Dual activities of an X-family DNA polymerase regulate CRISPR-induced insertional mutagenesis across species.

The canonical non-homologous end joining (c-NHEJ) repair pathway, generally viewed as stochastic, has recently been shown to produce predictable outcomes in CRISPR-Cas9 mutagenesis. This predictability, mainly in 1-bp insertions and small deletions, has led to the development of in-silico prediction programs for various animal species. However, the predictability of CRISPR-induced mutation profiles across species remained elusive. Comparing CRISPR-Cas9 repair outcomes between human and plant species reveals significant differences in 1-bp insertion profiles. The high predictability observed in human cells links to the template-dependent activity of human Polλ. Yet plant Polλ exhibits dual activities, generating 1-bp insertions through both templated and non-templated manners. Polλ knockout in plants leads to deletion-only mutations, while its overexpression enhances 1-bp insertion rates. Two conserved motifs are identified to modulate plant Polλ's dual activities. These findings unveil the mechanism behind species-specific CRISPR-Cas9-induced insertion profiles and offer strategies for predictable, precise genome editing through c-NHEJ.

Sexual dimorphism in neurobehavioural phenotype and gut microbial composition upon long-term exposure to structural analogues of bisphenol-A.

Bisphenol S (BPS) and Bisphenol F (BPF), the analogues of the legacy endocrine disrupting chemical, Bisphenol A (BPA) are ubiquitous in the environment and present in various consumer goods, and potentially neurotoxic. Here, we studied sex-specific responses of bisphenols on behavioural phenotypes, including their association with pro-inflammatory biomarkers and altered neurotransmitters levels, and the key gut microbial abundances. Neurobehavioural changes, using standard test battery, biochemical and molecular estimations for inflammatory cytokines, neurotransmitters, and oxido-nitrosative stress markers, gene expression analysis using qRT-PCR, H&E based histological investigations, gut permeability assays and Oxford Nanopore-based 16S-rRNA metagenomics sequencing for the gut microbial abundance estimations were performed. Bisphenol(s) exposure induces anxiety and depression-like behaviours, particularly in the male mice, with heightened pro-inflammatory cytokines levels and systemic endotoxemia, altered monoamine neurotransmitters levels/turnovers and hippocampal neuronal degeneration and inflammatory responses in the brain. They also increased gut permeability and altered microbial diversity, particularly in males. Present study provides evidence for sex-specific discrepancies in neurobehavioural phenotypes and gut microbiota, which necessitate a nuanced understanding of sex-dependent responses to bisphenols. The study contributes to ongoing discussions on the multifaceted implications of bisphenols exposure and underscores the need for tailored regulatory measures to mitigate potential health risks associated with them.

Correction: Anwer et al. Molecular and Morphological Characterization of Exserohilum turcicum (Passerini) Leonard and Suggs Causing Northern Corn Leaf Blight of Maize in Bihar. Bioengineering 2022, 9, 403.

In the original publication [...].

Prime editing in plants: prospects and challenges.

Prime editors are reverse transcriptase (RT)-based genome-editing tools that utilize double-strand break (DSB)-free mechanisms to decrease off-target editing in genomes and enhance the efficiency of targeted insertions. The multiple prime editors that have been developed within a short span of time are a testament to the potential of this technique for targeted insertions. This is mainly because of the possibility of generation of all types of mutations including deletions, insertions, transitions, and transversions. Prime editing reverses several bottlenecks of gene editing technologies that limit the biotechnological applicability to produce designer crops. This review evaluates the status and evolution of the prime editing technique in terms of the types of editors available up to prime editor 5 and twin prime editors, and considers the developments in plants in a systematic manner. The various factors affecting prime editing efficiency in plants are discussed in detail, including the effects of temperature, the prime editing guide (peg)RNA, and RT template amongst others. We discuss the current obstructions, key challenges, and available resolutions associated with the technique, and consider future directions and further improvements that are feasible to elevate the efficiency in plants.

Physiology, genomics, and evolutionary aspects of desert plants.

