Significance: Susceptibility (S) genes are plant genes that facilitate pathogen infection. Inactivation of S genes has been considered a promising strategy to obtain broad-spectrum and durable resistance in crops. We characterized two orthologs of the Arabidopsis S gene DMR6 in tomato: SlDMR6-1 and SlDMR6-2 . We show that SlDMR6-1, but not SlDMR6-2, is associated with plant immunity. Remarkably, Sldmr6-1 mutants display enhanced resistance to bacterial, oomycete, and fungal pathogens. This phenotype correlates with increased levels of the defense hormone salicylic acid (SA) and enhanced transcriptional activation of plant immune responses. We also demonstrate that SlDMR6-1 and SlDMR6-2 convert SA into its inactive form, 2,5-DHBA, indicating that they play a role in SA homeostasis.

Rice (Oryza sativa) is of paramount importance for global nutrition, supplying at least 20% of global calories. However, water scarcity and increased drought severity are anticipated to reduce rice yields globally. We explored stomatal developmental genetics as a mechanism for improving drought resilience in rice while maintaining yield under climate stress. CRISPR/Cas9-mediated knockouts of the positive regulator of stomatal development STOMAGEN and its paralog EPIDERMAL PATTERNING FACTOR-LIKE10 (EPFL10) yielded lines with ∼25% and 80% of wild-type stomatal density, respectively. epfl10 lines with moderate reductions in stomatal density were able to conserve water to similar extents as stomagen lines but did not suffer from the concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation observed in stomagen knockouts. Moderate reductions in stomatal density achieved by editing EPFL10 present a climate-adaptive approach for safeguarding yield in rice. Editing the paralog of STOMAGEN in other species may provide a means for tuning stomatal density in agriculturally important crops beyond rice.

Cassava (Manihot esculenta) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces the neurotoxin cyanide and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. This problem is further exacerbated by drought conditions which increase this toxin in the plant. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444; the West African, farmer-preferred cultivar TME 419; and the improved variety TMS 91/02324. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. The congruence of results across accessions indicates that our approach could readily be extended to other preferred or improved cultivars. This work demonstrates cassava genome editing for enhanced food safety and reduced processing burden, against the backdrop of a changing climate.

Wheat, an essential crop for global food security, is well adapted to a wide variety of soils. However, the gene networks shaping different root architectures remain poorly understood. We report here that dosage differences in a cluster of monocot-specific 12-OXOPHYTODIENOATE REDUCTASE genes from subfamily III (OPRIII) modulate key differences in wheat root architecture, which are associated with grain yield under water-limited conditions. Wheat plants with loss-of-function mutations in OPRIII show longer seminal roots, whereas increased OPRIII dosage or transgenic over-expression result in reduced seminal root growth, precocious development of lateral roots and increased jasmonic acid (JA and JA-Ile). Pharmacological inhibition of JA-biosynthesis abolishes root length differences, consistent with a JA-mediated mechanism. Transcriptome analyses of transgenic and wild-type lines show significant enriched JA-biosynthetic and reactive oxygen species (ROS) pathways, which parallel changes in ROS distribution. OPRIII genes provide a useful entry point to engineer root architecture in wheat and other cereals.

Understanding CRISPR-Cas9’s capacity to produce native overexpression (OX) alleles would accelerate agronomic gains achievable by gene editing. To generate OX alleles with increased RNA and protein abundance, we leveraged multiplexed CRISPR-Cas9 mutagenesis of noncoding sequences upstream of the rice PSBS1 gene. We isolated 120 gene-edited alleles with varying non-photochemical quenching (NPQ) capacity in vivo—from knockout to overexpression—using a high-throughput screening pipeline. Overexpression increased OsPsbS1 protein abundance two- to threefold, matching fold changes obtained by transgenesis. Increased PsbS protein abundance enhanced NPQ capacity and water-use efficiency. Across our resolved genetic variation, we identify the role of 5′UTR indels and inversions in driving knockout/knockdown and overexpression phenotypes, respectively. Complex structural variants, such as the 252-kb duplication/inversion generated here, evidence the potential of CRISPR-Cas9 to facilitate significant genomic changes with negligible off-target transcriptomic perturbations. Our results may inform future gene-editing strategies for hypermorphic alleles and have advanced the pursuit of gene-edited, non-transgenic rice plants with accelerated relaxation of photoprotection.

Development of anthers and pollen represents an important aspect of the life cycle in flowering plants. Genes contributing to anther and pollen development have been widely studied in many plant species. Ms26/CYP704B genes play an important role in pollen development through biosynthesis of sporopollenin for pollen exine formation. To investigate the role of Ms26/CYP704B genes in anther and pollen development of bread wheat, mutations in the A-, B-, and D-homeologs of the putative Ms26/CYP704B gene were analyzed. Single and double homozygous mutants in any of the homeologs did not affect pollen development and male fertility. Triple homozygous mutants resulted in completely male sterile plants that were defective in pollen and anther development. Additionally, double homozygous-single heterozygous mutants were also male sterile although with varying levels of residual fertility. The fertility of these triple mutants was dependent upon the homeolog contributing the wild-type allele. Two heterologous Ms26/CYP704B genes, when transformed into a triple homozygous mutant background, completely restored male fertility, whereas a single gene was unable to restore fertility. Functional analysis of Ms26/CYP704B furthers the understanding of male fertility genes which can be utilized for the development of novel hybrid seed production systems in wheat.