Development of Beet necrotic yellow vein virus‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

Abstract

Summary Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus ( BNYVV ) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive‐stranded RNA s. Here, we have established a BNYVV full‐length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV ‐based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co‐localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV ‐based vectors were used to deliver Nb PDS guide RNA s for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV ‐based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.

Application of a Reverse Genetic System for Beet Necrotic Yellow Vein Virus to Study Rz1 Resistance Response in Sugar Beet

“…Therefore, we recently developed a clone with an alternative marker insertion position following gene replacement strategy and labeled BNYVV by integrating the mRFP ORF into RNA2 through the replacement of the CP-RT (Laufer et al, 2018a). A similar strategy for fluorescence labeling was also used by Jiang et al (2019) for BNYVV, but systemic movement in sugar beet has not been demonstrated. We could observe a strong fluorescence in lateral roots and leaves indicative for a systemic spread of the labeled BNYVV clone.…”

Protein expression and gene editing in monocots using foxtail mosaic virus vectors

“…Viruses that carry fragments of plant genes in sense and antisense orientation cause virus-induced gene silencing (VIGS) of the targeted sequence, or microRNA inserts can initiate silencing with high specificity. Most recently, it has been demonstrated that plant viruses or their derivatives can be used to deliver CRISPR-Cas reagents consisting of single guide RNAs (gRNAs), DNA repair templates, and site-specific nucleases such as Cas9 (Ali et al, 2015;Ali, Eid, Ali, & Mahfouz, 2018;Butler, Baltes, Voytas, & Douches, 2016;Cody, Scholthof, & Mirkov, 2017;Dahan-Meir et al, 2018;Gao et al, 2019;Gil-Humanes et al, 2017;Jiang et al, 2019;Mahas, Ali, Tashkandi, & Mahfouz, 2019;Wang et al, 2017). These capabilities show that plant viruses can be useful biotechnological tools for gene function studies in plants, and they can have practical applications as well (Pasin et al, 2019;Zaidi & Mansoor, 2017).…”

A barley stripe mosaic virus‐based guide RNA delivery system for targeted mutagenesis in wheat and maize

“…TMV was also reported to be able to mediate target gene editing in the local leaves by partially substituting the CP ORF with a gRNA (Cody et al, 2017). Very recently, BNYVV has also been used for gRNA delivery in N. benthamiana (Jiang et al, 2019). BSMV-based VIGE described above provides an alternative tool for targeted gene editing in the model plant N. benthamiana.…”

“…Compared to the traditional gRNA delivery methods via Agrobacterium transformation, plant virus‐mediated gRNA delivery systems have several advantages: (1) the gRNAs can accumulate to high levels owing to viral replication and systemic spread in plants and may contribute to a higher genome editing efficiency, (2) multiple functional gRNAs can be expressed from a single viral genome, which provides the potential for multi‐targeted genome editing, (3) phenotypic alterations may appear in infected plants in a relatively short period of time after gene targeting by virus‐induced genome editing (VIGE), and (4) transformation and regeneration of agriculturally important crops such as wheat is laborious and time‐consuming, but VIGE may shorten this period and simplify operation and editing processes of a target gene in the specific tissues. Several plant RNA viruses including tobacco rattle virus (TRV) (Ali et al ., ), pea early‐browning virus (PEBV) (Ali et al ., ), tobacco mosaic virus (TMV) (Cody et al ., ) and beet necrotic yellow vein virus (BNYVV) (Jiang et al ., ) have been reported to enable targeted genome editing in the model plants Nicotiana benthamiana and Arabidopsis thaliana or both. The DNA virus cabbage leaf curl virus (CaLCuV) has also been engineered for plant genome editing in tobacco (Yin et al ., ).…”

“…Recent studies indicate that viruses can be developed to deliver gRNAs for targeted mutagenesis in plants (Ali et al, 2015(Ali et al, , 2018Cody et al, 2017;Gil-Humanes et al, 2017;Jiang et al, 2019;Yin et al, 2015). Compared to the traditional gRNA delivery methods via Agrobacterium transformation, plant virus-mediated gRNA delivery systems have several advantages: (1) the gRNAs can accumulate to high levels owing to viral replication and systemic spread in plants and may contribute to a higher genome editing efficiency, (2) multiple functional gRNAs can be expressed from a single viral genome, which provides the potential for multi-targeted genome editing, (3) phenotypic alterations may appear in infected plants in a relatively short period of time after gene targeting by virus-induced genome editing (VIGE), and (4) transformation and regeneration of agriculturally important crops such as wheat is laborious and time-consuming, but VIGE may shorten this period and simplify operation and editing processes of a target gene in the specific tissues.…”