BACKGROUND: Despite the exposure to arid environmental conditions across the globe ultimately hampering the sustainability of the living organism, few plant species are equipped with several unique genotypic, biochemical, and physiological features to counter such harsh conditions. Physiologically, they have evolved with reduced leaf size, spines, waxy cuticles, thick leaves, succulent hydrenchyma, sclerophyll, chloroembryo, and photosynthesis in nonfoliar and other parts. At the biochemical level, they are evolved to perform efficient photosynthesis through Crassulacean acid metabolism (CAM) and C4 pathways with the formation of oxaloacetic acid (Hatch-Slack pathway) instead of the C3 pathway. Additionally, comparative genomics with existing data provides ample evidence of the xerophytic plants' positive selection to adapt to the arid environment. However, adding more high-throughput sequencing of xerophyte plant species is further required for a comparative genomic study toward trait discovery related to survival. Learning from the mechanism to survive in harsh conditions could pave the way to engineer crops for future sustainable agriculture. AIM OF THE REVIEW: The distinct physiology of desert plants allows them to survive in harsh environments. However, the genomic composition also contributes significantly to this and requires great attention. This review emphasizes the physiological and genomic adaptation of desert plants. Other important parameters, such as desert biodiversity and photosynthetic strategy, are also discussed with recent progress in the field. Overall, this review discusses the different features of desert plants, which prepares them for harsh conditions intending to translate knowledge to engineer plant species for sustainable agriculture. KEY SCIENTIFIC CONCEPTS OF REVIEW: This review comprehensively presents the physiology, molecular mechanism, and genomics of desert plants aimed towards engineering a sustainable crop.

Innovative Approaches and Therapies to Enhance Neuroplasticity and Promote Recovery in Patients With Neurological Disorders: A Narrative Review.

Brain rehabilitation and recovery for people with neurological disorders, such as stroke, traumatic brain injury (TBI), and neurodegenerative diseases, depend mainly on neuroplasticity, the brain's capacity to restructure and adapt. This literature review aims to look into cutting-edge methods and treatments that support neuroplasticity and recovery in these groups. A thorough search of electronic databases revealed a wide range of research and papers investigating several neuroplasticity-targeting methods, such as cognitive training, physical activity, non-invasive brain stimulation, and pharmaceutical interventions. The results indicate that these therapies can control neuroplasticity and improve motor, mental, and sensory function. In addition, cutting-edge approaches, such as virtual reality (VR) and brain-computer interfaces (BCIs), promise to increase neuroplasticity and foster rehabilitation. However, many issues and restrictions still need to be resolved, including the demand for individualized treatments and the absence of defined standards. In conclusion, this review emphasizes the significance of neuroplasticity in brain rehabilitation. It identifies novel strategies and treatments that promise to enhance recovery in patients with neurological illnesses. Future studies should concentrate on improving these therapies and developing evidence-based standards to direct clinical practice and enhance outcomes for this vulnerable population.

The curious case of genome packaging and assembly in RNA viruses infecting plants.

Genome packaging is the crucial step for maturation of plant viruses containing an RNA genome. Viruses exhibit a remarkable degree of packaging specificity, despite the probability of co-packaging cellular RNAs. Three different types of viral genome packaging systems are reported so far. The recently upgraded type I genome packaging system involves nucleation and encapsidation of RNA genomes in an energy-dependent manner, which have been observed in most of the plant RNA viruses with a smaller genome size, while type II and III packaging systems, majorly discovered in bacteriophages and large eukaryotic DNA viruses, involve genome translocation and packaging inside the prohead in an energy-dependent manner, i.e., utilizing ATP. Although ATP is essential for all three packaging systems, each machinery system employs a unique mode of ATP hydrolysis and genome packaging mechanism. Plant RNA viruses are serious threats to agricultural and horticultural crops and account for huge economic losses. Developing control strategies against plant RNA viruses requires a deep understanding of their genome assembly and packaging mechanism. On the basis of our previous studies and meticulously planned experiments, we have revealed their molecular mechanisms and proposed a hypothetical model for the type I packaging system with an emphasis on smaller plant RNA viruses. Here, in this review, we apprise researchers the technical breakthroughs that have facilitated the dissection of genome packaging and virion assembly processes in plant RNA viruses.

Efficient protein tagging and cis-regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants.

Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient nonhomologous end-joining-mediated targeted insertion method was recently developed by combining clustered regularly interspaced short palindromic repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated nuclease 9 (SpCas9) gene editing with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet, this approach often leads to imprecise insertions with no control over the insertion direction. Here, we compared the influence of chemical protection of dsODNs on efficiency of targeted insertion. We observed that CRISPR/SpCas9 frequently induced staggered cleavages with 1-nucleotide 5' overhangs; we also evaluated the effect of donor end structures on the direction and precision of targeted insertions. We demonstrate that chemically protected dsODNs with 1-nucleotide 5' overhangs significantly improved the precision and direction control of target insertions in all tested CRISPR targeted sites. We applied this method to endogenous gene tagging in green foxtail (Setaria viridis) and engineering of cis-regulatory elements for disease resistance in rice (Oryza sativa). We directionally inserted 2 distinct transcription activator-like effector binding elements into the promoter region of a recessive rice bacterial blight resistance gene with up to 24.4% efficiency. The resulting rice lines harboring heritable insertions exhibited strong resistance to infection by the pathogen Xanthomonas oryzae pv. oryzae in an inducible and strain-specific manner.

An extensible vector toolkit and parts library for advanced engineering of plant genomes.

Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual-luciferase assays to measure contributions to expression from promoters, terminators, and from cross-cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

Genome-resolved metagenomics inferred novel insights into the microbial community, metabolic pathways, and biomining potential of Malanjkhand acidic copper mine tailings.

Mine tailing sites provide profound opportunities to elucidate the microbial mechanisms involved in ecosystem functioning. In the present study, metagenomic analysis of dumping soil and adjacent pond around India's largest copper mine at Malanjkhand has been done. Taxonomic analysis deciphered the abundance of phyla Proteobacteria, Bacteroidetes, Acidobacteria, and Chloroflexi. Genomic signatures of viruses were predicted in the soil metagenome, whereas Archaea and Eukaryotes were noticed in water samples. Mesophilic chemolithotrophs, such as Acidobacteria bacterium, Chloroflexi bacterium, and Verrucomicrobia bacterium, were predominant in soil, whereas, in the water sample, the abundance of Methylobacterium mesophilicum, Pedobacter sp., and Thaumarchaeota archaeon was determined. The functional potential analysis highlighted the abundance of genes related to sulfur, nitrogen, methane, ferrous oxidation, carbon fixation, and carbohydrate metabolisms. The genes for copper, iron, arsenic, mercury, chromium, tellurium, hydrogen peroxide, and selenium resistance were found to be predominant in the metagenomes. Metagenome-assembled genomes (MAGs) were constructed from the sequencing data, indicating novel microbial species genetically related to the phylum predicted through whole genome metagenomics. Phylogenetic analysis, genome annotations, functional potential, and resistome analysis showed the resemblance of assembled novel MAGs with traditional organisms used in bioremediation and biomining applications. Microorganisms harboring adaptive mechanisms, such as detoxification, hydroxyl radical scavenging, and heavy metal resistance, could be the potent benefactions for their utility as bioleaching agents. The genetic information produced in the present investigation provides a foundation for pursuing and understanding the molecular aspects of bioleaching and bioremediation applications.

Molecular and Morphological Characterization of Exserohilum turcicum (Passerini) Leonard and Suggs Causing Northern Corn Leaf Blight of Maize in Bihar.

Maize is considered the third most important cereal crop in Asia after rice and wheat. Many diseases affect this crop due to the cultivation of various hybrids. This research aimed to characterize the causative agent of northern corn leaf blight disease in Bihar, India, caused by Exserohilum turcicum (Passerini) Leonard and Suggs. Leaf samples were collected from infected fields in five maize growing districts of Bihar in 2020-2022. A total of 45 fungal isolates from 135 samples were examined for cultural, morphological, and molecular characteristics and were identified as E. turcicum. The isolates were grouped into four groups based on colony color, i.e., olivaceous brown, blackish brown, whitish black, and grayish, and into two groups based on regular and irregular margins. The conidial shapes were observed to be elongated and spindle-shaped with protruding hilum, with conidial septa ranging from 2-12. Similarly, conidial length varied from 52.94 µm to 144.12 µm. ß-tubulin gene sequences analysis made it possible to verify the identities of fungal strains and the phylogenetic relationships of all isolates, which were clustered in the same clade. The ß-tubulin gene sequences of all the isolates showed a high level of similarity (100%) with reference isolates from GenBank accession numbers KU670342.1, KU670344.1, KU670343.1, KU670341.1, and KU670340.1. The findings of this study will serve as a baseline for future studies and will help to minimize yield losses.

