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Summary Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best‐performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of 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.

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors. In this report, we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that AFB4 and AFB5 are targets of the picloram family of auxinic herbicides in addition to indole-3-acetic acid. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity.

Plant diseases cause significant reductions in agricultural productivity worldwide. Disease symptoms have deleterious effects on the growth and development of crop plants, limiting yields and making agricultural products unfit for consumption. For many plant-pathogen systems, we lack knowledge of the physiological mechanisms that link pathogen infection and the production of disease symptoms in the host. A variety of quantitative high-throughput image-based methods for phenotyping plant growth and development are currently being developed. These methods range from detailed analysis of a single plant over time to broad assessment of the crop canopy for thousands of plants in a field and employ a wide variety of imaging technologies. Application of these methods to the study of plant disease offers the ability to study quantitatively how host physiology is altered by pathogen infection. These approaches have the potential to provide insight into the physiological mechanisms underlying disease symptom development. Furthermore, imaging techniques that detect the electromagnetic spectrum outside of visible light allow us to quantify disease symptoms that are not visible by eye, increasing the range of symptoms we can observe and potentially allowing for earlier and more thorough symptom detection. In this review, we summarize current progress in plant disease phenotyping and suggest future directions that will accelerate the development of resistant crop varieties.

Plant disease symptoms exhibit complex spatial and temporal patterns that are challenging to quantify. Image-based phenotyping approaches enable multidimensional characterization of host-microbe interactions and are well suited to capture spatial and temporal data that are key to understanding disease progression. We applied image-based methods to investigate cassava bacterial blight, which is caused by the pathogen Xanthomonas axonopodis pv. manihotis (Xam). We generated Xam strains in which individual predicted type III effector (T3E) genes were mutated and applied multiple imaging approaches to investigate the role of these proteins in bacterial virulence. Specifically, we quantified bacterial populations, water-soaking disease symptoms, and pathogen spread from the site of inoculation over time for strains with mutations in avrBs2, xopX, and xopK as compared to wild-type Xam. ΔavrBs2 and ΔxopX both showed reduced growth in planta and delayed spread through the vasculature system of cassava. ΔavrBs2 exhibited reduced water-soaking symptoms at the site of inoculation. In contrast, ΔxopK exhibited enhanced induction of disease symptoms at the site of inoculation but reduced spread through the vasculature. Our results highlight the importance of adopting a multipronged approach to plant disease phenotyping to more fully understand the roles of T3Es in virulence. Finally, we demonstrate that the approaches used in this study can be extended to many host-microbe systems and increase the dimensions of phenotype that can be explored.

The plant hormone auxin is an important regulator of plant growth and development, as well as plant-pathogen interactions. I investigated the roles of auxin during infection of the bacterial pathogen Pseudomonas syringae strain DC3000 in one of its plant hosts Arabidopsis thaliana. Previous work indicated that treatment of plants with exogenous auxin promotes susceptibility to P. syringae and led to the hypothesis that auxin promotes pathogenesis by suppressing salicylic acid (SA)-mediated host defenses, the primary defenses against P. syringae infection. To investigate this hypothesis and further elucidate the mechanisms by which auxin promotes pathogenesis, I took advantage of a transgenic A. thaliana line that over-expresses the YUCCA1 (YUC1) auxin biosynthesis gene and thus accumulates elevated endogenous auxin levels. I demonstrated that this YUC1-overexpressing line is more susceptible to P. syringae, but this enhanced susceptibility is caused by a mechanism that is independent of suppression of SA-mediated defenses. These results support the idea that elevated auxin acts through alternative mechanisms, such as by promoting pathogen virulence or by influencing host physiology in ways that promote disease. I propose future experiments to investigate these potential mechanisms. Additionally, I investigated the roles of host auxin signaling during DC3000 pathogenesis by performing infection experiments with A. thaliana auxin receptor mutants. A mutant lacking four auxin receptors, which exhibits severely compromised auxin responses, supported normal levels of pathogen growth when inoculated with P. syringae. Thus, our experiments provide no evidence that host auxin signaling is required for susceptibility to P. syringae. Nonetheless, one auxin receptor mutant afb4-2, which appears to define a novel protein with altered function, exhibited enhanced susceptibility to P. syringae. In this mutant, enhanced susceptibility appears to be primarily due to reduced basal SA levels. The mechanism for how this mutation impacts SA accumulation is unknown but seems to be unrelated to its effects on auxin signaling. Quantification of the plant hormones abscisic acid (ABA) and jasmonic acid, as well as drought tolerance experiments, suggest that elevated ABA accumulation in the afb4-2 mutant may play a role in suppressing SA levels. This work provides a basis for future investigation of the afb4-2 mutation, including biochemical and genetic experiments that may provide key insights into interactions between a component of auxin signaling and other hormone signaling pathways. In summary, auxin does not promote P. syringae pathogenesis simply through suppression of SA-mediated host defenses but potentially through a range of novel mechanisms.

