Genome editing of an African elite rice variety confers resistance against endemic and emerging Xanthomonas oryzae pv. oryzae strains.

Bacterial leaf blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), threatens global food security and the livelihood of small-scale rice producers. Analyses of Xoo collections from Asia, Africa and the Americas demonstrated complete continental segregation, despite robust global rice trade. Here, we report unprecedented BB outbreaks in Tanzania. The causative strains, unlike endemic African Xoo, carry Asian-type TAL effectors targeting the sucrose transporter SWEET11a and iTALes suppressing Xa1. Phylogenomics clustered these strains with Xoo from Southern-China. African rice varieties do not carry effective resistance. To protect African rice production against this emerging threat, we developed a hybrid CRISPR-Cas9/Cpf1 system to edit all known TALe-binding elements in three SWEET promoters of the East African elite variety Komboka. The edited lines show broad-spectrum resistance against Asian and African strains of Xoo, including strains recently discovered in Tanzania. The strategy could help to protect global rice crops from BB pandemics.

Efficient protein tagging and cis-regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants.

Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient nonhomologous end-joining-mediated targeted insertion method was recently developed by combining clustered regularly interspaced short palindromic repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated nuclease 9 (SpCas9) gene editing with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet, this approach often leads to imprecise insertions with no control over the insertion direction. Here, we compared the influence of chemical protection of dsODNs on efficiency of targeted insertion. We observed that CRISPR/SpCas9 frequently induced staggered cleavages with 1-nucleotide 5' overhangs; we also evaluated the effect of donor end structures on the direction and precision of targeted insertions. We demonstrate that chemically protected dsODNs with 1-nucleotide 5' overhangs significantly improved the precision and direction control of target insertions in all tested CRISPR targeted sites. We applied this method to endogenous gene tagging in green foxtail (Setaria viridis) and engineering of cis-regulatory elements for disease resistance in rice (Oryza sativa). We directionally inserted 2 distinct transcription activator-like effector binding elements into the promoter region of a recessive rice bacterial blight resistance gene with up to 24.4% efficiency. The resulting rice lines harboring heritable insertions exhibited strong resistance to infection by the pathogen Xanthomonas oryzae pv. oryzae in an inducible and strain-specific manner.

A dolabralexin-deficient mutant provides insight into specialized diterpenoid metabolism in maize.

Two major groups of specialized metabolites in maize (Zea mays), termed kauralexins and dolabralexins, serve as known or predicted diterpenoid defenses against pathogens, herbivores, and other environmental stressors. To consider the physiological roles of the recently discovered dolabralexin pathway, we examined dolabralexin structural diversity, tissue-specificity, and stress-elicited production in a defined biosynthetic pathway mutant. Metabolomics analyses support a larger number of dolabralexin pathway products than previously known. We identified dolabradienol as a previously undetected pathway metabolite and characterized its enzymatic production. Transcript and metabolite profiling showed that dolabralexin biosynthesis and accumulation predominantly occur in primary roots and show quantitative variation across genetically diverse inbred lines. Generation and analysis of CRISPR-Cas9-derived loss-of-function Kaurene Synthase-Like 4 (Zmksl4) mutants demonstrated dolabralexin production deficiency, thus supporting ZmKSL4 as the diterpene synthase responsible for the conversion of geranylgeranyl pyrophosphate precursors into dolabradiene and downstream pathway products. Zmksl4 mutants further display altered root-to-shoot ratios and root architecture in response to water deficit. Collectively, these results demonstrate dolabralexin biosynthesis via ZmKSL4 as a committed pathway node biochemically separating kauralexin and dolabralexin metabolism, and suggest an interactive role of maize dolabralexins in plant vigor during abiotic stress.

Polarly localized WPR proteins interact with PAN receptors and the actin cytoskeleton during maize stomatal development.

