Genetic engineering tools have the potential to rapidly and precisely improve the genome of slow-to-breed cacao. We previously developed an efficient protocol for transforming cacao using cotyledonary explants derived from secondary somatic embryos via Agrobacterium tumefaciens. In this study, we demonstrate that our transformation protocol is successful in elite cultivars, INIAPG-038 and Matina 1-6, producing fertile seeds with stable visual marker inheritance regardless of whether the transgenic plants were used as the pollen or ovule donor. Three vectors were used in the transformations, each containing genes for enhanced yellow fluorescent protein (eyfp) and neomycin phosphotransferase II (nptII). Three transgenic INIAPG-038 events and one transgenic Matina 1-6 event were used to evaluate seed fertility and the stability of transgene inheritance in cacao seeds and plants. The T1 progeny of these four transgenic events were analyzed for YFP expression and transgene presence. YFP expression segregated at a 1:1 ratio in all events when the transgenic plants were crossed with non-transgenic plants, while a 3:1 segregation was observed when transgenic events were crossed with each other. The transgenic plants exhibited a normal phenotype compared to non-transgenic control plants, producing seeds with a 97% germination rate.

Peptide hormone signaling coordinates plant growth and osmotic stress responses, yet how the transition between these responses is regulated remains poorly understood. Here, we investigated the function of the rice PLANT PEPTIDES CONTAINING SULFATED TYROSINE 8 (OsPSY8) peptide in osmotic stress responses. OsPSY8 was predominantly expressed in root tissues under non-stress conditions, with preferential expression in lateral roots where it promoted root growth. Osmotic stress rapidly reduced OsPSY8 expression in roots through the OsWRKY24 transcription factor. Loss-of-function ospsy8 mutants exhibited enhanced osmotic stress tolerance, whereas OsPSY8 overexpression increased osmotic stress susceptibility. Transcriptomic analyses revealed that disruption of OsPSY8 activated stress-responsive pathways, including those associated with lignin biosynthesis, compatible solute production, cell wall remodeling, and reactive oxygen species (ROS) scavenging, and was accompanied by increased lignin accumulation in roots. In contrast, overexpression of OsPSY8 resulted in maintenance of growth-associated transcriptional programs while suppressing stress-responsive pathways under osmotic stress. Together, these findings identify OsPSY8 as an important regulator of the transition from growth to stress adaptation in rice and suggest that stress-induced repression of PSY signaling is required to disengage growth programs and activate adaptive responses during osmotic stress. Significance Statement:Crop survival during drought depends on the ability to transition from growth to stress adaptation. Plant peptide hormones have emerged as important regulators of this critical transition, highlighting the importance of investigating their roles and potential for improving crop resilience. We show that a rice peptide hormone regulates this transition. Under non-stress conditions, this peptide hormone, predominantly expressed in rice roots, promotes root growth while suppressing stress responses. During osmotic stress, expression of the peptide hormone decreases, resulting in activation of stress-responsive pathways, such as lignin biosynthesis and reactive oxygen species scavenging. These findings demonstrate that a peptide hormone coordinates the balance between growth and stress adaptation in rice, with broader implications for understanding and improving crop resilience.

TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Because of their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold great potential for genome editing. However, most TnpBs lack robust gene-editing activity. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein using deep mutational scanning and we discovered activating mutations in both the RNA and the protein. Leveraging the protein's mutational landscape, we constructed a combinatorial library of activating mutations, from which we identified two enhanced TnpB variants. These variants increased editing in human cells, Nicotania benthamiana, pepper and rice. While editing efficiencies varied by target site, engineered variants achieved up to 55% insertion and deletion frequencies (a 50-fold increase over wild type) in N. benthamiana, surpassing ISYmu1 (<7%), AsCas12f-HKRA (<9%) and other compact editors. These findings highlight elements critical for regulating TnpB endonuclease activity and demonstrate latent activity accessible through mutation.

