Kira M Veley, Ihuoma Okwuonu, Greg Jensen, Marisa Yoder, Nigel J Taylor, Blake C Meyers, Rebecca S Bart

G3 (Bethesda); 2021 Apr 15;11(4):jkab028.  doi: 10.1093/g3journal/jkab028.

Genes, Genomes and Genetics.

Abstract

Research on a few model plant-pathogen systems has benefitted from years of tool and resource development. This is not the case for the vast majority of economically and nutritionally important plants, creating a crop improvement bottleneck. Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is an important disease in all regions where cassava (Manihot esculenta Crantz) is grown. Here, we describe the development of cassava that can be used to visualize one of the initial steps of CBB infection in vivo. Using CRISPR-mediated homology-directed repair (HDR), we generated plants containing scarless insertion of GFP at the 3' end of CBB susceptibility (S) gene MeSWEET10a. Activation of MeSWEET10a-GFP by the transcription activator-like (TAL) effector TAL20 was subsequently visualized at transcriptional and translational levels. To our knowledge, this is the first such demonstration of HDR via gene editing in cassava.

See: https://pubmed.ncbi.nlm.nih.gov/33855431/

Figure 1: CRISPR-TA-coupled HDR strategy for tagging MeSWEET10a. (a) RT-PCR for MeSWEET10a expression in floral tissue. Analyses of male or female floral tissues are shown separately. Xe- and Xe+TAL20_Xam668-infiltrated leaf tissues, harvested 2 days post-infiltration (dpi), were analyzed as negative or positive controls, respectively. Three biological replicates are presented for each tissue type. Actin was used as a loading control. Genomic DNA (gDNA) and no template controls are also shown. (b) Summary of the CRISPR-TA-coupled HDR strategy for tagging MeSWEET10a. Row 1: the supplied T-DNA includes CRISPR-based gene editing components (gRNA(s) and a nuclease, Cas9), a GFP-containing repair template (homology arms shown in blue/white and purple/white patterns), and a TA system, consisting of a FEC tissue-specific promoter and the TAL20 gene. Row 2: targeting the 3’ end of the MeSWEET10a locus. Cas9 + gRNAs cut just before the stop codon. Sequences homologous to the arms of the repair template are indicated in matched colored patterns. The effector binding element (EBE) present in the promoter of MeSWEET10a is shown in orange. Row 3: Identification of repair-positive individuals using the TA system. TAL20 (orange circle) binds the EBE, activating MeSWEET10a expression (arrow). If the gene has been tagged in frame with GFP, fluorescence is detected through screening. Homology arms remain present (blue/white and purple/white patterns). (c) Expression pattern of gene from which the promoter was used in panel B. Y-axis: Fragments per kilobase of transcript per million mapped reads (FPKM) values. X-axis: tissue types included in tissue-specific RNA-seq data (Wilson et al. 2017). OES: organized embryogenic structures, FEC: friable embryogenic callus, SAM: shoot apical meristem, RAM: root apical meristem. Graph was produced using the available online Cassava Atlas tool (http://shiny.danforthcenter.org/cassava_atlas).