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.

Improving cassava bacterial blight resistance by editing the epigenome.

Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas phaseoli pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. In this work, we direct methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display decreased CBB symptoms while maintaining normal growth and development. This work therefore presents an epigenome editing approach useful for crop improvement.

Mutations in DNA polymerase δ subunit 1 co-segregate with CMD2-type resistance to Cassava Mosaic Geminiviruses.

Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for this loss of resistance, whole genome sequencing and genetic variant analysis was performed and the CMD2 locus was fine-mapped to a 190 kilobase interval. Collectively, these data indicate that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a CMD-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional candidate resistance alleles within MePOLD1. Genetic variation of MePOLD1, therefore, could represent an important genetic resource for resistance breeding and/or genome editing, and elucidating mechanisms of resistance to geminiviruses.

Increased signal-to-noise ratios within experimental field trials by regressing spatially distributed soil properties as principal components.

Environmental variability poses a major challenge to any field study. Researchers attempt to mitigate this challenge through replication. Thus, the ability to detect experimental signals is determined by the degree of replication and the amount of environmental variation, noise, within the experimental system. A major source of noise in field studies comes from the natural heterogeneity of soil properties which create microtreatments throughout the field. In addition, the variation within different soil properties is often nonrandomly distributed across a field. We explore this challenge through a sorghum field trial dataset with accompanying plant, microbiome, and soil property data. Diverse sorghum genotypes and two watering regimes were applied in a split-plot design. We describe a process of identifying, estimating, and controlling for the effects of spatially distributed soil properties on plant traits and microbial communities using minimal degrees of freedom. Importantly, this process provides a method with which sources of environmental variation in field data can be identified and adjusted, improving our ability to resolve effects of interest and to quantify subtle phenotypes.

A comparison of ImageJ and machine learning based image analysis methods to measure cassava bacterial blight disease severity.

BACKGROUND: Methods to accurately quantify disease severity are fundamental to plant pathogen interaction studies. Commonly used methods include visual scoring of disease symptoms, tracking pathogen growth in planta over time, and various assays that detect plant defense responses. Several image-based methods for phenotyping of plant disease symptoms have also been developed. Each of these methods has different advantages and limitations which should be carefully considered when choosing an approach and interpreting the results. RESULTS: In this paper, we developed two image analysis methods and tested their ability to quantify different aspects of disease lesions in the cassava-Xanthomonas pathosystem. The first method uses ImageJ, an open-source platform widely used in the biological sciences. The second method is a few-shot support vector machine learning tool that uses a classifier file trained with five representative infected leaf images for lesion recognition. Cassava leaves were syringe infiltrated with wildtype Xanthomonas, a Xanthomonas mutant with decreased virulence, and mock treatments. Digital images of infected leaves were captured overtime using a Raspberry Pi camera. The image analysis methods were analyzed and compared for the ability to segment the lesion from the background and accurately capture and measure differences between the treatment types. CONCLUSIONS: Both image analysis methods presented in this paper allow for accurate segmentation of disease lesions from the non-infected plant. Specifically, at 4-, 6-, and 9-days post inoculation (DPI), both methods provided quantitative differences in disease symptoms between different treatment types. Thus, either method could be applied to extract information about disease severity. Strengths and weaknesses of each approach are discussed.

Identification of beneficial and detrimental bacteria impacting sorghum responses to drought using multi-scale and multi-system microbiome comparisons.

Drought is a major abiotic stress limiting agricultural productivity. Previous field-level experiments have demonstrated that drought decreases microbiome diversity in the root and rhizosphere. How these changes ultimately affect plant health remains elusive. Toward this end, we combined reductionist, transitional and ecological approaches, applied to the staple cereal crop sorghum to identify key root-associated microbes that robustly affect drought-stressed plant phenotypes. Fifty-three Arabidopsis-associated bacteria were applied to sorghum seeds and their effect on root growth was monitored. Two Arthrobacter strains caused root growth inhibition (RGI) in Arabidopsis and sorghum. In the context of synthetic communities, Variovorax strains were able to protect plants from Arthrobacter-caused RGI. As a transitional system, high-throughput phenotyping was used to test the synthetic communities. During drought stress, plants colonized by Arthrobacter had reduced growth and leaf water content. Plants colonized by both Arthrobacter and Variovorax performed as well or better than control plants. In parallel, we performed a field trial wherein sorghum was evaluated across drought conditions. By incorporating data on soil properties into the microbiome analysis, we accounted for experimental noise with a novel method and were able to observe the negative correlation between the abundance of Arthrobacter and plant growth. Having validated this approach, we cross-referenced datasets from the high-throughput phenotyping and field experiments and report a list of bacteria with high confidence that positively associated with plant growth under drought stress. In conclusion, a three-tiered experimental system successfully spanned the lab-to-field gap and identified beneficial and deleterious bacterial strains for sorghum under drought.