Plant transformation efficiency is highly dependent on species, individual genotypes, and tissue types. In maize, immature embryos are regularly used for transformation. The process relies heavily on callus development, as it is intricately associated with somatic embryogenesis and subsequent plant regeneration, both of which directly affect transformation efficiency. Immature embryos of the segregation progeny derived from the two inbred parents, a transformation-amenable line A188 and a recalcitrant line B73, can be cultured to form two primary callus types: Type I and Type II. The Type II callus grows faster and is a favorable type for regeneration. Here, Type I and II calli from the B73xA188 F2 population were genotyped by Genotyping-By-Sequencing (GBS). Quantitative trait locus (QTL) analysis of the callus type identified QTLs at chromosomes 2, 5, 6, 8, and 9. The result was largely supported by the bulk segregant RNA-seq (BSR-seq) genetic analysis using RNA from separately pooled Type I and II calli. Both analyses revealed that an allele of A188 on chromosome 6 and B73 alleles on chromosomes 2, 5, 8, and 9 promoted the formation of the Type II callus. Differentially expressed genes (DEGs) between the Type II and I F2 calli were also identified. In addition, the A188 calli developed from the same immature embryos often exhibit heterogeneous morphology, including the fast- and slow-growing callus sectors. The transcriptional comparison between the two sectors was performed to identify DEGs. Both sets of DEGs were enriched in genes involved in cell-wall organization and wax biosynthesis pathways.

TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold significant potential for genome editing. However, most TnpBs lack robust gene editing activity, and unbiased profiling of mutational effects on editing activity has not been explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. One- and two-position RNA mutants outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the protein's mutational landscape, we identified enhanced TnpB variants from a combinatorial library of activating mutations. These variants enhanced editing in human cells, N. benthamiana, pepper, and rice, with up to a fifty-fold increase compared to wild-type TnpB. These findings highlight previously unknown elements critical for regulating TnpB endonuclease activity and reveal surprising latent activity accessible through mutation.

Reactive oxygen species (ROS) accumulation is required for effective plant defense. Accumulation of the Arabidopsis (Arabidopsis thaliana) NADPH oxidase respiratory burst oxidase homolog D (RBOHD) is regulated by phosphorylation of a conserved C-terminal residue (T912) leading to ubiquitination by the RING E3 ligase Pbl13-interacting RING domain E3 ligase (PIRE). Arabidopsis PIRE knockouts exhibit enhanced ROS production and resistance to the foliar pathogen Pseudomonas syringae. Here, we identified 170 PIRE homologs, which emerged in tracheophytes and expanded in angiosperms. We investigated the role of tomato (Solanum lycopersicum) PIRE homologs in regulating ROS production, RBOH stability, and disease resistance. Mutational analyses of residues corresponding to T912 in the tomato RBOHD ortholog, SlRBOHB, affected protein accumulation and ROS production in a PIRE-dependent manner. Using genome editing, we generated mutants in 2 S. lycopersicum PIRE (SlPIRE) homologs. SlPIRE1 edited lines (Slpire1) in the tomato cultivar M82 displayed enhanced ROS production upon treatment with flg22, an immunogenic epitope of flagellin. Furthermore, Slpire1 exhibited decreased disease symptoms and bacterial accumulation when inoculated with foliar bacterial pathogens P. syringae and Xanthomonas campestris. However, Slpire1 exhibited similar levels of colonization as wild type upon inoculation with diverse soil-borne pathogens. These results indicate that PIRE regulates RBOHs in multiple plant species and is a promising target for foliar disease control. This study also highlights the pathogen-specific role of PIRE, indicating its potential for targeted manipulation to enhance foliar disease resistance without affecting root-associated pathogenic interactions.

Here, we report the successful implementation of heritable virus-induced genome editing (VIGE) in tomato (Solanum lycopersicum). We generated three transgenic tomato lines expressing Streptococcus pyogenes Cas9 (SpCas9) under the control of Cauliflower mosaic virus 35S (35S), S. lycopersicum ribosomal protein S5A (SlRPS5A), or S. lycopersicum YAO promoters (SlYAO). These three lines were tested for somatic and heritable editing using the tobacco rattle virus (TRV)-based system carrying guide RNAs (gRNAs) fused with mobile RNA sequences. TRV with gRNA targeted to Phytoene desaturase (SlPDS) and Downy mildew resistance 6 (SlDMR6) genes fused to mobile RNA sequences showed significant somatic editing efficiency in all three tomato lines expressing SpCas9. However, the progenies from the SlYAO promoter-driven SpCas9 tomato infected with TRV with gRNA targeted to SlDMR6 fused to the mobile RNA sequence resulted in monoallelic mutations with a frequency of 3%. Optimization of environmental conditions, such as reduced light intensity, significantly increased heritable editing frequencies, from 0% to 86% at the SlPDS and from 3% to 100% at the SlDMR6, including biallelic mutations. These findings underscore the use of appropriate promoters to express Cas nucleases and optimized environmental conditions to enhance heritable genome editing efficiency in tomato using VIGE. Furthermore, our method enables the generation of mutants without additional tissue culture or transformation once a SpCas9-expressing tomato line is established.

Glucosinolates are plant-specialized metabolites that can be hydrolyzed by glycosyl hydrolases, called myrosinases, creating a variety of hydrolysis products that benefit human health. While cruciferous vegetables are a rich source of glucosinolates, they are often cooked before consumption, limiting the conversion of glucosinolates to hydrolysis products due to the denaturation of myrosinases. Here we screen a panel of glycosyl hydrolases for high thermostability and engineer the Brassica crop, broccoli (Brassica oleracea L.), for the improved conversion of glucosinolates to chemopreventive hydrolysis products. Our transgenic broccoli lines enabled glucosinolate hydrolysis to occur at higher cooking temperatures, 20 °C higher than in wild-type broccoli. The process of cooking fundamentally transforms the bioavailability of many health-relevant bioactive compounds in our diet. Our findings demonstrate the promise of leveraging genetic engineering to tailor crops with novel traits that cannot be achieved through conventional breeding and improve the nutritional properties of the plants we consume.

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.

Plant diseases pose a significant threat to global crop production. Most disease resistance genes used in crop breeding programs encode nucleotide-binding leucine-rich repeat receptors (NLRs) that are limited in pathogen specificity and durability. In this study, we leveraged synthetic biology to develop an inducible broad-spectrum resistance in tomatoes. Constitutive expression of autoactive NLRs in plants leads to robust resistance against multiple pathogens but significantly stunts growth. We expressed autoactive NLRs under the control of pathogen-inducible (PI) promoters to mitigate the fitness costs. Taking advantage of extensive, new genomic and transcriptomic resources, we identified PI promoters that responded to multiple pathogens but not abiotic stress. We further validated functionality of predicted elements through a promoter luciferase assay. We generated significant resistance in transgenic tomatoes but we also encountered unwanted expression induction of the native promoter regions in flowers which led to lethal fruit development. Thus, we pursued promoter engineering for fine-tuning the induction. We identified cis-regulatory regions responsible for pathogen-inducibility through promoter bashing experiments and recombined the native promoter with the inducible part and the core promoter. Furthermore, we rationally created synthetic promoters showing a gradient of expression levels, which will allow for selection for transgenic tomatoes with the best performance. We found that the spacing between functional sequences, repeat number of inducible sequences, and core promoters all influence the outcome of engineering. Our study outlines a framework for developing broad-spectrum synthetic immune constructs with reduced fitness cost and provides examples of pathogen-inducible promoter engineering. ### Competing Interest Statement The authors have declared no competing interest.

Cis-regulatory element editing can generate quantitative trait variation that mitigates extreme phenotypes and harmful pleiotropy associated with coding sequence mutations. Here, we applied a multiplexed CRISPR/Cas9 approach, informed by bioinformatic datasets, to generate genotypic variation in the promoter of OsSTOMAGEN, a positive regulator of rice stomatal density. Engineered genotypic variation corresponded to broad and continuous variation in stomatal density, ranging from 70% to 120% of wild-type stomatal density. This panel of stomatal variants was leveraged in physiological assays to establish discrete relationships between stomatal morphological variation and stomatal conductance, carbon assimilation and intrinsic water use efficiency in steady-state and fluctuating light conditions. Additionally, promoter alleles were subjected to vegetative drought regimes to assay the effects of the edited alleles on developmental response to drought. Notably, the capacity for drought-responsive stomatal density reprogramming in stomagen and two cis-regulatory edited alleles was reduced. Collectively our data demonstrate that cis-regulatory element editing can generate near-isogenic trait variation that can be leveraged for establishing relationships between anatomy and physiology, providing a basis for optimizing traits across diverse environments.

Bacterial spot, caused by Xanthomonas species, is a devastating disease of tomato (Solanum lycopersicum) and pepper (Capsicum annuum) (Schwartz et al., 2015). The recessively inherited resistance, bacterial spot 5 (bs5), in pepper (hereafter referred to as Cabs5) can confer resistance against different Xanthomonas strains (Jones et al., 2002). The Cabs5 resistance is characterized by the absence of disease symptoms, faint chlorosis at the site of infection, and reduced bacterial growth. Remarkably, commercial pepper varieties containing the bs5 allele show durable resistance, effectively impeding hypervirulent strain emergence in agricultural fields (Vallejos et al., 2010). The CaBs5 gene, together with its paralog CaBs5-like (CaBs5L), has recently been cloned (Sharma et al., 2023; Szabó et al., 2023). CaBs5 encodes a 92 amino acid long protein possessing a cysteine-rich transmembrane (CYSTM) domain, which is implicated in various biotic and abiotic responses. Typically, the CYSTM domain contains conserved residues composed of four consecutive cysteines, followed by two hydrophobic amino acids. A recent study suggested that Cabs5 mediating the resistance against bacterial spot lacks these two conserved leucine residues within the CYSTM domain (Szabó et al., 2023). Tomatoes and peppers are close relatives in the Solanaceae family and commonly susceptible to Xanthomonas infection. Based on the current findings in pepper, we hypothesized that modifying the ortholog of CaBs5 in tomato could confer resistance against Xanthomonas. Consequently, putative Bs5 (SlBs5) and Bs5L (SlBs5L) were identified in tomato based on homology to CaBs5. Both SlBs5 and SlBs5L were located on chromosome 9 with the same head-to-head orientation as their pepper homologues on chromosome 3 (Figure 1a). Despite short and highly similar amino acid sequences of SlBs5 and SlBs5L (Figure 1b), the conserved synteny and gene order in pepper and tomato genomes allowed the assignment of orthology for Bs5 and Bs5L. The mechanism by which the double leucine deletion in Cabs5 leads to resistance against Xanthomonas remains elusive (Figure 1b). Yet, this deletion in the conserved CYSTM domain could potentially impair CaBs5's native functionality (Abell and Mullen, 2011). Following this assumption, we postulated that knocking out SlBs5 would produce similar outcomes to Cabs5. We aimed to disrupt both SlBs5 and SlBs5L to prevent possible functional complementation by SlBs5L, given their greater amino acid sequence similarity compared to CaBs5 and CaBs5L (Figure 1b). We constructed a binary vector for Cas9 and a single-guide RNA (sgRNA) targeting conserved sequences present in both SlBs5 and SlBs5L (Figure 1c). Tomato variety Fla. 8000 was transformed with Agrobacterium. From the progeny of successful transformants, we selected two homozygous lines, Slbs5-1 and Slbs5-2, containing frameshift mutations in both genes (Figure 1c). These mutant lines were self-pollinated or backcrossed to the wild-type parent variety to segregate the T-DNA containing the Cas9-sgRNA cassette. The resistance of the two selected mutant lines was qualitatively evaluated against Xanthomonas perforans GE485 with dip inoculation assays (Figure 1d). At 21 days post-inoculation, the wild-type leaves were covered by black spots indicative of Xanthomonas infection, while both Slbs5-1 and Slbs5-2 retained green leaves with fewer visible symptoms. These phenotypes remained consistent in inoculations of X. perforans 4B and Xanthomonas gardneri 153 (Figure S1). Quantitative evaluation of bacterial growth further supported these findings. At 5 days post-infiltration with a low-density bacterial suspension, Slbs5-1 showed significant decreases in Xanthomonas populations compared to wild-type plants (Figure 1e). Such reductions were consistently observed for Slbs5-2 (Figure S2). However, Slbs5-1 could not significantly hinder Pseudomonas population growth. We additionally examined the growth penalty associated with Slbs5-1 and Slbs5-2 in controlled conditions (Figure 1f). The height of plants was measured at two different time points, but no significant differences were observed between the wild type and the two mutant lines (Figure 1f; Figure S3). This suggested that the resistance to Xanthomonas species comes at no developmental cost in the vegetative stage in the laboratory