Genome editing: a novel approach to manage insect vectors of plant viruses.

Insect vectors significantly threaten global agriculture by transmitting numerous viruses. Various measures, from conventional insecticides to genetic engineering, are used to mitigate this threat. However, none provide complete resistance. Therefore, researchers are looking for novel control options. In recent years with the advancements in genomic technologies, genomes and transcriptomes of various insect vectors have been generated. However, the lack of knowledge about gene function hinders the development of novel strategies to restrict virus spread. RNA interference (RNAi) is widely used to elucidate gene functions, but its variable efficacy hampers its use in insect vectors. Genome editing has the potential to overcome these challenges and has been extensively used in various insect pest species. This review summarizes the progress and potential of genome editing in plant virus vectors and its application as a functional genomic tool to elucidate virus-vector interactions. We also discuss the major challenges associated with editing insect vectors.

Dissecting the Genetic Diversity of USDA Cowpea Germplasm Collection Using Kompetitive Allele Specific PCR-Single Nucleotide Polymorphism Markers.

Cowpea (Vigna unguiculata L. Walp) is an important grain legume crop of the subtropics, particularly in West Africa, where it contributes to the livelihoods of small-scale farmers. Despite being a drought-resilient crop, cowpea production is hampered by insect pests, diseases, parasitic weeds, and various abiotic stresses. Genetic improvement can help overcome these limitations, and exploring diverse cowpea genetic resources is crucial for cowpea breeding. This study evaluated the genetic diversity of 361 cowpea accessions from the USDA core collection for the species using 102 Kompetitive Allele Specific PCR (KASP) single nucleotide polymorphism (SNP) markers. A total of 102 KASP-SNP was validated in the germplasm panel, and 72 showed polymorphism across the germplasm panel. The polymorphism information content (PIC) of all SNPs ranged from 0.1 to 0.37, with an average of 0.29, while the mean observed heterozygosity was 0.52. The population structure revealed three distinct populations that clustered into two major groups after phylogenetic analysis. Analysis of molecular variance (AMOVA) indicated greater genetic variation within populations than among populations. Although cowpea generally has a narrow genetic diversity, the accessions used in this study exhibited considerable variation across geographical regions, sub-species, and improvement status. These results indicated that the selected KASP genotyping assay can provide robust and accurate genotyping data for application in the selection and management of cowpea germplasm in breeding programs and genebanks.

Using single cell type proteomics to identify Al-induced proteomes in outer layer cells and interior tissues in the apical meristem/cell division regions of tomato root-tips.