Current status and impending progress for cassava structural genomics.

KEY MESSAGE: We demystify recent advances in genome assemblies for the heterozygous staple crop cassava (Manihot esculenta), and highlight key cassava genomic resources. Cassava, Manihot esculenta Crantz, is a crop of societal and agricultural importance in tropical regions around the world. Genomics provides a platform for accelerated improvement of cassava's nutritional and agronomic traits, as well as for illuminating aspects of cassava's history including its path towards domestication. The highly heterozygous nature of the cassava genome is widely recognized. However, the full extent and context of this heterozygosity has been difficult to reveal because of technological limitations within genome sequencing. Only recently, with several new long-read sequencing technologies coming online, has the genomics community been able to tackle some similarly difficult genomes. In light of these recent advances, we provide this review to document the current status of the cassava genome and genomic resources and provide a perspective on what to look forward to in the coming years.

Large structural variations in the haplotype-resolved African cassava genome.

Cassava (Manihot esculenta Crantz, 2n = 36) is a global food security crop. It has a highly heterozygous genome, high genetic load, and genotype-dependent asynchronous flowering. It is typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi-C to resolve each haplotype of the genome of a farmer-preferred cassava line, TME7 (Oko-iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON-Phase and Hi-C read data. The ultralong-range information from Hi-C sequencing was also used for scaffolding. Comparison of the two phases revealed >5000 large haplotype-specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele-specific expression was further explored. RNA-sequencing data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy-to-use web-based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype-specific structural variation, gene hemizygosity, and allele-specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.

Gene tagging via CRISPR-mediated homology-directed repair in cassava.

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.

Escalation in the host-pathogen arms race: A host resistance response corresponds to a heightened bacterial virulence response.

The zig-zag model of host-pathogen interaction describes the relative strength of defense response across a spectrum of pathogen-induced plant phenotypes. A stronger defense response results in increased resistance. Here, we investigate the strength of pathogen virulence during disease and place these findings in the context of the zig-zag model. Xanthomonas vasicola pv. holcicola (Xvh) causes sorghum bacterial leaf streak. Despite being widespread, this disease has not been described in detail at the molecular level. We divided diverse sorghum genotypes into three groups based on disease symptoms: water-soaked lesions, red lesions, and resistance. Bacterial growth assays confirmed that these three phenotypes represent a range of resistance and susceptibility. To simultaneously reveal defense and virulence responses across the spectrum of disease phenotypes, we performed dual RNA-seq on Xvh-infected sorghum. Consistent with the zig-zag model, the expression of plant defense-related genes was strongest in the resistance interaction. Surprisingly, bacterial virulence genes related to the type III secretion system (T3SS) and type III effectors (T3Es) were also most highly expressed in the resistance interaction. This expression pattern was observed at multiple time points within the sorghum-Xvh pathosystem. Further, a similar expression pattern was observed in Arabidopsis infected with Pseudomonas syringae for effector-triggered immunity via AvrRps4 but not AvrRpt2. Specific metabolites were able to repress the Xvh virulence response in vitro and in planta suggesting a possible signaling mechanism. Taken together, these findings reveal multiple permutations of the continually evolving host-pathogen arms race from the perspective of host defense and pathogen virulence responses.

Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence.

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive-sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, novel cap-binding protein-1 (nCBP-1), and nCBP-2. Protein-protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9-mediated genome editing was employed to generate ncbp-1, ncbp-2, and ncbp-1/ncbp-2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp-1/ncbp-2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild-type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.

An automated, high-throughput method for standardizing image color profiles to improve image-based plant phenotyping.