setting. Although Cabs5-mediated immunity is subtle, it has shown practical value in commercial pepper cultivation. To examine the commercial potential of Slbs5, field trials were conducted with both Slbs5-1 and Slbs5-2 lines at the Gulf Coast Research and Education Center in Florida, a major state for tomato production. Along with naturally occurring Xanthomonas populations, a two-isolate cocktail of X. perforans race T4 was inoculated in the field to heighten disease pressure. Plants were grown with recommended fertilizers and pest management programs, excluding the use of any bactericides or activators of systemic acquired resistance. Despite seasonal variations, Slbs5 mutant lines consistently maintained reduced disease symptoms (Figure 1g). Additionally, no developmental defects, such as stunting, were observed in these mutants (Figure 1h). Quantification of disease severity, based on visible symptoms caused by Xanthomonas infection on plant leaf surfaces, revealed higher percentages of Slbs5-2 leaves with reduced disease symptoms than wild-type leaves in all tested seasons (Figure 1i; Figure S4). Notably, the Slbs5-2 mutants demonstrated effective resistance during three periods of elevated disease pressure, Spring 2018, Fall 2019, and Fall 2023. The marketable yield of fruits is a critical consideration in tomato cultivation. We quantified total marketable yield across five seasonal trials, except for two seasons impacted by a hurricane (Fall 2022) and extremely dry weather (Spring 2023). Throughout all seasons, there was no statistically significant difference in marketable fruit yields between Slbs5-2 and the wild-type plants (Figure 1j; Figure S5). However, during the three periods of increased disease prevalence in Spring 2018, Fall 2019, and Fall 2023 (Figure 1i), the mutants consistently showed a tendency to produce a greater quantity of marketable tomatoes (Figure 1j). This possibly suggests a correlation between Xanthomonas resistance of the mutant lines and improved fruit yields. Overall, this study shows that a knockout of SlbBs5 and SlBs5L in tomatoes represents a promising strategy to achieve broad-spectrum resistance to bacterial spot disease. Compared to stronger sources of resistance, the resistance mediated by Slbs5 and Slbs5L may be considered subtle. However, our mutant lines consistently led to a reduced population of Xanthomonas in laboratory and field conditions. This decrease in pathogen populations could lessen the likelihood of hypervirulent strain emergence. Furthermore, when these mutants are combined with other sources of downstream resistance genes, they may serve as a prior layer of defence. This initial protection has the potential to diminish the probability of pathogen effectors directly interacting with and overcoming the resistance genes, possibly extending the efficacy of durable resistance in the agricultural field. This research was funded by the Innovative Genomics Institute Founders Fund and the 2Blades Foundation. A.O. and B.J.S. conceptualized the project. B.J.S. supervised the project. A.O., D.D. and B.J.S. designed the experiments and helped analyze the data. K.S. and E.S performed bioinformatics analyses. K.S. led statistical analyses and designed the figures. A.S. helped plan the project, designed and tested the guide RNAs and did preliminary genotyping and bacterial disease assays. A.O. did further genotyping, guide RNA testing and conducted disease and phenotype assays of progeny. D.P.T.T., J.V.W., J.B.J, G.M, E.S.O and D.D. performed supplemental bacterial growth assays. E.S. and S.H. conducted field trials. M.J.C. supervised the generation of tomato mutant lines. E.Z. and J.P. conducted tomato transformations. A.O., K.S. and D.P.T.T. analyzed the data and wrote the manuscript. The data that support the findings of this study are available on request from the corresponding author. Appendix S1 Materials and Methods. Figure S1 Qualitative evaluation of disease symptoms with a dip inoculation assay. Figure S2 Quantitative evaluation of bacterial growth after inoculation. Figure S3 Height comparison between wild type and mutant plants. Figure S4 The disease symptoms on wild type and Slbs5-1 plant leaves in the field trials. Figure S5 Fruit yields of wild type and Slbs5-1 plants in the Fall 2023 field trial. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

