Search

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.

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., Author summary The post-translational addition of SUMO to other proteins by the MMS21 SUMO ligase has been implicated in a plethora of biological processes in plants but the identit(ies) of its targets and the biological consequences of their modification remain poorly resolved. Here, we address this issue by characterizing a collection of maize mms21 mutants using genetic, biochemical, transcriptomic and proteomic approaches. Our results revealed that mms21 mutations substantially compromise pollen germination and seed/vegetative development, dysregulate the maize transcriptome, including the ectopic expression of seed storage protein mRNAs in leaves, increase DNA damage, and alter the proteome/transcriptome balance. Interaction studies showed that MMS21 associates in the nucleus with the NON-SMC-ELEMENT (NSE)-4 and STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC)-5 components of the chromatin organizer SMC5/6 complex responsible for DNA-damage repair and chromatin accessibility. Our data demonstrate that MMS21 is crucial for plant development likely through its maintenance of DNA repair, balanced transcription, and genome stability.

Summary 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.

SummarySWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by transcription activator-like effectors (TALe) of Xanthomonas ssp. is a key factor for bacterial leaf blight (BLB) infection of rice, cassava and cotton. Here, we identified the so far unknown OsSWEET11b with roles in male fertility and BLB susceptibility in rice. While single ossweet11a or b mutants were fertile, double mutants were sterile. Since clade III SWEETs can transport gibberellin (GA), a key hormone for rice spikelet fertility, sterility and BLB susceptibility might be explained by GA transport deficiencies. However, in contrast to the Arabidopsis homologs, OsSWEET11b did not mediate detectable GA transport. Fertility and susceptibility must therefore depend on SWEET11b-mediated sucrose transport. Ectopic induction of OsSWEET11b by designer TALe enables TALe-free Xanthomonas oryzae pv. oryzae (Xoo) to cause disease, identifying OsSWEET11b as a BLB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence of Xoo. Notably, only three of now six clade III SWEETs are targeted by known Xoo strains from Asia and Africa. The identification of OsSWEET11b has relevance in the context of fertility and for protecting rice against emerging Xoo strains that evolve TALes to exploit OsSWEET11b.

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.

Specialized metabolites constitute key layers of immunity underlying crop resistance; however, challenges in resolving complex pathways limit our understanding of their functions and applications. In maize (Zea mays) the inducible accumulation of acidic terpenoids is increasingly considered as a defense regulating disease resistance. To understand maize antibiotic biosynthesis, we integrated association mapping, pan-genome multi-omic correlations, enzyme structure-function studies, and targeted mutagenesis. We now define ten genes in three zealexin (Zx) gene clusters comprised of four sesquiterpene synthases and six cytochrome P450s that collectively drive the production of diverse antibiotic cocktails. Quadruple mutants blocked in the production of β-macrocarpene 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 utilizing diverse substrates and in vivo combinatorial chemistry to yield complex antibiotic blends. The elucidated genetic basis of biochemical phenotypes underlying disease resistance demonstrates a predominant maize defense pathway and informs innovative strategies for transferring chemical immunity between crops.

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 β subunit gene (Gβ) 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 Gβ phenotype and also identified a maize Gβ allele with weak autoimmune responses but strong development phenotypes. Using these tools, we show that Gβ controls meristem size in maize, acting epistatically with G protein α subunit gene (Gα), suggesting that Gβ and Gα function in a common signaling complex. Furthermore, we used an association study to show that natural variation in Gβ influences maize kernel row number, an important agronomic trait. Our results demonstrate the dual role of Gβ in immunity and development in a cereal crop and suggest that it functions in cross-talk between these competing signaling networks. Therefore, modification of Gβ has the potential to optimize the trade-off between growth and defense signaling to improve agronomic production.

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., Strategic deployment of blight-resistant rice lines is enabled by a molecular diagnostic kit.

Recently, the engineered CRISPR/Cas9 system has been applied to rapidly and efficiently modify the targeted gene(s) in a wide variety of plants. Recent studies of successful targeted mutagenesis using the CRISPR/Cas9 system with a single gRNA expression in rice plants have been reported. GA20ox-2 is a gene encoding an oxidase enzyme involved in the biosynthesis of gibberellin and linked to sd1 locus. A previous study revealed that mutation of this gene resulted in shorter stature of rice plant due to defects in the gibberellin’s signalling pathway. Here, we studied targeted mutation of OsGA20ox-2 gene in rice using the CRISPR/Cas9 system with the expression of two gRNAs. In this study, we introduced a single plasmid vector of CRISPR/Cas9 system harboring dual gRNAs to modify OsGA20ox-2 gene in a rice model cv. Kitaake via Agrobacterium-mediated transformation. Targeted mutagenesis of OsGA20ox-2 gene using CRISPR/Cas9 generated nine mutated rice lines with a mutation frequency of 90%. Most mutated lines (50%) had mutations in both OsGA20ox-2 gRNA. They resulted in homo-diallelic mutation type with 44 bp deletion, while three lines were heterozygous, one line was homo-diallelic with 2 bp insertion, and one line had no mutation. The K15 mutated rice line was identified as a homozygous two-nucleotide insertion and had the semi-dwarf phenotype, demonstrating that OsGA20ox-2 gene had been disrupted.

Bacterial blight of rice, caused by the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) in rice, represents one of the most well-studied crop diseases and is also well-known as a model for studying host/microbe interaction. TALEs (transcription activator-like effectors), as a group of pathogenesis factors and once translocated into the host cells from pathogen, recognize and activate host genes to condition disease susceptibility and also trigger host resistance responses dependent on the nature of target genes in plants. TALEs and their target genes have become the foci of the molecular battles between Xoo and rice. The continuing battles have led to incredibly diverse virulence mechanisms in pathogen and counteracting defense mechanisms in rice. Extensive efforts have been made to understand the TALE biology, identify host target genes, and elucidate their interaction and resulting physiological relevance to rice blight and other crop diseases. This review aims to summarize how much we have learned about TALEs and their role in bacterial blight of rice, as well as associated susceptibility and resistance genes in the host. The review also intends to provide a prospect of engineering genetic resistance by applying precise genome editing of TALE-associated target genes in rice.

Summary CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease‐based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium‐delivered CRISPR/Cas9 for high‐frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4‐reductase or anthocyaninless genes (a1 and a4). T0 transgenic events carrying mono‐ or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi‐II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target‐specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.

Transcription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize glossy2 (gl2) locus. Transgenic lines containing mono- or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (nine mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. In a majority of the events, the integrated TALEN T-DNA segregated independently from the new loss of function alleles, producing mutated null-segregant progeny in T1 generation. Our results demonstrate that TALENs are an effective tool for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement.