Showing papers by "Imran Amin published in 2022"

Dominance of Asia II 1 species of Bemisia tabaci in Pakistan and beyond

Abstract: Globally, Whitefly (Bemisia tabaci) is one of the most important insect pests of crops that causes huge economical losses. The current study was designed to exclusively screen the B. tabaci species in the cotton field of Pakistan during 2017-2020 and have to conduct comparative analysis of B. tabaci species in Asia where Asia II 1 has been reported. A total of 5142 B. tabaci sequences of mitochondrial cytochrome oxidase 1 (mtCO1) from Asian countries were analyzed to determine the species and their distribution in the region. Our analysis over time and space showed that Asia II 1 has gradually dominated over Asia 1 in Punjab Province and over both Asia 1 and MEAM1 in Sindh Province. Asia has been divided into three regions i.e., South Asia (2524 sequences), Southeast Asia (757 sequences) and East Asia (1569 sequences) and dominance of different species of B. tabaci has been determined by calculating the relative percentage of each species. Interestingly, Asia II 1 has been found dominant in the neighboring region (northern zone) of India and also being dominant in its central zone. The dominance of Asia II 1 in Pakistan and northern India explains whitefly epidemic being reported in recent years.

Broad-spectrum resistance against multiple PVY-strains by CRSIPR/Cas13 system in Solanum tuberosum crop

Abstract: ABSTRACT Potato virus Y (PVY) is a deadly environmental constraint that damages productivity of potato (Solanum tuberosum) around the globe. One of the major challenges is to develop resistance against PVY. Emerging clustered regularly short palindromic repeat (CRISPR)/Cas systems have the potential to develop resistance against PVY. In the current research, CRISPR-Cas13 has been exploited to target multiple strains of PVYN, PVYO, and PVYNTN. Multiple genes PI, HC-Pro, P3, Cl1, Cl2, and VPg genes of PVY were targeted by CRISPR/Cas13a. Multiplex gRNA cassettes were developed on the conserved regions of the PVY-genes. Three independent CRISPR/Cas13 transgenic potato lines were developed by applying an optimized concentration of trans-ribo zeatin and indole acetic acid at callus development, rooting, and shooting growth stages. The level of resistance in transgenic plants was confirmed through double-antibody sandwich enzyme-linked immunosorbent assay and real-time quantitative PCR. Our results have shown that efficiency of PVY inhibition was positively correlated with the Cas13a/sgRNA expression. Finding provides the specific functionality of Cas13 with specific gRNA cassette and engineering the potential resistance in potato crop against multiple strains of PVY.

Operational parameters optimization for remediation of crude oil-polluted water in floating treatment wetlands using response surface methodology

Abstract: The application of floating treatment wetlands (FTWs) is an innovative nature-based solution for the remediation of polluted water. The rational improvement of water treatment via FTWs is typically based on multifactorial experiments which are labor-intensive and time-consuming. Here, we used the response surface methodology (RSM) for the optimization of FTW's operational parameters for the remediation of water polluted by crude oil. The central composite design (CCD) of RSM was used to generate the experimental layout for testing the effect of the variables hydrocarbon, nutrient, and surfactant concentrations, aeration, and retention time on the hydrocarbon removal in 50 different FTW test systems planted with the common reed, Phragmites australis. The results from these FTW were used to formulate a mathematical model in which the computational data strongly correlated with the experimental results. The operational parameters were further optimized via modeling prediction plus experimental validation in test FTW systems. In the FTW with optimized parameters, there was a 95% attenuation of the hydrocarbon concentration, which was very close to the 98% attenuation predicted by the model. The cost-effectiveness ratio showed a reduction of the treatment cost up to $0.048/liter of wastewater. The approach showed that RSM is a useful strategy for designing FTW experiments and optimizing operational parameters.

CRISPR/Cas9-Mediated Targeting of Susceptibility Factor eIF4E-Enhanced Resistance Against Potato Virus Y

Abstract: Potato (Solanum tuberosum L.) is an important staple food around the world, and potato virus Y (PVY) is a major constraint for potato production. The VPg protein of PVY interacts with the translation initiation factor eIF4E of the host that works as a susceptibility factor during infection. The interaction between eIF4E and VPg was disrupted by CRISPR/Cas9. The homozygous conserved region of eIF4E of the potato variety “Kruda” was mutated by CRISPR/Cas9. Tracking of insertion, deletion, and conversion events was performed by Sanger sequencing with ∼15% editing efficiency. Truncated and mutated eIF4E proteins were unable to interact with VPg, and the virus was not able to exploit the host machinery for replication and systemic spreading. Mutated eIF4E lines showed enhanced resistance to PVYO strain. DAS-ELISA and RT-PCR were used for validation of the observed resistance. PVY resistance in tetraploid lines via CRISPR/Cas9 provides a route to develop novel resistant potato cultivars.