In flowering plants, rapid activation of the zygotic genome occurs after fertilization1-3, but there is limited knowledge of the molecular pathways underlying embryo initiation4. In rice, a key role is played by the transcription factor BABY BOOM 1 (OsBBM1), initially expressed from the paternal genome1. Ectopic OsBBM1 expression in the egg cell can override the fertilization requirement, giving rise to parthenogenetic progeny5. Here we show that the WOX-family transcription factor DWARF TILLER1 (OsDWT1)/WUSCHEL-LIKE HOMEODOMAIN 9 (OsWOX9A)6, another gene paternally expressed in zygotes, is a strong enhancer of embryo initiation by OsBBM1. Co-expression of OsWOX9A and OsBBM1 in egg cells results in 86-91% parthenogenesis, representing 4- to 15-fold increases over OsBBM1 alone. These results suggest that embryo initiation is promoted by the synergistic action of paternal-genome-expressed transcription factors in the fertilized egg cell. These findings can be utilized for the efficient production of haploids, as well as clonal hybrid seeds in crop plants7,8. The efficient induction of embryos without fertilization, which is important for crop breeding and hybrid seed production, can be achieved by combined expression in the egg of BBM1 and WOX9A, two rice transcription factors normally expressed from the male genome.

Reactive oxygen species (ROS) accumulation is required for effective plant defense. Accumulation of the Arabidopsis NADPH oxidase RBOHD is regulated by phosphorylation of a conserved C-terminal residue (T912) leading to ubiquitination by the RING E3 ligase PIRE. Arabidopsis PIRE knockouts exhibit enhanced ROS production and resistance to the foliar pathogen Pseudomonas syringae. Here, we identified 170 PIRE homologs, which emerged in Tracheophytes and expanded in Angiosperms. We investigated the role of Solanum lycopersicum (tomato) PIRE homologs in regulating ROS production, RBOH stability, and disease resistance. Mutational analyses of residues corresponding to T912 in the tomato RBOHD ortholog, SlRBOHB, affected protein accumulation and ROS production in a PIRE-dependent manner. Using CRISPR-cas9, we generated mutants in two S. lycopersicum PIRE homologs (SlPIRE). SlPIRE1 edited lines (Slpire1) in the tomato cultivar M82 displayed enhanced ROS production upon treatment with flg22, an immunogenic epitope of flagellin. Furthermore, Slpire1 exhibited decreased disease symptoms and bacterial accumulation when inoculated with foliar bacterial pathogens Pseudomonas syringae and Xanthomonas campestris. However, Slpire1 exhibited similar levels of colonization as wild type upon inoculation with diverse soilborne pathogens. These results indicate that phosphorylation and ubiquitination crosstalk regulate RBOHs in multiple plant species, and PIRE is a promising target for foliar disease control. This study also highlights the pathogen-specific role of PIRE, indicating its potential for targeted manipulation to enhance foliar disease resistance without affecting root-associated interactions, positioning PIRE as a promising target for improving overall plant health.

The advancement of precision engineering for crop trait improvement is important in the face of rapid population growth, climate change, and disease. To this end, targeted double-stranded break technology using RNA-guided Cas9 has been adopted widely for genome editing in plants. Agrobacterium or particle bombardment-based delivery of plasmids encoding Cas9 and guide RNA (gRNA) is common, but requires optimization of expression and often results in random integration of plasmid DNA into the plant genome. Recent advances have described gene editing by the delivery of Cas9 and gRNA as pre-assembled ribonucleoproteins (RNPs) into various plant tissues, but with moderate efficiency in resulting regenerated plants. In this report we describe significant improvements to Cas9-RNP mediated gene editing in wheat. We demonstrate that Cas9-RNP assays in protoplasts are a fast and effective tool for rational selection of optimal gRNAs for gene editing in regenerable immature embryos (IEs), and that high temperature treatment enhances gene editing rates in both tissue types. We also show that Cas9-mediated editing persists for at least 14 days in gold particle bombarded wheat IEs. The regenerated edited wheat plants in this work are recovered at high rates in the absence of exogenous DNA and selection. With this method, we produce knockouts of a set of three homoeologous genes and two pathogenic effector susceptibility genes, engineering insensitivity to corresponding necrotrophic effectors produced by Parastagonospora nodorum. The establishment of highly efficient, exogenous DNA-free gene editing technology holds promise for accelerated trait diversity production in an expansive array of crops.

The maize BABY BOOM 1 gene, when ectopically expressed in egg cells, induces parthenogenetic haploid progeny at high frequency, suggesting a promising route for producing clonal hybrid seeds in maize.

Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.

