I-SceI Endonuclease-Mediated Plant Genome Editing by Protein Transport through a Bacterial Type III Secretion System

Abstract

Xanthomonas campestris is one of bacteria carrying a type III secretion system which transports their effector proteins into host plant cells to disturb host defense system for their infection. To establish a genome editing system without introducing any foreign gene, we attempted to introduce genome editing enzymes through the type III secretion system. In a test of protein transfer, X. campestris pv. campestris (Xcc) transported a considerable amount of a reporter protein sGFP-CyaA into tobacco plant cells under the control of the type III secretion system while maintaining cell viability. For proof of concept for genome editing, we used a reporter tobacco plant containing a luciferase (LUC) gene interrupted by a meganuclease I-SceI recognition sequence; this plant exhibits chemiluminescence of LUC only when a frameshift mutation is introduced at the I-SceI recognition site. Luciferase signal was observed in tobacco leaves infected by Xcc carrying an I-SceI gene which secretes I-SceI protein through the type III system, but not leaves infected by Xcc carrying a vector control. Genome-edited tobacco plant could be regenerated from a piece of infected leaf piece by repeated selection of LUC positive calli. Sequence analysis revealed that the regenerated tobacco plant possessed a base deletion in the I-SceI recognition sequence that activated the LUC gene, indicating genome editing by I-SceI protein transferred through the type III secretion system of Xcc.

Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma

“…However, there are limitations on the tissues that can be used, such as shoot meristems, and/or need for pretreatment for introduction of genome editing enzymes. A bacterium carrying a type III secretion system is useful for protein- based genome editing enzymes, such as TALEN [ 11 ]. However, it can transfer only proteins into the cells and have demonstrated certain limitations with respect to its application in host plants.…”

“…Therefore, in most plants, genes encoding specific genome editing enzymes are introduced via genetic transformation, and the introduced genes are removed after genome editing. Recently, several methods have been reported for introduction of proteins into plant cells, such as the use of a cell penetrating peptide (CPP) [ 8 , 9 ], in planta particle bombardment [ 10 ], and protein transportation by a bacterium carrying a type III secretion system [ 11 ]. Although they work for genome editing enzymes, they have limitations with respect to the tissues where they can be used, such as shoot meristems, host plants against bacterial infection, and/or the need for pretreatment for protein introduction [ 10 – 12 ].…”

Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

“…However, the sizes of these nucleases, approximately 165 amino acids (aa) for meganucleases and 300 aa per ZFN monomer (Baltes & Voytas, 2015), allow for delivery via vector systems that require shorter coding sequences, including viruses (Marton et al., 2010). Meganuclease‐ and ZFN‐mediated gene editing have been reported both for proof‐of‐concept and trait development in tobacco (Baltes et al., 2014; Chujo et al., 2017; Petolino et al., 2010; Townsend et al., 2009; Yanagawa et al., 2020) and wheat (Bilichak et al., 2020; Cigan et al., 2017). TALENs are the most specific of all the genome‐editing tools in terms of site recognition (Mussolino et al., 2014), but their assembly for targeted edits is far more time consuming compared to CRISPR/Cas, and their large size (∼950 aa per monomer) limits multiplexed gene editing (Baltes & Voytas, 2015).…”

Plant genome editing: ever more precise and wide reaching

“…Besides Agrobacterium , SSN protein can be introduced via other types of secretion systems (Schmitz et al, 2020). Most recently, the I‐ Sce I meganuclease was reported to be introduced via the type III secretion system of Xanthomonas campestris (Yanagawa et al, 2020), suggesting that various kinds of bacteria have the potential as “carriers” to deliver SSN proteins into plant cells.…”

“…The single-stranded T-DNA is converted into double-stranded T-DNA in the plant nucleus; thus, genes located on the double-stranded T-DNA can be expressed without integration of T-DNA into the plant genome (for a review, see Gelvin 2017). Transient expression of T-DNA can be applied to targeted mutagenesis in tobacco and potato (Chen et al, 2018;Yasumoto et al, 2020).…”