Aluminum (Al) toxicity primarily targets the root tips, inhibiting root growth and function and leading to crop yield losses on acidic soils. Previously we reported using laser capture microdissection (LCM) proteomics to identify Al-induced proteins in the outer layer cells in the transitional zone of tomato root-tips. This study aims to further characterize Al-induced proteomic dynamics from the outer to interior tissues, thus providing a panoramic view reflecting Al resistance in the root tip as a whole in tomatoes. Three types of cells were isolated via LCM from the basal 350-400 µm (below cell elongation regions) of root tips using tomato (Solanum lycopersicum) 'Micro-Tom' plants. Type I and Type II were from Al-treated plants. Type I included cells of the outer three layers, i.e., the epidermis and cortex initials and the quiescent center (QC) in root apical meristem (RAM), and Type II possessed the interior tissues of the same region. Type III contained cells from the non-Al-treated root tips collected in the same region as Type I. Two tandem mass tag (TMT) proteomics analyses with three biological replicates for each sample type were conducted. The TMTexp1 (comparing Type I and Type II) identified 6575 quantifiable proteins and 178 different abundance proteins (DAPs). The TMTexp2 (comparing Type I and Type III) identified 7197 quantifiable proteins and 162 DAPs. Among all quantified proteins (7685) from the two TMT experiments, 6088 (79%) proteins, including 313 DAPs (92% of the 340 total), were identified in all tissues. A model reflecting the tissue-specific Al-resistance mechanism was proposed, in which the level of the citrate transporter MATE protein, involved in Al exclusion, accumulated to the highest level in the outer-layer cells but decreased toward the interior of root-tips (which concurs with the tissue-specific importance in Al resistance). Proteins for biosynthesis of ethylene and jasmonic acid, proteolytic enzymes, stress-responsive proteins, and cell wall modeling were affected by Al treatment, some in a cell type-specific manner. The KEGG metabolite pathways enriched with these DAPs changed depending on the cell types. This study demonstrated the advantage of using the tissue/cell-specific analysis for identifying proteins and their dynamic changes directly associated with Al resistance in the root-tip region. The proteomics datasets have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (https://www.ebi.ac.uk/pride/) with the dataset identifier as PXD021994 under project title: Proteomics studies of outer and inner cellular layers of tomato root-tips for Al stress, Project DOI: 10.6019/ PXD021994; and PXD018234 under Project title: Al-induced root proteomics changes in stress-acclimated tomato plant, Project DOI: https://doi.org/10.6019/PXD018234. SIGNIFICANCE: This paper presents the method of using laser capture microdissection (LCM) to collect homogenous cell-type specific tissue samples from the outer layers and inner central regions of tomato root-tips. The tandem mass tag-proteomics analysis showed that the outer-layer cells expressed proteomes that were different from the inner tissues of Al-treated root-tips; proteins related to resistance/tolerance to Al toxicity were highly accumulated in the outer-layer cells. Furthermore, the Al-treated outer-layer cells expressed proteomes which were different from the non-Al treated counterpart cells. This study has provided the first dataset of proteins differentiating from the outer to inner layers of cells in Al-treated root-tips. It provided convincing experimental evidences demonstrating the single-cell type proteomics as a powerful analytical approach to identify Al tolerance mechanisms in plants. The analytical procedure of LCM-tandem mass tag-quantitative proteomics analysis has a broad application for proteomics analysis of spatially separated cells in complex tissues.

Distributions of Microdamage Are Altered Between Trabecular Rods and Plates in Cancellous Bone From Men With Type 2 Diabetes Mellitus.

Individuals with type 2 diabetes mellitus (T2DM) have an increased risk of fragility fracture despite exhibiting normal to high bone mineral density (BMD). Conditions arising from T2DM, such as reduced bone turnover and alterations in microarchitecture, may contribute to skeletal fragility by influencing bone morphology and microdamage accumulation. The objectives of this study were (i) to characterize the effect of T2DM on microdamage quantity and morphology in cancellous bone, and (ii) relate the accumulation of microdamage to the cancellous microarchitecture. Cancellous specimens from the femoral neck were collected during total hip arthroplasty (T2DM: n = 22, age = 65 ± 9 years, glycated hemoglobin [HbA1c] = 7.00% ± 0.98%; non-diabetic [non-DM]: n = 25, age = 61 ± 8 years, HbA1c = 5.50% ± 0.4%), compressed to 3% strain, stained with lead uranyl acetate to isolate microdamage, and scanned with micro-computed tomography (µCT). Individual trabeculae segmentation was used to isolate rod-like and plate-like trabeculae and their orientations with respect to the loading axis. The T2DM group trended toward a greater BV/TV (+27%, p = 0.07) and had a more plate-like trabecular architecture (+8% BVplates , p = 0.046) versus non-DM specimens. Rods were more damaged relative to their volume compared to plates in the non-DM group (DVrods /BVrods versus DVplates /BVplates : +49%, p < 0.0001), but this difference was absent in T2DM specimens. Longitudinal rods were more damaged in the non-DM group (DVlongitudinal rods /BVlongitudinal rods : +73% non-DM versus T2DM, p = 0.027). Total damage accumulation (DV/BV) and morphology (DS/DV) did not differ in T2DM versus non-DM specimens. These results provide evidence that cancellous microarchitecture does not explain fracture risk in T2DM, pointing to alterations in material matrix properties. In particular, cancellous bone from men with T2DM may have an attenuated ability to mitigate microdamage accumulation through sacrificial rods. © 2022 American Society for Bone and Mineral Research (ASBMR).