Knockdown of capsid protein encoding novel ATPase domain inhibits genome packaging in potato leafroll virus.

Potato leafroll virus (PLRV) uses powerful molecular machines to package its genome into a viral capsid employing ATP as fuel. Although, recent bioinformatics and structural studies have revealed detailed mechanism of DNA packaging, little is known about the mechanochemistry of genome packaging in small plant viruses such as PLRV. We have identified a novel P-loop-containing ATPase domain with two Walker A-like motifs, two arginine fingers, and two sensor motifs distributed throughout the polypeptide chain of PLRV capsid protein (CP). The composition and arrangement of the ATP binding and hydrolysis domain of PLRV CP is unique and rarely reported. The discovery of the system sheds new light on the mechanism of viral genome packaging, regulation of viral assembly process, and evolution of plant viruses. Here, we used the RNAi approach to suppress CP gene expression, which in turn prevented PLRV genome packaging and assembly in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with siRNA constructs against the CP with ATPase domain exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of CP gene expression is an efficient method for generating PLRV-resistant potato plants. In addition, molecular docking study reveals that the PLRV CP protein has ATP-binding pocket at the interface of each monomer. This further confirms that knockdown of the CP harboring ATP-binding domain could hamper the process of viral genome packaging and assembly. Moreover, our findings provide a robust approach to generate PLRV-resistant potato plants, which can be further extended to other species. Finally, we propose a new mechanism of genome packaging and assembly in plant viruses.

Exserohilum turcicum (Passerini) Leonard and Suggs: Race Population Distribution in Bihar, India.

Northern corn leaf blight (NCLB) of maize, caused by Exserohilum turcicum (Pass.) Leonard and Suggs., is an important foliar disease common across maize-producing areas of the world, including Bihar, India. In this study, virulence and distribution of races were observed against Ht-resistant genes and also identified the E. turcicum race population distribution in Bihar. For that, 45 E. turcicum isolates were collected from maize fields in Bhagalpur, Begusarai, Khagaria, Katihar and Samastipur districts between 2020 and 2022. These isolates were screened on maize differential lines containing Ht1, Ht2, Ht3 and HtN1 resistance genes. Five different physiological races were observed based on the symptoms response of the differential maize lines. These races are race 0, race 1, race 3, race 23N and race 123N. E. turcicum race 3 was the most prevalent race having 26.6% frequency followed by race 0 (24.4%) and race 1 (22.2%) and the least prevalent races were race 23N and 123N having 13.3% each. Varied resistance response of different isolates was observed on differential lines having different resistant genes. Despite the fact that virulence was seen against all Ht resistance genes, NCLB control might be increased by combining qualitative Ht resistance genes with quantitative resistance.

Frequency of Electrolyte Imbalance in Patients Presenting With Acute Stroke.

Introduction Electrolyte disturbances are commonly reported in acute stroke in studies conducted in the western world. Presently, the data available about the prevalence of electrolyte disturbance in patients with stroke are not sufficient, especially from developing countries. The purpose of our study is to determine the frequency of occurrence of electrolyte imbalance in patients presenting with acute stroke in a tertiary care hospital. Methods This descriptive cross-sectional study was conducted in the department of internal medicine and neurology in a tertiary care hospital, Pakistan, from December 2019 to March 2021. A total of 300 patients, aged between 30 and 70 years, with either ischemic or hemorrhagic stroke, as diagnosed on contrast tomography (CT) scan of the head or magnetic resonance imaging (MRI) of the brain, were enrolled in the study. The biochemical analysis of the stroke patients was done. Results Out of the 300 participants, 139 (46.3%) participants were from the ischemic stroke group while 161 (53.7%) were from the hemorrhagic stroke group. The mean sodium level was significantly lower in the ischemic group as compared to the hemorrhagic group (129.41 ± 3.12 mEq/L vs. 134.42 ± 3.46 mEq/L; p-value: <0.0001). Potassium level was significantly higher in the hemorrhagic group compared to the ischemic group (6.27 ± 1.12 mmol vs. 4.31 ± 0.71 mmol; p-value: <0.0001). Conclusion Patients coming to emergency with stroke should be screened immediately for electrolyte imbalance. Early identification of rapid imbalances of serum electrolytes may aid in prompt medical intervention and resultant improved outcomes in stroke patients. It is crucial that electrolyte imbalances in these patients are closely monitored to avoid any complications.