Designing Synergistic Biostimulants Formulation Containing Autochthonous Phosphate-Solubilizing Bacteria for Sustainable Wheat Production

Abstract: Applying phosphate-solubilizing bacteria (PSB) as biofertilizers has enormous potential for sustainable agriculture. Despite this, there is still a lack of information regarding the expression of key genes related to phosphate-solubilization (PS) and efficient formulation strategies. In this study, we investigated rock PS by Ochrobactrum sp. SSR (DSM 109610) by relating it to bacterial gene expression and searching for an efficient formulation. The quantitative PCR (qPCR) primers were designed for PS marker genes glucose dehydrogenase (gcd), pyrroloquinoline quinone biosynthesis protein C (pqqC), and phosphatase (pho). The SSR-inoculated soil supplemented with rock phosphate (RP) showed a 6-fold higher expression of pqqC and pho compared to inoculated soil without RP. Additionally, an increase in plant phosphorous (P) (2%), available soil P (4.7%), and alkaline phosphatase (6%) activity was observed in PSB-inoculated plants supplemented with RP. The root architecture improved by SSR, with higher root length, diameter, and volume. Ochrobactrum sp. SSR was further used to design bioformulations with two well-characterized PS, Enterobacter spp. DSM 109592 and DSM 109593, using the four organic amendments, biochar, compost, filter mud (FM), and humic acid. All four carrier materials maintained adequate survival and inoculum shelf life of the bacterium, as indicated by the field emission scanning electron microscopy analysis. The FM-based bioformulation was most efficacious and enhanced not only wheat grain yield (4–9%) but also seed P (9%). Moreover, FM-based bioformulation enhanced soil available P (8.5–11%) and phosphatase activity (4–5%). Positive correlations were observed between the PSB solubilization in the presence of different insoluble P sources, and soil available P, soil phosphatase activity, seed P content, and grain yield of the field grown inoculated wheat variety Faisalabad-2008, when di-ammonium phosphate fertilizer application was reduced by 20%. This study reports for the first time the marker gene expression of an inoculated PSB strain and provides a valuable groundwork to design field scale formulations that can maintain inoculum dynamics and increase its shelf life. This may constitute a step-change in the sustainable cultivation of wheat under the P-deficient soil conditions.

Insights into performance evaluation of com-pound-protein interaction prediction methods

Abstract: MOTIVATION Machine-learning-based prediction of compound-protein interactions (CPIs) is important for drug design, screening and repurposing. Despite numerous recent publication with increasing methodological sophistication claiming consistent improvements in predictive accuracy, we have observed a number of fundamental issues in experiment design that produce overoptimistic estimates of model performance. RESULTS We systematically analyze the impact of several factors affecting generalization performance of CPI predictors that are overlooked in existing work: (i) similarity between training and test examples in cross-validation; (ii) synthesizing negative examples in absence of experimentally verified negative examples and (iii) alignment of evaluation protocol and performance metrics with real-world use of CPI predictors in screening large compound libraries. Using both state-of-the-art approaches by other researchers as well as a simple kernel-based baseline, we have found that effective assessment of generalization performance of CPI predictors requires careful control over similarity between training and test examples. We show that, under stringent performance assessment protocols, a simple kernel-based approach can exceed the predictive performance of existing state-of-the-art methods. We also show that random pairing for generating synthetic negative examples for training and performance evaluation results in models with better generalization in comparison to more sophisticated strategies used in existing studies. Our analyses indicate that using proposed experiment design strategies can offer significant improvements for CPI prediction leading to effective target compound screening for drug repurposing and discovery of putative chemical ligands of SARS-CoV-2-Spike and Human-ACE2 proteins. AVAILABILITY AND IMPLEMENTATION Code and supplementary material available at https://github.com/adibayaseen/HKRCPI. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Smart breeding approaches in post-genomics era for developing climate-resilient food crops

