Summary: CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9‐guide RNA (gRNA) and LbCas12a‐CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium‐mediated transformation. On‐target mutation analysis showed that 90%–100% of the Cas9‐edited T0 plants carried indel mutations and 63%–77% of them were homozygous or biallelic mutants. In contrast, 0%–60% of Cas12a‐edited T0 plants had on‐target mutations. We then conducted CIRCLE‐seq analysis to identify genome‐wide potential off‐target sites for Cas9. A total of 18 and 67 potential off‐targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off‐target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.

We have generated 47 DNA pools and 235 subpools from 21,049 T-DNA insertion lines of rice. DNA pools of 500–1,000 lines were adequate for screening a T-DNA insertion within a 2-kb region. To examine the efficacy of the DNA pools, we selected MADS-box genes, which play an important role in controlling various aspects of plant development. A total of 34 MIKC-type MADS-box genes have now been identified from rice sequence databases. Our PCR screening for T-DNA insertions within 12 MADS-box genes resulted in the identification of five insertions in four different genes. These DNA pools will be valuable when isolating T-DNA insertional mutants in various rice genes. The DNA pool screening service and the mutant seeds are available upon request to genean@postech.ac.kr.

Endophytic fungi represent diverse taxa that inhabit plant hosts without causing disease symptoms. We used endophytic isolates of Fusarium verticillio

The metabolome and transcriptome of the maize-infecting fungi Ustilago maydis and Fusarium verticillioides were analyzed as the two fungi interact. Both fungi were grown for 7 days in liquid medium alone or together in order to study how this interaction changes their metabolomic and transcriptomic profiles. When grown together, decreased biomass accumulation occurs for both fungi after an initial acceleration of growth compared to the biomass changes that occur when grown alone. The biomass of U. maydis declined most severely over time and may be attributed to the action of F. verticillioides , which secretes toxic secondary metabolites and expresses genes encoding adhesive and cell wall-degrading proteins at higher levels than when grown alone. U. maydis responds to cocultivation by expressing siderophore biosynthetic genes and more highly expresses genes potentially involved in toxin biosynthesis. Also, higher expression was noted for clustered genes encoding secreted proteins that are unique to U. maydis and that may play a role during colonization of maize. Conversely, decreased gene expression was seen for U. maydis genes encoding the synthesis of ustilagic acid, mannosylerythritol D, and another uncharacterized metabolite. Ultimately, U. maydis is unable to react efficiently to the toxic response of F. verticillioides and proportionally loses more biomass. This in vitro study clarifies potential mechanisms of antagonism between these two fungi that also may occur in the soil or in maize, niches for both fungi where they likely interact in nature.

SUMMARY: Plant genetic transformation is essential for understanding gene functions and developing improved crop varieties. Traditional methods, often genotype‐dependent, are limited by plants' recalcitrance to gene delivery and low regeneration capacity. To overcome these limitations, new approaches have emerged that greatly improve efficiency and genotype flexibility. This review summarizes key strategies recently developed for plant transformation, focusing on groundbreaking technologies enhancing explant‐ and genotype flexibility. It covers the use of morphogenic regulators (MRs), stem cell‐based methods, and in planta transformation methods. MRs, such as maize Babyboom (BBM) with Wuschel2 (WUS2), and GROWTH‐REGULATING FACTORs (GRFs) with their cofactors GRF‐interacting factors (GIFs), offer great potential for transforming many monocot species, including major cereal crops. Optimizing BBM/WUS2 expression cassettes has further enabled successful transformation and gene editing using seedling leaves as starting material. This technology lowers the barriers for academic laboratories to adopt monocot transformation systems. For dicot plants, tissue culture‐free or in planta transformation methods, with or without the use of MRs, are emerging as more genotype‐flexible alternatives to traditional tissue culture‐based transformation systems. Additionally, the discovery of the local wound signal peptide Regeneration Factor 1 (REF1) has been shown to enhance transformation efficiency by activating wound‐induced regeneration pathways in both monocot and dicot plants. Future research may combine these advances to develop truly genotype‐independent transformation methods.

Keunsub Lee, Minjeong Kang, Qing Ji, Sehiza Grosic, Kan Wang; New T-DNA binary vectors with NptII selection and RUBY reporter for efficient maize transformation