Frequency of Cutaneous Disorders in Patients With Celiac Disease.

Background and objective Celiac disease is an autoimmune multisystem disorder that is triggered by dietary gluten sensitivity in genetically susceptible individuals. It presents with extraintestinal cutaneous manifestations including dermatitis herpetiformis (DH), atopic dermatitis, psoriasis, urticaria, and alopecia areata. Due to the insufficient availability of data, this study aimed to estimate the frequency of cutaneous manifestation in a Pakistani population with celiac disease. Methods This cross-sectional study was conducted from January 2020 to July 2021, and 300 patients with a confirmed diagnosis of celiac disease were enrolled in the study from the internal medicine department of a tertiary care hospital in Pakistan. Celiac disease was confirmed by the presence of immunoglobulin A (IgA) endomysial antibody and IgA tissue transglutaminase antibody. The presence of cutaneous manifestations was assessed with the assistance of a qualified dermatologist and noted in a self-structured questionnaire. Results Overall, the most common cutaneous manifestation was DH (16.0%), whereas the second most common cutaneous manifestation was psoriasis (13.8%). DH was most commonly found among males (18.9%), while psoriasis was more common among females (14.12%). Conclusion Among the various cutaneous presentations in patients with celiac disease, the most common dermatological manifestation was DH. Therefore, patients with cutaneous manifestations should undergo screening for celiac disease.

Gastrointestinal Manifestation of COVID-19 in Hospitalized Patients.

INTRODUCTION: During the initial phase of the pandemic, gastrointestinal (GI) symptoms were less frequent but during the later stages, GI manifestations have become more frequent. This study aims to explore the prevalence of GI symptoms in COVID-19 patients, and also focuses on the frequency of these symptoms. METHODS: This longitudinal study was conducted in a COVID-19 unit of a tertiary care hospital, Pakistan. Data of patients hospitalized with COVID-19 infection between June 2021 and July 2021 was included in the study. A total of 412 participants were enrolled in the study via consecutive convenient non-probability sampling. Participants' symptoms and demographics were noted in a self-structured questionnaire. The collected data were analyzed using Statistical Package for Social Sciences (SPSS), version 23.0 (IBM Corp., Armonk, NY). RESULTS: A total of 261 (63.3%) participants had a minimum of one GI symptom. The most common symptom was anorexia (43.9%), followed by diarrhea (24.7%) and nausea/vomiting (17.9%). CONCLUSION:  Our study indicates high frequency of COVID-19 patients reporting GI symptoms. Anorexia, diarrhea, nausea, and vomiting were commonly reported symptoms. Therefore, COVID 19 testing should be considered with patients presenting with GI symptoms.

Long-Term Neurological Impact of COVID-19.

INTRODUCTION: Recent research has observed the ability of coronavirus disease 2019 (COVID-19) to spread in the brain from the respiratory system. The associated neurological disorder includes encephalopathies, inflammatory syndromes, stroke, peripheral neuropathies, and various other central nervous system disorders. This study aims to highlight the long-term neurological sequelae in patients with COVID-19 disease. METHODS: This long-term study was carried out in the COVID-19 unit of a tertiary care hospital in Pakistan from July 2020 to July 2021. After obtaining informed consent, we enrolled 1000 patients who recovered from COVID-19 and were discharged. The participants were followed up after 30 and 90 days. RESULTS: At the time of enrollment, there were 602 (60.2%) males and 398 (39.8%) females. The most common neurological symptom on 30-day follow-up was headache (8.8%), followed by insomnia. The most common neurological symptom on day 90 follow-up was insomnia (5.07%), followed by an altered sense of smell (3.3%). CONCLUSION: COVID-19 tends to produce a wide range of neurological symptoms, ranging from headache to anosmia to increased risk of stroke, that complicates clinical management. Potential neurologic effects and drug interactions have been reported secondary to the medications used to treat COVID-19. In light of the aforementioned facts, COVID-19 could potentially have a long-term effect on the brain. Therefore, it is important that the clinicians must be aware of the potential neurologic complications. Lastly, proper follow-up is recommended that would aid in timely recognition and management of the neurological disorder.