Abstract: Improving the crop traits is highly required for the development of superior crop varieties to deal with climate change and the associated abiotic and biotic stress challenges. Climate change-driven global warming can trigger higher insect pest pressures and plant diseases thus affecting crop production sternly. The traits controlling genes for stress or disease tolerance are economically imperative in crop plants. In this scenario, the extensive exploration of available wild, resistant or susceptible germplasms and unraveling the genetic diversity remains vital for breeding programs. The dawn of next-generation sequencing technologies and omics approaches has accelerated plant breeding by providing the genome sequences and transcriptomes of several plants. The availability of decoded plant genomes offers an opportunity at a glance to identify candidate genes, quantitative trait loci (QTLs), molecular markers, and genome-wide association studies that can potentially aid in high throughput marker-assisted breeding. In recent years genomics is coupled with marker-assisted breeding to unravel the mechanisms to harness better better crop yield and quality. In this review, we discuss the aspects of marker-assisted breeding and recent perspectives of breeding approaches in the era of genomics, bioinformatics, high-tech phonemics, genome editing, and new plant breeding technologies for crop improvement. In nutshell, the smart breeding toolkit in the post-genomics era can steadily help in developing climate-smart future food crops.

Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance

Abstract: Cotton is an international agricultural commodity and the main cash crop of Pakistan of which quality and quantity are subject to various whims of nature. Climate change, insect pest complex, and weeds are reducing its productivity. Here, we have developed triple gene cotton containing EPSPS gene along with two Bt toxin genes Cry1Ac and Cry2Ab using a strategy where all three genes are cloned in the same T-DNA, followed by successful cotton transformation via Agrobacterium-mediated transformation. This strategy has been developed to help cotton breeders in developing new cultivars by incorporating these genes into the non-transgenic or single Bt (Cry1Ac) gene cotton background where all three genes will inherit together. The expression of all three proteins was confirmed through immunostrips and was quantified through enzyme-linked immunosorbent assay (ELISA). The spatio-temporal expression of Bt protein in different parts of triple gene NIBGE cotton plants was determined. Maximum expression was found in leaves followed by seeds and boll rinds. Insect bioassays with cotton bollworms (Helicoverpa armigera), armyworms (Spodoptera litura), and pink bollworms (Pectinophora gossypiella) showed more than 90% mortality. The best performing line (NIBGE-E2) on the basis of spatiotemporal expression, glyphosate assays, and insect mortality data, was used for event characterization by using the genome sequencing approach. The event was successfully characterized and named NIBGE 20-01. A diagnostics test based on event-specific PCR was developed and its ability to distinguish NIBGE 20-01 event from other commercial transgenic cotton events was confirmed. To confirm stable expression of all three proteins in the field conditions, homozygous transgenic lines were grown in the field and the expression was confirmed through immunostrip assays. It was found that all three genes are expressed under field conditions. To show that all three genes are inherited together upon crossing with local elite cotton lines, the F1 generation was grown under glasshouse and field conditions. The expression of all three genes was confirmed under field conditions. Our results showed that transgenic cotton with three genes cloned in the same T-DNA can express all genes and can be conveniently transferred into elite cotton lines through a single cross.

Fully Transient CRISPR/Cas12f system in plants capable of broad-spectrum resistance against Begomovirus

Abstract: CRISPR/Cas system has emerged as the most efficient genome editing technology for both eukaryotic and prokaryotic cells. Recently, biologist have started using CRISPR/Cas9 as a defence machinery in plants against DNA viruses by targeting conserve regions of their genome. Considerable resistance requires formation of stable transgenic lines with multiple gRNAs, targeting specific viruses. Development of such transgenic plants is not only time consuming but also there will always be some uncertainty of their efficiency and off targets in plant genome. Newly discovered miniature CRISPR/Cas12f (Cas14a) system has unique ability to chop down every ssDNA fragment once activated through targeted cleavage. Here we report first fully functional transient CRISPR/Cas12f system in plants. We also show that Cas12f with just one gRNA is enough for substantial broad-spectrum resistance against Gemini viruses. Plant phenotype showed nearly complete recovery and qPCR results showed multifold decrease infection of CLCuV in CRISPR/Cas12f treated plants compare to the infected plants (infected with CLCuMuV and CLCuKV). Smaller size, broad range and more efficiency make Cas12f a superior alternative of Cas9 against diverse group of ssDNA viruses.

