Search for: All records

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.

 

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.

 

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 imprinteddosage-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 whended1is transmitted through the male, while homozygous mutants are defective with a 70–90% seed weight reduction.Ded1encodes 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 ofDed1causes the paternal allele to set the pace of endosperm development thereby influencing grain set and size.

 

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. 

SWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by Transcription Activator‐Like effectors (TALe) ofXanthomonasssp. 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 singleossweet11aor11bmutants 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 ofOsSWEET11bby designer TALe enabled TALe‐freeXanthomonas oryzaepv.oryzae(Xoo) to cause disease, identifyingOsSWEET11bas a potential BB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence ofXoo. Notably, only three of six clade III SWEETs are targeted by knownXoostrains from Asia and Africa.

The identification of OsSWEET11b is relevant for fertility and for protecting rice against emergingXoostrains that targetOsSWEET11b.

 

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,mir390mutant showed a severe defect in the shoot apical meristem (SAM), a shootless phenotype, which could be rescued by the wild‐typeMIR390. Small RNA sequencing showed the two base pair deletion inmir390substantially 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 generatedmir160anull mutants with fragment deletions, at a higher efficiency than a single‐guide RNA. The difference between the phenotypic severity ofmiR160amutants 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.