Peripheral Artery Disease as a Risk Factor for Myocardial Infarction.

INTRODUCTION:  Atherosclerosis contributes to the underlying pathophysiology for peripheral arterial disease (PAD), coronary artery disease (CAD), and cerebrovascular disease. Several studies have been conducted to demonstrate PAD as a major risk factor for cardiovascular (CV) events, however, the regional data are limited. This study aims to highlight PAD as a major risk factor in CV events in a local setting. METHODS:  In this longitudinal study, 400 hypertensive patients with a confirmed diagnosis of PAD were enrolled from the outpatient department of the cardiology unit. Diagnosis of PAD was made using the ankle brachial index (ABI). ABI less than 0.9 was labeled as participants with PAD. Another group of 400 without PAD was also enrolled as the control group from the outpatient department of cardiology unit. Patients were followed up for 12 months or for the development of myocardial infarction (MI). RESULT:  Participants with PAD had a significant increased risk of total MI events with a relative risk (RR) of 1.67 (confidence interval, CI 95%: 1.05-2.66; p-value: 0.02). The RR for fatal MI was 2.62 (CI 95%: 0.94-7.29; p-value: 0.06) compared to the participants without PAD, however, it was not significant. CONCLUSION:  This study has focused on the risk factors of PAD and has suggested that the patients who have any of the mentioned risk factors should be treated with caution under strict instructions given by doctors. A variety of treatment options is available, but the initial changes should be made in the lifestyle of these patients, making sure the risk factors are being treated.

Effect of Cigarette Smoking on Hearing Levels in Young and Middle-Aged Males.

INTRODUCTION: Smoking is a well-recognized risk factor for many health issues; however, its association with hearing loss has been a debate. Some studies have shown a positive association while others did not. In this study, we aim to identify the effect of cigarette smoking on hearing in our population. METHODS: This cross-sectional study was conducted in a tertiary care hospital in Pakistan from August 2020 to March 2021. Five hundred male smokers (n = 500), with a history of smoking for more than three years between the ages of 21 and 50, were enrolled in the study via consecutive convenient non-probability sampling after informed consent. Five hundred male non-smokers (n = 500) were enrolled as a reference group. Audiometry was performed in a soundproof room. RESULTS: The hearing levels in audiometry were significantly higher in smokers compared to non-smokers (22.8 ± 8.12 decibels vs 18.7 ± 6.12; p-value < 0.0001). Participants who had been smoking for more than 10 years had higher hearing levels in the audiometry test compared to the participants with less than 10 years of smoking history (24.21 ± 8.91 decibels vs. 21.1 ± 8.01 decibels: p-value < 0.0001). CONCLUSION: In this study, smokers were associated with greater loss in hearing compared to non-smokers. In addition to other adverse events associated with smoking, smokers should be counselled about hearing loss related to it.

Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein.

Viruses cause many severe plant diseases, resulting in immense losses of crop yield worldwide. Therefore, developing novel approaches to control plant viruses is crucial to meet the demands of a growing world population. Recently, RNA interference (RNAi) has been widely used to develop virus-resistant plants. Once genome replication and assembly of virion particles is completed inside the host plant, mature virions or sometimes naked viral genomes spread cell-to-cell through plasmodesmata by interacting with the virus-encoded movement protein (MP). We used the RNAi approach to suppress MP gene expression, which in turn prevented potato leafroll virus (PLRV) systemic infection in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with MP siRNA constructs exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of MP gene expression is an efficient method for generating PLRV-resistant potato plants. Further, we identified novel ATPase motifs in MP that may be involved in DNA binding and translocation through plasmodesmata. We also showed that the ATPase activity of MP was stimulated in the presence of DNA/RNA. Overall, our findings provide a robust technology to generate PLRV-resistant potato plants, which can be extended to other species. Moreover, this approach also contributes to the study of genome translocation mechanisms of plant viruses.