CRISPR/Cas9-generated mutations in a sugar transporter gene reduce cassava susceptibility to bacterial blight.

Bacteria from the genus Xanthomonas are prolific phytopathogens that elicit disease in over 400 plant species. Xanthomonads carry a repertoire of specialized proteins called transcription activator-like (TAL) effectors that promote disease and pathogen virulence by inducing expression of host susceptibility (S) genes. Xanthomonas phaseoli pv. manihotis (Xpm) causes bacterial blight on the staple food crop cassava (Manihot esculenta Crantz). The Xpm effector TAL20 induces ectopic expression of the S gene Manihot esculenta Sugars Will Eventually be Exported Transporter 10a (MeSWEET10a), which encodes a sugar transporter that contributes to cassava bacterial blight susceptibility. We used CRISPR/Cas9 to generate multiple cassava lines with edits to the MeSWEET10a TAL20 effector binding site and/or coding sequence. In several of the regenerated lines, MeSWEET10a expression was no longer induced by Xpm, and in these cases, we observed reduced cassava bacterial blight (CBB) disease symptoms post Xpm infection. Because MeSWEET10a is expressed in cassava flowers, we further characterized the reproductive capability of the MeSWEET10a promoter and coding sequence mutants. Lines were crossed to themselves and to wild-type plants. The results indicated that expression of MeSWEET10a in female, but not male, flowers, is critical to produce viable F1 seed. In the case of promoter mutations that left the coding sequence intact, viable F1 progeny were recovered. Taken together, these results demonstrate that blocking MeSWEET10a induction is a viable strategy for decreasing cassava susceptibility to CBB and that ideal lines will contain promoter mutations that block TAL effector binding while leaving endogenous expression of MeSWEET10a unaltered.

Evaluation of Gene Knockouts by CRISPR as Potential Targets for the Genetic Engineering of the Mosquito Culex quinquefasciatus.

Culex quinquefasciatus mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people and, separately, poses a threat to the conservation of insular birds. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations. Yet, research into Cx. quinquefasciatus has been lagging compared to other disease vectors. Here, we use this tool to disrupt a set of five pigmentation genes in Cx. quinquefasciatus that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of Cx. quinquefasciatus that are relevant to potential future public health and bird conservation applications. We generate a double-mutant line, demonstrating the possibility of sequentially combining multiple such mutations in a single individual. Lastly, we target two loci, doublesex in the sex-determination pathway and proboscipedia, a hox gene, demonstrating the flexibility of these methods applied to novel targets. Our work provides a platform of seven validated loci that could be used for targeted mutagenesis in Cx. quinquefasciatus and the future development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily scored transgenesis marker.

Taro Genome Assembly and Linkage Map Reveal QTLs for Resistance to Taro Leaf Blight.

Taro (Colocasia esculenta) is a food staple widely cultivated in the humid tropics of Asia, Africa, Pacific and the Caribbean. One of the greatest threats to taro production is Taro Leaf Blight caused by the oomycete pathogen Phytophthora colocasiae Here we describe a de novo taro genome assembly and use it to analyze sequence data from a Taro Leaf Blight resistant mapping population. The genome was assembled from linked-read sequences (10x Genomics; ∼60x coverage) and gap-filled and scaffolded with contigs assembled from Oxford Nanopore Technology long-reads and linkage map results. The haploid assembly was 2.45 Gb total, with a maximum contig length of 38 Mb and scaffold N50 of 317,420 bp. A comparison of family-level (Araceae) genome features reveals the repeat content of taro to be 82%, >3.5x greater than in great duckweed (Spirodela polyrhiza), 23%. Both genomes recovered a similar percent of Benchmarking Universal Single-copy Orthologs, 80% and 84%, based on a 3,236 gene database for monocot plants. A greater number of nucleotide-binding leucine-rich repeat disease resistance genes were present in genomes of taro than the duckweed, ∼391 vs. ∼70 (∼182 and ∼46 complete). The mapping population data revealed 16 major linkage groups with 520 markers, and 10 quantitative trait loci (QTL) significantly associated with Taro Leaf Blight disease resistance. The genome sequence of taro enhances our understanding of resistance to TLB, and provides markers that may accelerate breeding programs. This genome project may provide a template for developing genomic resources in other understudied plant species.