Application of CRISPR-Based C-to-G Base editing in rice protoplasts

Recently, new types of base editors, C-to-G base editors (CGBEs), that enable cytosine transversions that are unachievable with cytosine base editors (CBEs) and adenosine base editors (ABEs), have been developed in human cells. However, despite their importance in crop genome editing, the efficacy of CGBEs has not yet been extensively evaluated. In our study, based on the previously reported plant-compatible CBE and human CGBE, we demonstrated that our monocot plant-compatible CGBEs (PcCGBEs) enable cytosine transversions (C-to-G) in rice protoplasts. For all targets tested, PcCGBEs (monocot plant-compatible CGBEs) appeared to have substantial levels of C-to-G editing activity. PcCGBE showed a much higher C-to-G base editing activity and C-to-G specificity among C-to-D conversions than the mini-version of PcCGBE. Our demonstration of PcCGBE could provide a platform for the further development of enhanced CGBEs for reliable application as a new crop breeding technology.This work was supported by the Creative-Pioneering Researchers Program of Seoul National University, National Research Foundation of Korea (NRF) Grant (2019R1F1A1046305), and New Breeding Technologies Development Program (PJ016542) through the Rural Development Administration (RDA), Republic of Korea

Tomato ARPC1 regulates trichome morphology and density and terpene biosynthesis

Main conclusion Based on transcriptomic analysis of wild-type and mutant tomato plants, ARPC1 was found to be important for trichome formation and development and it plays a key role in terpene synthesis. Trichomes are protruding epidermal cells in plant species. They function as the first defense layer against biotic and abiotic stresses. Despite the essential role of tomato trichomes in defense against herbivores, the understanding of their development is still incomplete. Therefore, the aim of this study was to identify genes involved in trichome formation and morphology and terpene synthesis, using transcriptomic techniques. To achieve this, we examined leaf morphology and compared the expression levels of some putative genes involved in trichome formation between wild-type (WT) and hairless-3 (hl-3) tomato mutant. The hl-3 plants displayed swollen and distorted trichomes and reduced trichome density (type I and IV) and terpene synthesis compared with that of the WT plants. Gene expression analysis showed that Actin-Related Protein Component1 (ARPC1) was expressed more highly in the WT than in the hl-3 mutant, indicating its critical role in trichome morphology and density. Additionally, the expression of MYC1 and several terpene synthase genes (TPS9, 12, 20), which are involved in type VI trichome initiation and terpene synthesis, was lower in the hl-3 mutant than in the WT plants. Moreover, transformation of the hl-3 mutant with WT ARPC1 restored normal trichome structure and density, and terpene synthesis. Structural and amino acid sequence analysis showed that there was a missplicing mutation in the hl-3 mutant, which was responsible for the abnormal trichome structure and density, and impaired terpene synthesis. Overall, the findings of this study demonstrated that ARPC1 is involved in regulating trichome structure and terpene synthesis in tomato.N

Molecular insights into plant cell proliferation disturbance by Agrobacterium protein 6b

The Agrobacterium Ti plasmid (T-DNA) 6b proteins interact with many different host proteins implicated in plant cell proliferation. Here, we show that Arabidopsis plants overexpressing 6b display microRNA (miRNA) deficiency by directly targeting SERRATE and AGO1 via a specific loop fragment (residues 40–55). In addition, we report the crystal structures of Agrobacterium tumefaciens AK6b at 2.1 Å, Agrobacterium vitis AB6b at 1.65 Å, and Arabidopsis ADP ribosylation factor (ARF) at 1.8 Å. The 6b structure adopts an ADP-ribosylating toxin fold closely related to cholera toxin. In vitro ADP ribosylation analysis demonstrates that 6b represents a new toxin family, with Tyr 66, Thr 93, and Tyr 153 as the ADP ribosylation catalytic residues in the presence of Arabidopsis ARF and GTP. Our work provides molecular insights, suggesting that 6b regulates plant cell growth by the disturbance of the miRNA pathway through its ADP ribosylation activity

Data from: Genome-wide analysis uncovers regulation of long intergenic noncoding RNAs in Arabidopsis

Long intergenic noncoding RNAs (lincRNAs) transcribed from intergenic regions of yeast and animal genomes play important roles in key biological processes. Yet, plant lincRNAs remain poorly characterized and how lincRNA biogenesis is regulated is unclear. Using a reproducibility-based bioinformatics strategy to analyze 200 Arabidopsis transcriptome datasets, we identified 13,230 intergenic transcripts of which 6,480 can be classified as lincRNAs. Expression of 2,708 lincRNAs was detected by RNA-seq experiments. Transcriptome profiling by custom microarrays revealed that the majority of these lincRNAs are expressed at a level between those of mRNAs and pri-miRNAs. A subset of lincRNA genes show organ-specific expression whereas others are responsive to biotic and/or abiotic stresses. Further analysis of transcriptome data in 11 mutants uncovered SERRATE, CBP20, and CBP80 as regulators of lincRNA expression and biogenesis. RT-PCR experiments confirmed these 3 proteins are also needed for splicing of a small group of intron-containing lincRNAs