Polarization of cells prior to asymmetric cell division is crucial for correct cell divisions, cell fate, and tissue patterning. In maize (Zea mays) stomatal development, the polarization of subsidiary mother cells (SMCs) prior to asymmetric division is controlled by the BRICK (BRK)-PANGLOSS (PAN)-RHO FAMILY GTPASE (ROP) pathway. Two catalytically inactive receptor-like kinases, PAN2 and PAN1, are required for correct division plane positioning. Proteins in the BRK-PAN-ROP pathway are polarized in SMCs, with the polarization of each protein dependent on the previous one. As most of the known proteins in this pathway do not physically interact, possible interactors that might participate in the pathway are yet to be described. We identified WEAK CHLOROPLAST MOVEMENT UNDER BLUE LIGHT 1 (WEB1)/PLASTID MOVEMENT IMPAIRED 2 (PMI2)-RELATED (WPR) proteins as players during SMC polarization in maize. WPRs physically interact with PAN receptors and polarly accumulate in SMCs. The polarized localization of WPR proteins depends on PAN2 but not PAN1. CRISPR-Cas9-induced mutations result in division plane defects in SMCs, and ectopic expression of WPR-RFP results in stomatal defects and alterations to the actin cytoskeleton. We show that certain WPR proteins directly interact with F-actin through their N-terminus. Our data implicate WPR proteins as potentially regulating actin filaments, providing insight into their molecular function. These results demonstrate that WPR proteins are important for cell polarization.

Paternal imprinting of dosage-effect defective1 contributes to seed weight xenia in maize.

Historically, xenia effects were hypothesized to be unique genetic contributions of pollen to seed phenotype, but most examples represent standard complementation of Mendelian traits. We identified the imprinted dosage-effect defective1 (ded1) locus in maize (Zea mays) as a paternal regulator of seed size and development. Hypomorphic alleles show a 5-10% seed weight reduction when ded1 is transmitted through the male, while homozygous mutants are defective with a 70-90% seed weight reduction. Ded1 encodes an R2R3-MYB transcription factor expressed specifically during early endosperm development with paternal allele bias. DED1 directly activates early endosperm genes and endosperm adjacent to scutellum cell layer genes, while directly repressing late grain-fill genes. These results demonstrate xenia as originally defined: Imprinting of Ded1 causes the paternal allele to set the pace of endosperm development thereby influencing grain set and size.

OsSWEET11b, a potential sixth leaf blight susceptibility gene involved in sugar transport-dependent male fertility.

SWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by Transcription Activator-Like effectors (TALe) of Xanthomonas ssp. is key for virulence in rice, cassava and cotton. We identified OsSWEET11b with roles in male fertility and potential bacterial blight (BB) susceptibility in rice. While single ossweet11a or 11b mutants were fertile, double mutants were sterile. As clade III SWEETs can transport gibberellin (GA), a key hormone for spikelet fertility, sterility and BB susceptibility might be explained by GA transport deficiencies. However, in contrast with the Arabidopsis homologues, OsSWEET11b did not mediate detectable GA transport. Fertility and susceptibility therefore are likely to depend on sucrose transport activity. Ectopic induction of OsSWEET11b by designer TALe enabled TALe-free Xanthomonas oryzae pv. oryzae (Xoo) to cause disease, identifying OsSWEET11b as a potential BB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence of Xoo. Notably, only three of six clade III SWEETs are targeted by known Xoo strains from Asia and Africa. The identification of OsSWEET11b is relevant for fertility and for protecting rice against emerging Xoo strains that target OsSWEET11b.

The SUMO ligase MMS21 profoundly influences maize development through its impact on genome activity and stability.

The post-translational addition of SUMO plays essential roles in numerous eukaryotic processes including cell division, transcription, chromatin organization, DNA repair, and stress defense through its selective conjugation to numerous targets. One prominent plant SUMO ligase is METHYL METHANESULFONATE-SENSITIVE (MMS)-21/HIGH-PLOIDY (HPY)-2/NON-SMC-ELEMENT (NSE)-2, which has been connected genetically to development and endoreduplication. Here, we describe the potential functions of MMS21 through a collection of UniformMu and CRISPR/Cas9 mutants in maize (Zea mays) that display either seed lethality or substantially compromised pollen germination and seed/vegetative development. RNA-seq analyses of leaves, embryos, and endosperm from mms21 plants revealed a substantial dysregulation of the maize transcriptome, including the ectopic expression of seed storage protein mRNAs in leaves and altered accumulation of mRNAs associated with DNA repair and chromatin dynamics. Interaction studies demonstrated that MMS21 associates in the nucleus with the NSE4 and STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC)-5 components of the chromatin organizer SMC5/6 complex, with in vitro assays confirming that MMS21 will SUMOylate SMC5. Comet assays measuring genome integrity, sensitivity to DNA-damaging agents, and protein versus mRNA abundance comparisons implicated MMS21 in chromatin stability and transcriptional controls on proteome balance. Taken together, we propose that MMS21-directed SUMOylation of the SMC5/6 complex and other targets enables proper gene expression by influencing chromatin structure.

Single-cell RNA sequencing of developing maize ears facilitates functional analysis and trait candidate gene discovery.

Crop productivity depends on activity of meristems that produce optimized plant architectures, including that of the maize ear. A comprehensive understanding of development requires insight into the full diversity of cell types and developmental domains and the gene networks required to specify them. Until now, these were identified primarily by morphology and insights from classical genetics, which are limited by genetic redundancy and pleiotropy. Here, we investigated the transcriptional profiles of 12,525 single cells from developing maize ears. The resulting developmental atlas provides a single-cell RNA sequencing (scRNA-seq) map of an inflorescence. We validated our results by mRNA in situ hybridization and by fluorescence-activated cell sorting (FACS) RNA-seq, and we show how these data may facilitate genetic studies by predicting genetic redundancy, integrating transcriptional networks, and identifying candidate genes associated with crop yield traits.

Differential activities of maize plant elicitor peptides as mediators of immune signaling and herbivore resistance.

Plant elicitor peptides (Peps) are conserved regulators of defense responses and models for the study of damage-associated molecular pattern-induced immunity. Although present as multigene families in most species, the functional relevance of these multigene families remains largely undefined. While Arabidopsis Peps appear largely redundant in function, previous work examining Pep-induced responses in maize (Zm) implied specificity of function. To better define the function of individual ZmPeps and their cognate receptors (ZmPEPRs), activities were examined by assessing changes in defense-associated phytohormones, specialized metabolites and global gene expression patterns, in combination with heterologous expression assays and analyses of CRISPR/Cas9-generated knockout plants. Beyond simply delineating individual ZmPep and ZmPEPR activities, these experiments led to a number of new insights into Pep signaling mechanisms. ZmPROPEP and other poaceous precursors were found to contain multiple active Peps, a phenomenon not previously observed for this family. In all, seven new ZmPeps were identified and the peptides were found to have specific activities defined by the relative magnitude of their response output rather than by uniqueness. A striking correlation was observed between individual ZmPep-elicited changes in levels of jasmonic acid and ethylene and the magnitude of induced defense responses, indicating that ZmPeps may collectively regulate immune output through rheostat-like tuning of phytohormone levels. Peptide structure-function studies and ligand-receptor modeling revealed structural features critical to the function of ZmPeps and led to the identification of ZmPep5a as a potential antagonist peptide able to competitively inhibit the activity of other ZmPeps, a regulatory mechanism not previously observed for this family.

Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity.

Specialized metabolites constitute key layers of immunity that underlie disease resistance in crops; however, challenges in resolving pathways limit our understanding of the functions and applications of these metabolites. In maize (Zea mays), the inducible accumulation of acidic terpenoids is increasingly considered to be a defence mechanism that contributes to disease resistance. Here, to understand maize antibiotic biosynthesis, we integrated association mapping, pan-genome multi-omic correlations, enzyme structure-function studies and targeted mutagenesis. We define ten genes in three zealexin (Zx) gene clusters that encode four sesquiterpene synthases and six cytochrome P450 proteins that collectively drive the production of diverse antibiotic cocktails. Quadruple mutants in which the ability to produce zealexins (ZXs) is blocked exhibit a broad-spectrum loss of disease resistance. Genetic redundancies ensuring pathway resiliency to single null mutations are combined with enzyme substrate promiscuity, creating a biosynthetic hourglass pathway that uses diverse substrates and in vivo combinatorial chemistry to yield complex antibiotic blends. The elucidated genetic basis of biochemical phenotypes that underlie disease resistance demonstrates a predominant maize defence pathway and informs innovative strategies for transferring chemical immunity between crops.

Use of CRISPR/Cas9 for Targeted Mutagenesis in Sorghum.

