trimmomatic 0.39 with default parameters. Subsequently, Trinity 2.13.0 was used for de novo assembly, resulting in 179,910 contigs, which, when annotated as previously described (Bak et al., 2024), revealed the presence of tomato spotted wilt virus (TSWV) and pepper chlorosis-associated virus (PepCaV). Three PepCaV-related contigs, representing a near-complete genome, were submitted to NCBI GenBank (PP502423-PP502425). BLASTn analysis showed 95.21% (7275/7641 bp), 95.89% (1680/1752 bp), and 96.18% (1461/1519 bp) identities with the L, M, and S segments of the PepCaV isolate Higashitsuno_2021 (LC719619-LC719621), respectively. RT-PCR was performed to confirm the presence of viruses using specific primers for PepCaV (5'-CACCTTGATTATAGACATGACAG/GTTCTCAAATCTTCCCAATCG-3') targeting the coat protein region, and TSWV (5'-GCAACAACTTGCAAGAAGATG/CATGACCTTCAGAAGGCTTG-3'; Kim et al., 2021). Initial results showed both samples were positive for TSWV, and one was positive for PepCaV. Additionally, 26 symptomatic and 8 asymptomatic samples were collected from the same field. PepCaV was positive in 3 samples, TSWV in 12, and TSWV+PepCaV in 11 by RT-PCR. All asymptomatic samples were negative. Leaves infected with PepCaV, whether solely or co-infected with TSWV, initially exhibited interveinal chlorosis and vein contraction on one side of the main vein (Fig. S1). In plants infected with PepCaV alone, these symptoms were mild or less noticeable in the initial phases. The majority of PepCaV-infected samples were co-infected with TSWV, during which symptoms appeared more severe. Following this, an amplicon, derived from the sample that initially tested positive for PepCaV in the first RT-PCR was cloned using the All in OneTM Cloning Kit (BioFact, Daejeon, Korea) and sequenced by Bionner (Daejeon, Korea). The resulting sequence, submitted to NCBI GenBank (PP928956), demonstrated 96.87% (805/831 bp) identity with the PepCaV isolate Higashitsuno_2021 (LC719600). Moreover, sap inoculation was performed on A. lappa, Nicotiana benthamiana, N. clevelandii, N. debneyi, N. rustica, N. tabacum, Chenopodium amaranticolor, and C. quinoa, but no transmission was identified in any case. PepCaV, recently identified and likely belonging to the genus Ophiovirus within the family Ophioviridae, is known to infect pepper and tobacco (Shimomoto et al., 2023; Wang et al., 2024). This is the first report of PepCaV in Korea and the first identification of burdock as a natural host. Although studies on its vectors and pathogenicity are lacking, PepCaV's presence suggests potential risks. Given the widespread cultivation of burdock in Korea, PepCaV could significantly impact local agriculture by affecting crop yield and health.

A Self-Amplifying Human Papillomavirus 16 Vaccine Candidate Delivered by Tobacco Mosaic Virus-Like Particles.

Virus-like particles (VLPs) are naturally occurring delivery platforms with potential for mRNA vaccines that can be used as an alternative to lipid nanoparticles. Here we describe a self-amplifying mRNA vaccine based on tobacco mosaic virus (TMV) expressing a mutated E7 protein from human papillomavirus 16 (HPV16). E7 is an early gene that plays a central role in viral replication and the oncogenic transformation of host cells, but nononcogenic mutant E7 proteins can suppress this activity. Immunization studies involving the delivery of self-amplifying mutant E7 mRNA packaged with TMV coat proteins confirmed the elicitation of E7-specific IgG antibodies. Additional in vitro splenocyte proliferation and cytokine profiling assays indicated the activation of humoral and cellular immune responses. We conclude that TMV particles are suitable for the delivery of mRNA vaccines and can preserve their integrity and functionality in vitro and in vivo.

Cryo-electron tomography reveals how COPII assembles on cargo-containing membranes.