Cis-regulatory element editing can generate quantitative trait variation while mitigating against extreme phenotypes and harmful pleiotropy associated with coding sequence mutations. Here, we applied a multiplexed guide RNA design approach, informed by bioinformatic datasets, to generate genotypic variation in the promoter of OsSTOMAGEN, a positive regulator of stomatal density in rice. Engineered genotypic variation corresponded to broad and continuous variation in stomatal density, ranging from 70% to 120% of wild-type stomatal density. This near-isogenic panel of stomatal variants was leveraged in physiological assays to establish discrete relationships between stomatal morphological variation and stomatal conductance, carbon assimilation, and intrinsic water use efficiency in steady-state and fluctuating light conditions. Additionally, promoter alleles were subjected to vegetative drought regimes to assay the effects of the edited alleles on developmental response to drought. Notably, the capacity for drought-responsive stomatal density reprogramming in stomagen and two cis-regulatory edited alleles was reduced. Collectively our data demonstrate that cis-regulatory element editing can generate near-isogenic trait variation that can be leveraged for establishing relationships between anatomy, physiology, and crop improvement along diverse environmental clines.### Competing Interest StatementThe authors have declared no competing interest.

Cassava (Manihot esculenta) is a globally important staple crop, particularly in sub-Saharan Africa. Its widespread consumption may be attributed to its hardiness. Relative to other staple crops such as maize, wheat and rice, cassava produces more energy per unit area in periods of drought and in marginal soils (Amelework et al., 2021). Furthermore, cassava roots can be stored below ground for extended periods of time prior to harvest, enabling greater management flexibility for producers (Amelework et al., 2021). Thus, cassava cultivation can safeguard against food insecurity, especially as climate change imposes severe threats to agricultural productivity. Despite its global importance, cassava accumulates human-toxic metabolites in the form of cyanogenic glucosides (CGs), chemical precursors to cyanide, which must be removed prior to safe human consumption (Ernesto et al., 2002). During periods of environmental or sociopolitical stress, the risk of improper cassava processing increases (Ernesto et al., 2002). Chronic cyanide exposure as a result of insufficient processing can result in damage to the central nervous system and, in severe cases, paralysis (Ernesto et al., 2002). To mitigate the human health impacts of CGs, researchers have developed strategies to attenuate their accumulation in cassava. CG levels have been successfully reduced by gene editing to knockout CG biosynthesis (Gomez et al., 2023; Juma et al., 2022). Some evidence indicates that acyanogenic varieties generated through gene editing suffered from greater herbivory, which may have implications for yield (Juma et al., 2022). To substantiate the relationship between CGs and yield, we analysed publicly available data from CassavaBase (Fernandez-Pozo et al., 2015) (Data S1). Data aggregated from Nigerian research trials show an association of greater CG levels with increased fresh storage root weight (Figure 1a, Figure S1). This trend remained consistent when the data were aggregated by two individual field sites in Nigeria (Figure S1). Previous literature has suggested that CGs are synthesized in the shoot apex and transported to roots (Jørgensen et al., 2005). Interruption of this transport could lower levels of CGs in the roots. However, evidence of de novo biosynthesis of CGs in roots of cassava raises questions regarding the extent to which basipetal transport or de novo biosynthesis supplies cassava roots with CGs (Du et al., 1995; Jørgensen et al., 2005). Jørgensen et al. validated a putative, high-affinity transporter of CGs from the cassava genome through heterologous expression in Xenopus laevis oocytes. Only a single queried transcript, hereafter referred to as MeCGTR1, was found to have transport capacity for linamarin, the most abundant CG in cassava (Jørgensen et al., 2017). The expression profile of MeCGTR1 is consistent with its role as a transporter (Figure S2) (Wilson et al., 2017). These findings provided a discrete target for gene editing to affect cassava CG transport. Leveraging the ability to make CRISPR-Cas9-mediated edits in cassava, we generated knockouts of MeCGTR1 (Figure 1b). Two unique events, each resulting in an early stop codon, were produced (Figure 1b). Cyanide levels of wildtype (WT) and cgtr1 events were measured in roots, stems, top leaves and bottom leaves using a picrate assay (Gomez et al., 2023). Lower levels of CGs were detected in the top leaves and stems of cgtr1. No difference was found between genotypes