Sorghum (Sorghum bicolor) fulfills the demand for bioenergy resources and also provides substantial diet calories to the world's population. Therefore, many biological studies use sorghum as a research model for improvement of the domesticated food and bioenergy crops. Furthermore, leveraging genome editing systems in a plethora of grass plant species has been extensively studied with no exception in sorghum. However, a protocol that details the genome editing strategies using CRISPR/Cas9 and that combines an efficient tissue culture and transformation platform in sorghum based on Agrobacterium-mediated DNA transfer has yet to be reported. This protocol outlines the steps and workflow from design of sorghum CRISPR target sites using BTx623 as a reference genome, construction of sorghum CRISPR/Cas9 plasmids, tissue culture, to Agrobacterium-mediated transformation followed by genotyping of CRISPR/Cas9 induced mutants. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Construction of CRISPR/Cas9 expression vector to analysis of CRISPR-edited plants Basic Protocol 2: Stable transformation of sorghum Support Protocol: Management of sorghum plants in a greenhouse.

Genome editing in grass plants.

Cereal crops including maize, rice, wheat, sorghum, barley, millet, oats and rye are the major calorie sources in our daily life and also important bioenergy sources of the world. The rapidly advancing and state-of-the-art genome-editing tools such as zinc finger nucleases, TAL effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (CRISPR-Cas9-, CRISPR-Cas12a- and CRISPR/Cas-derived base editors) have accelerated the functional genomics and have promising potential for precision breeding of grass crops. With the availability of annotated genomes of the major cereal crops, application of these established genome-editing toolkits to grass plants holds promise to increase the nutritional value and productivity. Furthermore, these easy-to-use and robust genome-editing toolkits have advanced the reverse genetics for discovery of novel gene functions in crop plants. In this review, we document some of important progress in development and utilization of genome-editing tool sets in grass plants. We also highlight present and future uses of genome-editing toolkits that can sustain and improve the quality of cereal grain for food consumption.

Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production.

MicroRNAs (miRNAs) are 20-24 nucleotides (nt) small RNAs functioning in eukaryotes. The length and sequence of miRNAs are not only related to the biogenesis of miRNAs but are also important for downstream physiological processes like ta-siRNA production. To investigate these roles, it is informative to create small mutations within mature miRNA sequences. We used both TALENs (transcription activator-like effector nucleases) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to introduce heritable base pair mutations in mature miRNA sequences. For rice, TALEN constructs were built targeting five different mature miRNA sequences and yielding heritable mutations. Among the resulting mutants, mir390 mutant showed a severe defect in the shoot apical meristem (SAM), a shootless phenotype, which could be rescued by the wild-type MIR390. Small RNA sequencing showed the two base pair deletion in mir390 substantially interfered with miR390 biogenesis. In Arabidopsis, CRISPR/Cas9-mediated editing of the miR160* strand confirmed that the asymmetric structure of miRNA is not a necessary determinant for secondary siRNA production. CRISPR/Cas9 with double-guide RNAs successfully generated mir160a null mutants with fragment deletions, at a higher efficiency than a single-guide RNA. The difference between the phenotypic severity of miR160a mutants in Col-0 versus Ler backgrounds highlights a diverged role for miR160a in different ecotypes. Overall, we demonstrated that TALENs and CRISPR/Cas9 are both effective in modifying miRNA precursor structure, disrupting miRNA processing and generating miRNA null mutant plants.

The maize heterotrimeric G protein ß subunit controls shoot meristem development and immune responses.

Heterotrimeric G proteins are important transducers of receptor signaling, functioning in plants with CLAVATA receptors in controlling shoot meristem size and with pathogen-associated molecular pattern receptors in basal immunity. However, whether specific members of the heterotrimeric complex potentiate cross-talk between development and defense, and the extent to which these functions are conserved across species, have not yet been addressed. Here we used CRISPR/Cas9 to knock out the maize G protein ß subunit gene (Gß) and found that the mutants are lethal, differing from those in Arabidopsis, in which homologous mutants have normal growth and fertility. We show that lethality is caused not by a specific developmental arrest, but by autoimmunity. We used a genetic diversity screen to suppress the lethal Gß phenotype and also identified a maize Gß allele with weak autoimmune responses but strong development phenotypes. Using these tools, we show that Gß controls meristem size in maize, acting epistatically with G protein α subunit gene (Gα), suggesting that Gß and Gα function in a common signaling complex. Furthermore, we used an association study to show that natural variation in Gß influences maize kernel row number, an important agronomic trait. Our results demonstrate the dual role of Gß in immunity and development in a cereal crop and suggest that it functions in cross-talk between these competing signaling networks. Therefore, modification of Gß has the potential to optimize the trade-off between growth and defense signaling to improve agronomic production.