First report of Cotton leaf curl Multan virus infecting Millettia pinnata in Pakistan

Abstract: Millettia pinnata (Pongame oiltree, Sukh chain; family Fabaceae) is an important leguminous tree widely grown and utilised as a herb in Southeast Asia and the Indian subcontinent. In October 2020, symptoms of stunted growth, leaf curling (upward and downward) and vein thickening (Figures 1 and 2), which are associated with infection by Begomovirus species in other hosts, were observed on M. pinnata, growing near cotton fields (30.3°N, 73.0°E), in Faisalabad, Punjab Province, Pakistan. DNA was isolated from ten leaves (S1 to S10), one each from ten diseased plants, and one leaf from two asymptomatic plants (AS11 and AS12) using a cetyl trimethyl ammonium bromide protocol (Doyle & Doyle, 1990). Rolling circle amplification in combination with PCR using begomovirus universal primers (BegomoF/BegomoR) (Briddon & Markham, 1994) amplified a c. 2.8 kb product from all the diseased samples. PCR assays for the Begomovirus alphasatellite (primer pair DNA101F/DNA101R) (Bull et al., 2003) and betasatellite (primer pair Beta01F/Beta02F) (Briddon et al., 2002) each amplified a c. 1.4 kb product from all the diseased samples. Neither begomovirus genome nor satellite DNA was detected in the asymptomatic samples. Amplicons from the begomovirus genome (AV-2 and AV-3 from S1 and S2; MZ268122-MZ268123), alphasatellite (AV-36 from S1; MZ057253) and betasatellite (AV-8 and AV-10 from S1, AV-11 and AV-12 from S2, and AV-13 from S3; MZ057231- MZ057235), were cloned in a general purpose PTZ57R/T cloning vector (Thermo Fisher Scientific, USA), Sanger sequenced, and the sequences deposited in GenBank. The genome sequences shared >99% nucleotide identity with Cotton leaf curl Multan virus (CLCuMuV) and were most closely related to CLCuMuV strain NAS-14 (MK357257), which was found in cotton in the same region of Faisalabad (Ahmed et al., 2021). They also had 94% and 87% nt similarity with the CLCuMuV-Raj (AF363011) and CLCuMuV-Dar strains (EU365613) respectively. Moreover, the alphasatellite and betasatellite sequences shared 98% similarity with Cotton leaf curl Multan alphasatellite (CLCuMuA; MK357286.1) and betasatellite (CLCuMuB; MT920327.1), respectively. The five CLCuMuB betasatellite sequences (MZ057231-MZ057235) shared 99% identity and the two Begomovirus genomic sequences (MZ268122-MZ268123) from the M. pinnata samples had 99% identity with each other. Southern hybridization (Zubair et al., 2017) confirmed the detection of Begomovirus genomic DNA and quantitative real-time PCR (Shafiq et al., 2017) assays confirmed the detection of CLCuMuV in three samples, S1, S2 and S3, selected for further analysis. To the best of our knowledge, this is the first report of CLCuMuV in Millettia pinnata which may serve as an alternative host of the virus in the off season when cotton is not grown. Routine monitoring and detection of CLCuMuV is essential to prevent the devastating leaf curl disease it causes from spreading worldwide. The authors acknowledge support in Sanger sequencing from Dr. Brian Scheffler and his group from USDA-ARS.

Corrigendum: CRISPR/Cas9-mediated targeting of susceptibility factor eIF4E-enhanced resistance against Potato virus Y

Abstract: [This corrects the article DOI: 10.3389/fgene.2022.922019.].

Role of Suprapubic Cystostomy in BPH Patients Presented with first time for Acute Retention of Urine

Abstract: The study compares the role of suprapubic cystostomy versus per urethral catheterization in BPH patients with acute retention of urine. Majority 80% patients were 50-70 years old. The higher number of patients in Group A (18/25) voided normally with good flow rate of almost 15ml/sec and non-significant residual urine, compared to Group B (13/25). In Group B, 12 patients required re- catheterization while only 7 in Group A needed to have their suprapubic catheter opened. All patients took Tamsulosin 0.4mg daily. Patients in Group A had their suprapubic catheter clamped and tested for voiding through the urethra; while Group B was tested without a catheter. The flow rate and PMRV noted. In group A, 7 patients had retention relieved by a suprapubic catheter. Two patients pulled out per urethral catheterization and was excluded from the study. The results showed the effectiveness of suprapubic cystostomy for benign prostatic hyperplasia patients with first-time urine retention.