for roots or bottom leaves (Figure 1c–g). Bottom leaves had the lowest overall CG levels, followed by roots, stems and top leaves in WT (Figure S3). The presence of CGs in stems is consistent with their reported phloematic transport. The very low detected levels of CGs in stems of knockout events therefore provide evidence for the function of MeCGTR1 as a systemic transporter of CGs (Figure 1f). CGs detected in stems of cgtr1 events may be a result of the activity of an alternative transporter. A putative paralog of MeCGTR1, Manes.17G021100, is a probable candidate. cgtr1 events would hypothetically have lower root CGs if CGs are transported basipetally. However, no difference existed in cyanide levels between the roots of WT and cgtr1. Notably, there was a reduction of cyanide in the top leaves of cgtr1 (Figure 1d). The findings of the picrate assay suggest that cgtr1 is indeed a systemic transporter of CGs in cassava, and begins to suggest an acropetal mode of CG flow as an alternative to previously established evidence of exclusively basipetal movement. A root-upwards mode of CG transport was further substantiated by a publicly available expression database captured from 3-month-old cassava plants (Wilson et al., 2017). Expression of CYP79D1 and CYP79D2, genes that encode CG biosynthesis enzymes, were found to be highest in fibrous roots by many fold relative to shoot tissues (Figure S2) (Wilson et al., 2017). We undertook a phloem girdling approach to provide further resolution of the directionality of CG movement. Incisions to the phloem to prevent movement of CGs were made, and measurements of CGs above and below the incision zone were subsequently taken (Jørgensen et al., 2005). Overall, lower levels of CGs were found in cgtr1 events, consistent with the stem picrate assay (Figure 1h). Comparisons of CG levels below and above the incision point were calculated. In WT plants, ratios were greater than one, indicating an acropetal direction of CG movement (Figure 1i). A higher ratio was observed in WT relative to cgtr1 events, consistent with MeCGTR1's function as a transporter (Figure 1i). Efforts to improve the safety of cassava by editing cyanogenesis genes may be an effective approach in some contexts. Transporter editing as an alternative approach was considered in this work. Tissue-specific metabolite levels have been successfully modulated by transporter engineering in other organisms (Nour-Eldin and Halkier, 2013). Our work extends these findings to cassava and demonstrates the first in vivo validation of a systemic transporter in this crop. Leveraging the newly characterized function of MeCGTR1 as a systemic transporter of CGs, we sought to probe the extent to which de novo biosynthesis of CGs contributes to total CG content in cassava storage roots relative to transport from shoot apex tissues. Our work suggests that the primary source of cassava storage root CGs is root biosynthesis. It is possible that greater production of root CGs is induced by mutations in MeCGTR1, but unlikely, considering the high root expression levels of biosynthesis genes in wild type plants. The reduction of top leaf CGs in edited events suggests a root-to-shoot method of CG movement. Lower CG levels in the top leaves of cgtr1, accumulation of CGs below the incision point of the phloem girdle and high expression of biosynthesis genes in fibrous roots, all indicate a root-upward mode of CG movement. Multiple directions of CG movement are possible (Nour-Eldin and Halkier, 2013). Shifts between basipetal and acropetal movements of CGs may be contingent upon developmental stage, environmental status or other conditions. It is also possible that a basipetal mode of transport may be confined to tissues vicinal to the shoot apex. It is known that CG levels in cassava storage roots increase during drought stress (Ernesto et al., 2002). Further investigation is required to determine the source of elevated CGs in these conditions. In the case that basipetal shuttling is the primary mode of enrichment in this condition, cgtr1 events could prove an effective strategy for limiting storage root CG content. We would like to thank Christina Wistrom and all the greenhouse staff for their excellent plants. We would also like to thank Brian J. Staskawicz for providing us with laboratory space. NGK developed and led the project. GAG and NGK designed guides and prepared plasmids. BKG transformed cassava and maintained in vitro plantlets in the lab of MJC. SAL genotyped transformed plants and phenotyped them alongside NGK with assistance from LL, AGC and JBL. NGK and SAL wrote the manuscript with feedback from JBL. Data S1 Methods. Figure S1 Yield and cyanogenic glucoside levels in Nigerian field trials. Figure S2 Expression levels of MeCGTR1, CYP79D1 and CYP79D1 across cassava tissues. Figure S3 Comparison of cyanide levels among tissues. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.