Diagnostic kit for rice blight resistance.

Blight-resistant rice lines are the most effective solution for bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo). Key resistance mechanisms involve SWEET genes as susceptibility factors. Bacterial transcription activator-like (TAL) effectors bind to effector-binding elements (EBEs) in SWEET gene promoters and induce SWEET genes. EBE variants that cannot be recognized by TAL effectors abrogate induction, causing resistance. Here we describe a diagnostic kit to enable analysis of bacterial blight in the field and identification of suitable resistant lines. Specifically, we include a SWEET promoter database, RT-PCR primers for detecting SWEET induction, engineered reporter rice lines to visualize SWEET protein accumulation and knock-out rice lines to identify virulence mechanisms in bacterial isolates. We also developed CRISPR-Cas9 genome-edited Kitaake rice to evaluate the efficacy of EBE mutations in resistance, software to predict the optimal resistance gene set for a specific geographic region, and two resistant 'mega' rice lines that will empower farmers to plant lines that are most likely to resist rice blight.

Multiple genes recruited from hormone pathways partition maize diterpenoid defences.

Duplication and divergence of primary pathway genes underlie the evolution of plant specialized metabolism; however, mechanisms partitioning parallel hormone and defence pathways are often speculative. For example, the primary pathway intermediate ent-kaurene is essential for gibberellin biosynthesis and is also a proposed precursor for maize antibiotics. By integrating transcriptional coregulation patterns, genome-wide association studies, combinatorial enzyme assays, proteomics and targeted mutant analyses, we show that maize kauralexin biosynthesis proceeds via the positional isomer ent-isokaurene formed by a diterpene synthase pair recruited from gibberellin metabolism. The oxygenation and subsequent desaturation of ent-isokaurene by three promiscuous cytochrome P450s and a new steroid 5α reductase indirectly yields predominant ent-kaurene-associated antibiotics required for Fusarium stalk rot resistance. The divergence and differential expression of pathway branches derived from multiple duplicated hormone-metabolic genes minimizes dysregulation of primary metabolism via the circuitous biosynthesis of ent-kaurene-related antibiotics without the production of growth hormone precursors during defence.

Creating Large Chromosomal Deletions in Rice Using CRISPR/Cas9.

Engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) is an efficient and the most popularly used tool for genome engineering in eukaryotic organisms including plants, especially in crop plants. This system has been effectively used to introduce mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. CRISPR/Cas9 hence presents great value in basic and applied research for improving the performance of crop plants in various aspects such as increasing grain yields, improving nutritional content, and better combating biotic and abiotic stresses. Besides above applications, CRISPR/Cas9 system has been shown to be very effective in creating large chromosomal deletions in plants, which is useful for genetic analysis of chromosomal fragments, functional study of gene clusters in biological processes, and so on. Here, we present a protocol of creating large chromosomal deletions in rice using CRISPR/Cas9 system, including detailed information about single-guide RNA design, vector construction, plant transformation, and large deletion screening processes in rice.

CRISPR/Cas9 for Mutagenesis in Rice.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) provides a workhorse for genome editing biotechnology. CRISPR/Cas9 tailored for enabling genome editing has been extensively interrogated and widely utilized for precise genomic alterations in eukaryotic organisms including in plant species. The technology holds the great promise to better understand gene functions, elucidate networks, and improve the performance of crop plants such as increasing grain yields, improving nutritional content, and better combating the biotic and abiotic stresses. Various methods or protocols specific for different plant species have been established. Here, we present a CRISPR/Cas9-mediated genome editing protocol in rice, including detailed information about single-guide RNA design, vector construction, plant transformation, and mutant screening processes.