Proteins traverse the eukaryotic secretory pathway through membrane trafficking between organelles. The coat protein complex II (COPII) mediates the anterograde transport of newly synthesized proteins from the endoplasmic reticulum, engaging cargoes with a wide range of size and biophysical properties. The native architecture of the COPII coat and how cargo might influence COPII carrier morphology remain poorly understood. Here we reconstituted COPII-coated membrane carriers using purified Saccharomyces cerevisiae proteins and cell-derived microsomes as a native membrane source. Using cryo-electron tomography with subtomogram averaging, we demonstrate that the COPII coat binds cargo and forms largely spherical vesicles from native membranes. We reveal the architecture of the inner and outer coat layers and shed light on how spherical carriers are formed. Our results provide insights into the architecture and regulation of the COPII coat and advance our current understanding of how membrane curvature is generated.

Self-Assembly of Microstructured Protein Coatings with Programmable Functionality for Fluorescent Biosensors.

Proteins, as genetically programmable functional macromolecules, hold immense potential as biocompatible self-assembling building blocks, owing to their versatility in building coating materials and programming their functionality genetically. In this study, we demonstrate a modular self-assembly of protein coatings that are genetically programmable for a biosensor application. We designed and produced recombinant fusion protein building blocks to form microstructured coatings on diverse substrates, such as glass or polymers, through thermally triggered liquid-liquid phase separation and an orthogonal high-affinity coiled-coil interaction. We incorporated fluorescence proteins into coatings and controlled the protein density to enable fluorescence imaging and quantification in a low-resource setting. Then, we created a coating for a calcium biosensor using a genetically engineered calcium indicator protein. This protein coating served as the foundation for our smartphone-based fluorescent biosensor, which successfully measured free calcium concentrations in the millimolar range at which extracellular calcium homeostasis is maintained. Using this fluorescent biosensor, we were able to detect abnormal physiological conditions, such as mild or moderate hypercalcemia. We envision that this modular and genetically programmable functional protein coating platform could be extended to the development of highly accessible, low-cost fluorescent biosensors for a variety of targets.

Identification of a novel mycovirus belonging to the “flexivirus”-related family with icosahedral virion

Abstract The order Tymovirales currently comprises five viral families with positive-sense RNA ((+)RNA) genomes that infect plants, fungi, and insects. Virion morphologies within the order Tymovirales differ between families, with icosahedral virions in the Tymoviridae and filamentous virions in the other “flexi”viridae families. Despite their different morphologies, these viruses are placed in the same order based on phylogenetic analyses of replicase-associated polyproteins. However, one of the families in the Tymovirales, Deltaflexiviridae, is considered to be capsidless because there have been no published reports of virion isolation. Here we report that a new “flexivirus”-related (+)RNA virus, prospectively named Fusarium oxysporum icosahedral virus 1 (FoIV1), is icosahedral, and that most deltaflexiviruses may have icosahedral virions. Phylogenetic analyses based on replicase-associated polyproteins indicated that FoIV1 forms a distinct group in the Tymovirales with some viruses originally assigned to the Deltaflexiviridae. Electron microscopy, protein analysis, and protein structure predictions indicate that FoIV1 ORF4 encodes a single jelly-roll (SJR)-like coat protein (CP) that constitutes the icosahedral virions. Results of clustering analyses based on amino acid sequences and predicted CP structures suggested that most of the deltaflexiviruses have icosahedral virions composed of SJR-like CPs as in FoIV1, rather than having filamentous virions or capsidless. These results challenge the conventional understanding of viruses in the order Tymovirales, with important implications for revising its taxonomic framework and providing insights into the evolutionary relationships within this diverse and broad host range group of (+)RNA viruses.