Plant disease symptoms exhibit complex spatial and temporal patterns that are challenging to quantify. Image-based phenotyping approaches enable multi-dimensional characterization of host-microbe interactions and are well suited to capture spatial and temporal data that are key to understanding disease progression. We applied image-based methods to investigate cassava bacterial blight, which is caused by the pathogen Xanthomonas axonopodis pv. manihotis (Xam). We generated Xam strains in which individual predicted type III effector (T3E) genes were mutated and applied multiple imaging approaches to investigate the role of these proteins in bacterial virulence. Specifically, we quantified bacterial populations, water-soaking disease symptoms, and pathogen spread from the site of inoculation over time for strains with mutations in avrBs2, xopX, and xopK as compared to wild-type Xam. avrBs2 and xopX both showed reduced growth in planta and delayed spread through the vasculature system of cassava. avrBs2 exhibited reduced water-soaking symptoms at the site of inoculation. In contrast, xopK exhibited enhanced induction of disease symptoms at the site of inoculation but reduced spread through the vasculature. Our results highlight the importance of adopting a multi-pronged approach to plant disease phenotyping to more fully understand the roles of T3Es in virulence. Finally, we demonstrate that the approaches used in this study can be extended to many host-microbe systems and increase the dimensions of phenotype that can be explored.

Cassava (Manihot esculenta) feeds approximately 800 million people worldwide. 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 eleven cassava tissue types through RNA- sequencing and develop an open access, web-based interface for further interrogation of the data. Through this dataset, we report novel insight into the physiology of cassava and identify promoters able to drive specified tissue expression profiles. 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). The information gained from this study is of value for both conventional and biotechnological improvement programs.

A phenomenon known as magnetic fragmentation is observed by means of neutron scattering in the spin ice candidate Nd 2 Zr 2 O 7 . Fractionalized excitations that emerge from a many-body system have revealed rich physics and concepts, from composite fermions in two-dimensional electron systems, revealed through the fractional quantum Hall effect 1 , to spinons in antiferromagnetic chains 2 and, more recently, fractionalization of Dirac electrons in graphene 3 and magnetic monopoles in spin ice 4 . Even more surprising is the fragmentation of the degrees of freedom themselves, leading to coexisting and a priori independent ground states. This puzzling phenomenon was recently put forward in the context of spin ice, in which the magnetic moment field can fragment, resulting in a dual ground state consisting of a fluctuating spin liquid, a so-called Coulomb phase 5 , on top of a magnetic monopole crystal 6 . Here we show, by means of neutron scattering measurements, that such fragmentation occurs in the spin ice candidate Nd 2 Zr 2 O 7 . We observe the spectacular coexistence of an antiferromagnetic order induced by the monopole crystallization and a fluctuating state with ferromagnetic correlations. Experimentally, this fragmentation manifests itself through the superposition of magnetic Bragg peaks, characteristic of the ordered phase, and a pinch point pattern, characteristic of the Coulomb phase. These results highlight the relevance of the fragmentation concept to describe the physics of systems that are simultaneously ordered and fluctuating.