High-throughput genomic sequencing of cassava bacterial blight strains identifies conserved effectors to target for durable resistance

Abstract

Cassava bacterial blight (CBB), incited by Xanthomonas axonopodis pv. manihotis (Xam), is the most important bacterial disease of cassava, a staple food source for millions of people in developing countries. Here we present a widely applicable strategy for elucidating the virulence components of a pathogen population. We report Illumina-based draft genomes for 65 Xam strains and deduce the phylogenetic relatedness of Xam across the areas where cassava is grown. Using an extensive database of effector proteins from animal and plant pathogens, we identify the effector repertoire for each sequenced strain and use a comparative sequence analysis to deduce the least polymorphic of the conserved effectors. These highly conserved effectors have been maintained over 11 countries, three continents, and 70 y of evolution and as such represent ideal targets for developing resistance strategies.

Fig. 1.

Geographical/temporal representation of sequenced Xam strains. Represented countries are colored green. NR: no record. Map was created with the maps platform of R (18). Specific collection location of older strains is not known and therefore, for the purpose of this study, origin is limited to country.

Fig. 2.

Full-genome SNP identification allows phylogenetic analysis of closely related Xam strains. A vs. B shows difference in phylogenetic relationships when INDELs are excluded (A) or included (B). (A) CLC Genomics Workbench was used for reference-based assemblies, SNP identification, and neighbor-joining analysis (1,000 bootstraps; Fig. S1). Xam strains cluster primarily by geographic region with a few outliers. Colored groups are supported by 1,000/1,000 bootstrap replicates. Yellow, Brazil; blue, Colombia; purple, Africa; white, other/geographic outliers. (B) SNP-based phylogeny of Brazilian strains including INDELs. A total of 55,927 variable positions were identified, concatenated, and applied to neighbor-joining phylogenetic analysis (100 bootstraps, * denotes less than 100-bootstrap support for node). Date of collection for each strain is shown.

Fig. 3.

Xam strains contain diverse effector repertoires. Effector repertoires were deduced on the basis of homology to known animal and plant type three effector proteins. (Upper) Eighteen geographically/temporally diverse strains are shown (see Fig. S4 for all strains). Sixty-five Xam strains contain nine core effectors (red box). TAL effector copy number was estimated from Southern blot analysis after genomic DNA digestion with EcoRI (Fig. S6). Note that these data are estimates as this technique would not resolve multiple copies contained in a single EcoRI fragment or multiple copies with conserved EcoRI sites. Symptom development was assessed 10 d postinoculation. Symptoms are representative of six leaves and were scored on the basis of level of water soaking on a scale of 1–3. +, presence of homolog; /, absence of homolog; Ψ, premature stop codon or frameshift mutation. (Lower Left) Growth assay comparing select Xam strains. Day 0, average of two inoculations; days 6 and 10, SD of at least four inoculations. (Lower Right) Bacterial growth 10 d postinoculation. Effector arsenal size, TAL effector count, and symptom development are compared. Additional virulence assays are in Fig. S6.

Fig. 4.

Sequence comparison among Xam effectors identifies the least polymorphic effectors. Effector sequences were obtained from Xam genome assemblies as described in Materials and Methods and Fig. S5. Clustal sequence alignments were created for each effector and sequence variance was calculated by representing each sequence as a set of multidimensional vectors. A standard mathematical measure of variance was then applied (Materials and Methods). Gene models (solid black line) are displayed directly above the x axis and the number of strains with the given gene model is displayed to the right of each gene model. Additional effectors are displayed in Fig. S7.

Fig. P1.

Cassava is a staple crop and is of particular importance in developing nations. Depicted is a healthy cassava plant grown in the field. Image credit: Hervé Vanderschuren.