Prokaryotic Expression of Coat Protein Gene of Grapevine Berry Inner Necrosis Virus and Preparation of Its Polyclonal Antibody

Grapevine berry inner necrosis virus (GINV) and grapevine Pinot gris virus (GPGV) are prevalent viral diseases affecting viticulture, posing significant threats in grape-producing regions of China. Previous studies have emphasized the harmful effects of grape viruses on the grape industry all over the world. However, few reports have focused specifically on GINV. In wild grapevines, GINV infection frequently leads to grapevine fanleaf degeneration disease (GFDD). GINV often co-occurs with other grape viruses, exacerbating its harmful effects on the grapevine industry in China. In this study, we collected grapevine samples from Taizhou city, Jiangsu Province, where GINV infection was confirmed. Based on the GINV coat protein (CP) gene, we developed a high-throughput and high-sensitivity direct antigen-coated ELISA and Dot blot assay for field diagnosis of GINV CP in grape samples. The CP gene was cloned from GINV-infected grape samples, and the GINV CP was expressed using the pET30(a) vector. Specific polyclonal antiserum CPGINV was generated by immunizing rabbits with the purified protein, and its sensitivity was determined to be satisfactory. Leveraging the high accuracy and sensitivity of the CPGINV antiserum, we developed a rapid, precise, and scalable diagnostic method for GINV in the grapevine industry. The established ELISA and Dot blot assays successfully detected GINV-infected grapevine samples. The occurrence of GINV is relatively common in China, which poses a risk of transmission and threatens the healthy development of the grape industry. Therefore, this study prepared CPGINV antiserum and established an efficient, rapid, sensitive, accurate, and high-throughput diagnostic method, providing a foundational approach for the prevention and control of vitis viral diseases.

A survey on the occurrence of papaya ringspot virus-P (PRSV-P) in Malaysia and genetic diversity assessment of the coat protein region.

Papaya ringspot virus (PRSV) is a plant virus transmitted by aphids that has spread throughout many countries, including Malaysia, causing yield losses and economic impacts to the papaya industry worldwide. PRSV infection in papaya-distinctive ring-shaped patterns on papaya leaves resulted in stunted growth and reduced fruit quality. Management strategies such as the use of resistant varieties, cultural practices, and vector control are employed to mitigate the spread of PRSV. However, the evolution of new virus strains and the uncertainties posed by climate change pose ongoing challenges for the management of PRSV worldwide. Therefore, in this present study, we aim to confirm the presence of PRSV in symptomatic papaya leaves, to depict the current status of PRSV in Malaysia. Using reverse-transcription PCR (RT-PCR) targeting PRSV partial nuclear inclusion b protein (NIb) and coat protein (CP), 13 out of 40 papaya leaves collected were found positive for the PRSV strain-P (PRSV-P). Nucleotide analysis revealed a high similarity with strains from Taiwan and India, showing 96.83%, 97.03%, and 97.03% identity with the Taiwan strains (DQ340771, AY027810) and the India strain (KJ755852), respectively. Compared to the CP gene of Malaysian isolates reported in 2016 (EU082207), several nonsynonymous mutations have been discovered suggesting genetic diversity within the PRSV population in Malaysia. Overall, this study confirms the current circulation of PRSV infection in Malaysia since it was first identified in Johore in 1991. The re-occurrence of PRSV-P in this study highlights the need for continuous monitoring and targeted management strategies to prevent the further spread of PRSV-P in Malaysia.

Fish Snx27 promotes viral products by modulating the innate immune response and exosomal machinery.

