A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus

Abstract

The difficulty of genetic transformation has restricted research on functional genomics in cotton. Thus, a rapid and efficient method for gene overexpression that does not rely on genetic transformation is needed. Virus-based vectors offer a reasonable alternative for protein expression, as viruses can infect the host systemically to achieve expression and replication without transgene integration. Previously, a novel four-component Barley stripe mosaic virus (BSMV) was reported to overexpress large fragments of target genes in plants over a long period of time, which greatly simplified the study of gene overexpression. However, whether this system can infect cotton and stably overexpress target genes has not yet been studied. In this study, we verified that this new BSMV system can infect cotton through seed imbibition and systemically overexpress large fragments of genes (up to 2340 bp) in cotton. The target gene that was fused with GFP was expressed at a high level in the roots, stems, and cotyledons of cotton seedlings, and stable fluorescence signals were detected in the cotton roots and leaves even after 4 weeks. Based on the BSMV overexpression system, the subcellular localization marker line of endogenous proteins localized in the nucleus, endoplasmic reticulum, plasma membrane, Golgi body, mitochondria, peroxisomes, tonoplast, and plastids were quickly established. The overexpression of a cotton Bile Acid Sodium Symporter GhBASS5 using the BSMV system indicated that GhBASS5 negatively regulated salt tolerance in cotton by transporting Na+ from underground to the shoots. Furthermore, multiple proteins were co-delivered, enabling co-localization and the study of protein–protein interactions through co-transformation. We also confirmed that the BSMV system can be used to conduct DNA-free gene editing in cotton by delivering split-SpCas9/sgRNA. Ultimately, the present work demonstrated that this BSMV system could be used as an efficient overexpression system for future cotton gene function research.

Barley stripe mosaic virus-mediated somatic and heritable gene editing in barley (Hordeum vulgare L.)

“…Further, as demonstrated for wheat ( Budhagatapalli et al., 2020 ), the development of Cas9 expressing haploid inducer barley lines infected with BSMV carrying gRNA(s) of interest could be utilized to induce mutations in the genome of the desired genotypes. Recently, a modified four-component BSMV system with increased cargo capacity ( Cheuk and Houde, 2018 ) enabled transient somatic editing in cotton using split SpCas9 ( Chen et al., 2022b ). Whether reliable and frequent heritable editing based on a similar strategy can be achieved, needs to be addressed.…”

“…VIGE has been applied in dicot plants including Nicotiana benthamiana, Arabidopsis thaliana , and Glycine max using plant RNA viruses such as Tobacco rattle virus (TRV) ( Ellison et al., 2020 ; Nagalakshmi et al., 2022 ), Pea early browning virus (PEBV) ( Ali et al., 2018 ), Beet necrotic yellow vein virus (BNYVV) ( Jiang et al., 2019 ), Potato virus X (PVX) ( Ariga et al., 2020 ), Barley yellow striate mosaic virus (BYSMV) ( Gao et al., 2019 ) or Sonchus yellow net rhabdovirus (SYNV) ( Ali et al., 2015 ; Zaidi and Mansoor, 2017 ; Cody and Scholthof, 2019 ; Ellison et al., 2020 ; Ma et al., 2020 ; Nagalakshmi et al., 2022 ). In monocots, Foxtail mosaic virus in maize (FoMV) ( Mei et al., 2019 ) or BSMV in wheat, cotton, and maize ( Hu et al., 2019 ; Li et al., 2021 ; Chen et al, 2022a ; Chen et al, 2022b ; Wang et al., 2022 ) were employed for VIGE.…”

“…BSMV was harnessed to deliver sgRNAs into plants that ectopically express Cas9 , such as monocots (wheat and maize) and the dicot N. benthamiana , for eliciting Cas9-mediated targeted genome editing in somatic tissues ( Hu et al., 2019 ; Li et al., 2021 ; Chen et al., 2022a ; Wang et al., 2022 ). In wheat, different frequencies of BSMV-mediated heritable gene editing were observed, ranging from 0.8 to 3.0% ( Chen et al, 2022a ; Chen et al, 2022b ), 12.9 to 100% ( Li et al., 2021 ), and 0 to 19% ( Wang et al., 2022 ), depending on the genotype, type of sgRNA (with/without mobile RNA elements), and the target site ( Notaguchi et al., 2014 ; Zhang et al., 2016 ; Ellison et al., 2020 ; Nagalakshmi et al., 2022 ).…”

Editorial: CRISPR tools, technology development, and application

“…In addition, the authors also achieved successful editing of the meiosis-related candidate genes ASY1 encoding axis-localized HORMA domain protein, MUS81 encoding a DNA structure-selective endonuclease, and ZYP1 encoding a transverse filament protein of the synaptonemal complex) in barley. The BSMV mediated VIGE gene editing has also shown applicability in wheat (Chen et al, 2022a) and cotton (Chen et al, 2022b) suggesting that BSMV with its broad host range can find significant application in crop plants.…”