DataSheet1_CRISPR/Cas9-Mediated Targeting of Susceptibility Factor eIF4E-Enhanced Resistance Against Potato Virus Y.docx

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.</p

Mapping global biodiversity connections with DNA barcodes: Lepidoptera of Pakistan

<div><p>Sequences from the DNA barcode region of the mitochondrial COI gene are an effective tool for specimen identification and for the discovery of new species. The Barcode of Life Data Systems (BOLD) (<a href="http://www.boldsystems.org" target="_blank">www.boldsystems.org</a>) currently hosts 4.5 million records from animals which have been assigned to more than 490,000 different Barcode Index Numbers (BINs), which serve as a proxy for species. Because a fourth of these BINs derive from Lepidoptera, BOLD has a strong capability to both identify specimens in this order and to support studies of faunal overlap. DNA barcode sequences were obtained from 4503 moths from 329 sites across Pakistan, specimens that represented 981 BINs from 52 families. Among 379 species with a Linnaean name assignment, all were represented by a single BIN excepting five species that showed a BIN split. Less than half (44%) of the 981 BINs had counterparts in other countries; the remaining BINs were unique to Pakistan. Another 218 BINs of Lepidoptera from Pakistan were coupled with the 981 from this study before being compared with all 116,768 BINs for this order. As expected, faunal overlap was highest with India (21%), Sri Lanka (21%), United Arab Emirates (20%) and with other Asian nations (2.1%), but it was very low with other continents including Africa (0.6%), Europe (1.3%), Australia (0.6%), Oceania (1.0%), North America (0.1%), and South America (0.1%). This study indicates the way in which DNA barcoding facilitates measures of faunal overlap even when taxa have not been assigned to a Linnean species.</p></div

K2P sequence divergence in the COI barcode region for moth species from Pakistan with more than three specimens, genera with three or more species, and families with three or more genera.

<p>This analysis only considers specimens that were assigned to a Linnaean species.</p

Intraspecific variation (K2P) at the COI gene against geographical extent (km) of moth species from Pakistan and their conspecifics from other regions.

<p>Vertical downward arrow (red) indicates the percentage of species with less or more than 2% divergence across their range.</p

Map of Pakistan showing collection localities for specimens examined in this study.

<p>Map of Pakistan showing collection localities for specimens examined in this study.</p

Barcode index numbers and sequence divergence values (K2P) for the DNA barcode of COI for 52 families of moths collected in Pakistan.

<p>Sequence divergences are only reported for families with more than one BIN.</p

Heat map showing the percentage of Lepidoptera BINs from Pakistan (shaded gray) shared with other countries.

<p>Sharing (range) with other countries is colour-coded with varying circle sizes.</p

Primers used for PCR and RT-PCR amplification and <i>in vitro</i> transcription.

<p>The T7 promoter sequence is underlined.</p

Barcode gap analysis for species of moths with three or more specimens collected in Pakistan.

<p>NN = nearest neighbor.</p

Knock down of Whitefly Gut Gene Expression and Mortality by Orally Delivered Gut Gene-Specific dsRNAs

<div><p>Control of the whitefly <i>Bemisia tabaci</i> (Genn.) agricultural pest and plant virus vector relies on the use of chemical insecticides. RNA-interference (RNAi) is a homology-dependent innate immune response in eukaryotes, including insects, which results in degradation of the corresponding transcript following its recognition by a double-stranded RNA (dsRNA) that shares 100% sequence homology. In this study, six whitefly ‘gut’ genes were selected from an <i>in silico</i>-annotated transcriptome library constructed from the whitefly alimentary canal or ‘gut’ of the B biotype of <i>B</i>. <i>tabaci</i>, and tested for knock down efficacy, post-ingestion of dsRNAs that share 100% sequence homology to each respective gene target. Candidate genes were: <i>Acetylcholine receptor subunit α</i>, <i>Alpha glucosidase 1</i>, <i>Aquaporin 1</i>, <i>Heat shock protein 70</i>, <i>Trehalase1</i>, and <i>Trehalose transporter1</i>. The efficacy of RNAi knock down was further tested in a gene-specific functional bioassay, and mortality was recorded in 24 hr intervals, six days, post-treatment. Based on qPCR analysis, all six genes tested showed significantly reduced gene expression. Moderate-to-high whitefly mortality was associated with the down-regulation of osmoregulation, sugar metabolism and sugar transport-associated genes, demonstrating that whitefly survivability was linked with RNAi results. Silenced <i>Acetylcholine receptor subunit α</i> and <i>Heat shock protein 70</i> genes showed an initial low whitefly mortality, however, following insecticide or high temperature treatments, respectively, significantly increased knockdown efficacy and death was observed, indicating enhanced post-knockdown sensitivity perhaps related to systemic silencing. The oral delivery of gut-specific dsRNAs, when combined with qPCR analysis of gene expression and a corresponding gene-specific bioassay that relates knockdown and mortality, offers a viable approach for functional genomics analysis and the discovery of prospective dsRNA biopesticide targets. The approach can be applied to functional genomics analyses to facilitate, species-specific dsRNA-mediated control of other non-model hemipterans.</p></div