Viral nervous necrosis caused by the nervous necrosis virus (NNV) poses a significant threat to the global aquaculture industry. Developing preventive methods to minimize economic losses due to NNV infections is crucial. This study explored the role of the sorting nexin 27 (Snx27) gene, encoded by the orange-spotted grouper (Epinephelus coioides) and referred to as EcSnx27, as an immune regulator affecting red-spotted grouper nervous necrosis virus (RGNNV) infection in vitro. Our findings revealed that EcSnx27 negatively regulates interferon (IFN)-related cytokines and the promoter activities of fish ISRE and NF-κB. Furthermore, we identified the SNX-FERM and SNX-FERM-like domains as responsible for the interaction between EcSnx27 and RGNNV coat protein. Through the detection of viable virions associated with EcSnx27-containing exosomes, we propose that EcSnx27 may contribute to the release process of RGNNV by influencing the apoptosis-linked gene 2-interacting protein X (ALIX)-associated exosomal pathway. Consequently, our study suggests that EcSnx27 promotes RGNNV replication by inhibiting the IFN immune response and facilitating virus production and release through ALIX-mediated exosomal machinery.IMPORTANCERed grouper nervous necrosis virus (RGNNV), a member of the Nodaviridae family, has emerged as a significant cause of fish diseases worldwide, leading to high morbidity and mortality rates. This study investigated the sorting nexin 27 (Snx27) gene encoded by the orange-spotted grouper (Epinephelus coioides) on RGNNV infection in grouper kidney cells. Our findings revealed that EcSnx27 negatively regulated the interferon pathway, resulting in the promotion of RGNNV replication. Additionally, we observed that EcSnx27 could interact with apoptosis-linked gene 2-interacting protein X (ALIX) and the RGNNV coat protein, suggesting its potential involvement in viral release processes through modulation of the exosomal pathway. Our study identified EcSnx27 as a key target that RGNNV exploits to enhance viral production. This finding offers valuable insights into the immune evasion and viral release mechanisms of non-enveloped RNA viruses.

Fiber-based Biomaterial Scaffolds for Cell Support towards the Production of Cultivated Meat

The in vitro production of animal-derived foods via cellular agriculture is emerging as a key solution to global food security challenges. Here, the potential for fiber-based scaffolds, including silk and cotton, in the cultivation of muscle cells for tissue formation was pursued. Mechanical properties and cytocompatibility with the mouse myoblast cell line C2C12 and immortalized bovine muscle satellite cells (iBSCs) were assessed, as well as pre-digestion options for the materials due to their resilience within the human digestive track. The fibers supported cell adhesion, proliferation, and guided muscle cell orientation, facilitating myotube formation per differentiation. A progressive increase in biomass was also documented. Interestingly, iBSC proliferation was enhanced with coatings of recombinant proteins while C2C12 cells showed minimal response. Thus, both cotton and silk yarns were suitable as fiber-based scaffolds towards cell supportive goals, suggesting an alternative path toward structured protein-rich foods via this initial stage of textile engineering for food. Biomass prediction models were generated, enabling forecasts of cell growth and maturation across various scaffold conditions and cell types. This capability enhances the precision of the cultivation process towards an engineering approach, building on the inherent benefits of hierarchical muscle tissue structure, but here via textile engineering with these initial muscle-coated edible fibers. Further, the approach offers to reduce costs by optimizing cultivation time and media needs. These approaches are part of a foundation for future scalable and sustainable cultivated meat production. Statement of SignificanceThis research investigates the use of one-dimensional fiber-based scaffolds for cultivated meat production, contributing to advancements in cellular agriculture. It introduces a method to measure changes in biomass and scaffold degradation throughout the cultivation process. Additionally, our development of biomass prediction models improves the precision and predictability of cultivated meat production. This research not only aids in scaling up cultivated meats but also enhances the use of textile engineering techniques in tissue engineering, paving the way for producing complex, three-dimensional meat structures more sustainably.

TMEM39A and TMEM131 facilitate bulk transport of ECM proteins through large COPII vesicle formation

The growth of Caenorhabditis elegans involves multiple molting processes, during which old cuticles are shed, and new cuticles are rapidly formed. This process requires the regulated bulk secretion of cuticle components. The transmembrane protein-39 (TMEM-39) mutant exhibits distinct dumpy and ruptured phenotypes characterized by notably thin cuticles. TMEM-39 primarily co-localizes with the coat protein II complex (COPII) in large vesicles rather than small COPII vesicles. These TMEM-39-associated large vesicles (TMEM-39-LVs) form robustly during the molting period and co-localize with various extracellular matrix (ECM) components, including BLI-1 collagen, BLI-3 dual oxidase, and carboxypeptidases. Through immunoprecipitation using TMEM39A-FLAG and proteomics analysis in human sarcoma cells, we identify TMEM39A-associated proteins, including TMEM131. Knockdown of TMEM131 results in reduced TMEM39A-LV formation and collagen secretion in both C. elegans and human sarcoma cells, indicating a cooperative role between TMEM39A and TMEM131 in the secretion of extracellular components through the formation of large COPII vesicles. Given the conservation of TMEM39A and its associated proteins between C. elegans and humans, TMEM39A-LV may represent a fundamental machinery for rapid and extensive secretion across metazoans.