High-throughput phenotyping has emerged as a powerful method for studying plant biology. Large image-based datasets are generated and analyzed with automated image analysis pipelines. A major challenge associated with these analyses is variation in image quality that can inadvertently bias results. Images are made up of tuples of data called pixels, which consist of R, G, and B values, arranged in a grid. Many factors, for example image brightness, can influence the quality of the image that is captured. These factors alter the values of the pixels within images and consequently can bias the data and downstream analyses. Here, we provide an automated method to adjust an image-based dataset so that brightness, contrast, and color profile is standardized. The correction method is a collection of linear models that adjusts pixel tuples based on a reference panel of colors. We apply this technique to a set of images taken in a high-throughput imaging facility and successfully detect variance within the image dataset. In this case, variation resulted from temperature-dependent light intensity throughout the experiment. Using this correction method, we were able to standardize images throughout the dataset, and we show that this correction enhanced our ability to accurately quantify morphological measurements within each image. We implement this technique in a high-throughput pipeline available with this paper, and it is also implemented in PlantCV.

Genomics-enabled analysis of the emergent disease cotton bacterial blight.

Cotton bacterial blight (CBB), an important disease of (Gossypium hirsutum) in the early 20th century, had been controlled by resistant germplasm for over half a century. Recently, CBB re-emerged as an agronomic problem in the United States. Here, we report analysis of cotton variety planting statistics that indicate a steady increase in the percentage of susceptible cotton varieties grown each year since 2009. Phylogenetic analysis revealed that strains from the current outbreak cluster with race 18 Xanthomonas citri pv. malvacearum (Xcm) strains. Illumina based draft genomes were generated for thirteen Xcm isolates and analyzed along with 4 previously published Xcm genomes. These genomes encode 24 conserved and nine variable type three effectors. Strains in the race 18 clade contain 3 to 5 more effectors than other Xcm strains. SMRT sequencing of two geographically and temporally diverse strains of Xcm yielded circular chromosomes and accompanying plasmids. These genomes encode eight and thirteen distinct transcription activator-like effector genes. RNA-sequencing revealed 52 genes induced within two cotton cultivars by both tested Xcm strains. This gene list includes a homeologous pair of genes, with homology to the known susceptibility gene, MLO. In contrast, the two strains of Xcm induce different clade III SWEET sugar transporters. Subsequent genome wide analysis revealed patterns in the overall expression of homeologous gene pairs in cotton after inoculation by Xcm. These data reveal important insights into the Xcm-G. hirsutum disease complex and strategies for future development of resistant cultivars.

Cassava haplotype map highlights fixation of deleterious mutations during clonal propagation.

Cassava (Manihot esculenta Crantz) is an important staple food crop in Africa and South America; however, ubiquitous deleterious mutations may severely decrease its fitness. To evaluate these deleterious mutations, we constructed a cassava haplotype map through deep sequencing 241 diverse accessions and identified >28 million segregating variants. We found that (i) although domestication has modified starch and ketone metabolism pathways to allow for human consumption, the concomitant bottleneck and clonal propagation have resulted in a large proportion of fixed deleterious amino acid changes, increased the number of deleterious alleles by 26%, and shifted the mutational burden toward common variants; (ii) deleterious mutations have been ineffectively purged, owing to limited recombination in the cassava genome; (iii) recent breeding efforts have maintained yield by masking the most damaging recessive mutations in the heterozygous state but have been unable to purge the mutation burden; such purging should be a key target in future cassava breeding.

Gene expression atlas for the food security crop cassava.

Cassava (Manihot esculenta) feeds c. 800 million people world-wide. Although this crop displays high productivity under drought and poor soil conditions, it is susceptible to disease, postharvest deterioration and the roots contain low nutritional content. Here, we provide molecular identities for 11 cassava tissue/organ types through RNA-sequencing and develop an open access, web-based interface for further interrogation of the data. Through this dataset, we consider the physiology of cassava. Specifically, we focus on identification of the transcriptional signatures that define the massive, underground storage roots used as a food source and the favored target tissue for transgene integration and genome editing, friable embryogenic callus (FEC). Further, we identify promoters able to drive strong expression in multiple tissue/organs. The information gained from this study is of value for both conventional and biotechnological improvement programs.

Correction: A Genetic Screen Identifies a Requirement for Cysteine-Rich-Receptor-Like Kinases in Rice NH1 (OsNPR1)-Mediated Immunity.

[This corrects the article DOI: 10.1371/journal.pgen.1006049.].