EIF2Bβ confers resistance to Turnip mosaic virus by recruiting ALKBH9B to modify viral RNA methylation.

Eukaryotic translation initiation factors (eIFs) are the primary targets for overcoming RNA virus resistance in plants. In a previous study, we mapped a BjeIF2Bβ from Brassica juncea representing a new class of plant virus resistance genes associated with resistance to Turnip mosaic virus (TuMV). However, the mechanism underlying eIF2Bβ-mediated virus resistance remains unclear. In this study, we discovered that the natural variation of BjeIF2Bβ in the allopolyploid B. juncea was inherited from one of its ancestors, B. rapa. By editing of eIF2Bβ, we were able to confer resistance to TuMV in B. juncea and in its sister species of B. napus. Additionally, we identified an N6-methyladenosine (m6A) demethylation factor, BjALKBH9B, for interaction with BjeIF2Bβ, where BjALKBH9B co-localized with both BjeIF2Bβ and TuMV. Furthermore, BjeIF2Bβ recruits BjALKBH9B to modify the m6A status of TuMV viral coat protein RNA, which lacks the ALKB homologue in its genomic RNA, thereby affecting viral infection. Our findings have applications for improving virus resistance in the Brassicaceae family through natural variation or genome editing of the eIF2Bβ. Moreover, we uncovered a non-canonical translational control of viral mRNA in the host plant.

Immunodiagnostic detection of banana bract mosaic virus (BBrMV) infecting abaca through the recombinant coat protein and its sequence diversity analysis in the Philippines

Abaca is an indigenous fiber crop in the Philippines and is an important source of raw fiber worldwide. Banana bract mosaic virus (BBrMV) causes the abaca bract mosaic disease which results in decreased fiber yield and quality. Out of the 261 abaca samples tested, 175 tested positive via ELISA using raw antiserum against the recombinant BBrMV coat protein (CP). To further validate the antisera produced, a total of 48 samples were screened for BBrMV CP fragment via RT-PCR. Out of the 26 positives for RT-PCR, 22 were cloned and sequenced to validate the RT-PCR amplified fragments. Nucleotide analysis showed a high percentage identity (97–98%) towards BBrMV CP Pacific and Indian isolates while phylogenetic analysis revealed clustering of BBrMV CP Philippine abaca isolates with the Pacific group. The results validate the application of polyclonal antibodies against the recombinant coat protein of BBrMV (rCP) for its serological detection in infected abaca.

The first report of cotton leafroll dwarf virus infecting upland cotton plants in Arizona.

Cotton leafroll dwarf virus (CLRDV) represents a persistent threat to cotton production in the United States (U.S.) (Edula et al. 2023). Initially detected in Alabama (Avelar et al. 2019), CLRDV occurs in almost all the states in the U.S. cotton belt, extending from Virginia through Texas. The symptoms associated with CLRDV includes interveinal chlorosis, leaf rolling, stunting, and reduced boll sets. However, asymptomatic CLRDV-infected plants have also been reported (Edula et al. 2023). In the 2023 growing season, upland cotton plants (Gossypium hirsutum) presenting terminal splitting symptoms in the blooming stage were observed in a commercial field in Pinal County, Arizona, at 60% incidence. To evaluate if CLRDV may be associated with these symptoms, young fully expanded terminal leaves were collected from 20 symptomatic and ten asymptomatic plants. Moreover, ten samples were collected from a second asymptomatic block in the same field. Samples were shipped on dry ice to Cornell University in hermetic bags under APHIS PPQ permit 526-23-256-14384. Midribs and petioles were used to extract total nucleic acids from each sample using OPS Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics) per the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using an iScript Reverse transcriptase kit (BioRad) and used for the detection of a partial sequence of the coat protein (CP) of CLRDV using PCR assays as described previously (Mahas et al. 2022). The expected 309-bp product was obtained from only one symptomatic sample. CLRDV presence in the samples was further evaluated using CLRDV-specific primers targeting the RNA-dependent RNA polymerase (RdRp) gene. Primers CLRDV-Pol_innerF1 (5'- ACCCTCCAAGGAACAGAG -3') / R1 (5'- CGAATAATCTGATYGGGTCAC -3') and CLRDV-Pol_outerF1 (5'- AACGCGCCCAGTCCGCACAAATACC-3') / R1 (5'-ACCGGGTTTACTGGGGATTGCACGC-3'), designed based on virus isolates available in GenBank as of September 2022, were used to implement a single-tube nested RT-PCR as detailed by Dey et al. (2012) and to index the presence of CLRDV in all the samples. Two additional symptomatic samples and three asymptomatic samples (two from field one and one from field two) were positive for the virus. Direct Sanger sequencing of the one CP and two RdRp amplicons from symptomatic and asymptomatic samples (PP482918-20) demonstrated they shared >99% nucleotide identity to an isolate from South Carolina (OQ300129). To further evaluate the presence of CLRDV in Arizona, we performed double antibody sandwich (DAS)-ELISA on midrib and petiole samples using camelid single-chain antibodies against the CP as the capture antibody (Filed Patent 18/436,287) and the commercially available Anti-PLRV conjugate (Agdia, Elkhart, IN) as the secondary antibody. DAS-ELISA detected CLRDV in eight symptomatic and three asymptomatic samples, with the positive and negative controls testing positive and negative, respectively. Considering all the diagnostic approaches, ten symptomatic and five asymptomatic samples were positive for CLRDV (Table S1). To the best of our knowledge, this is the first report of CLRDV in Arizona. Future studies are required to evaluate the possible incidence and impact on the crop yield in the state and develop a reliable diagnostic assay to detect the virus in all infected samples. Since our study did not associate CLRDV with the terminal abortion or splitting symptoms, ongoing efforts are underway to elucidate if other viruses may be associated with the symptoms.

Selection of Force Sensors for In Situ Measurement of Neotissue Microenvironments.

Mechanical forces are a critical stimulus in both native and engineered tissues. Direct measurement of these microenvironmental forces has been challenging, particularly for cell-dense models. To address this, we previously developed hydrogel-based force sensors that are approximately the size of a cell and can be imaged over time to computationally assess the forces exerted by surrounding cells and matrix. The goal of this project was to identify how the physical characteristics of force sensors impact measurements. Sensors were varied in size, elastic modulus, and surface coating before being included in stem cell suspensions that then spontaneously self-assembled into spheroidal neotissues. Using this model of early mesenchymal condensation, we hypothesized that larger, softer sensors would provide greater sensitivity and precision, whereas protein coatings would influence the directionality of applied forces (tensile vs. compressive). These experiments were conducted using a high-content imaging system that allowed analysis of over a thousand sensors to evaluate the various conditions. Results indicated that measurement fidelity was highest for force sensors that had a diameter >20 µm and modulus ∼0.2 kPa. Extremely soft sensors deformed too much, whereas stiffer sensors deformed too little. Collagen and N-cadherin coatings, which replicated cell-matrix or cell-cell binding, respectively, allowed for tensile forces to be exerted on the sensors, with greater forces being observed for N-cadherin sensors in these highly cellular neotissue constructs. Uncoated sensors were universally compressed due to the lack of cell-sensor adhesion. Disruption of the actin cytoskeleton lessened microenvironmental forces, whereas disruption of microtubules had no measurable effect. Potential future applications of the technology include studies of in situ forces in developing tissues as well as a real-time sensor for monitoring the growth of engineered constructs.