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

ABSTRACT: Genome editing strategies in barley (Hordeum vulgare L.) typically rely on Agrobacterium-mediated genetic transformation for the delivery of required genetic reagents involving tissue culture techniques. These approaches are genotype-dependent, time-consuming, and labor-intensive, which hampers rapid genome editing in barley. More recently, plant RNA viruses have been engineered to transiently express short guide RNAs facilitating CRISPR/Cas9-based targeted genome editing in plants that constitutively express Cas9. Here, we explored virus-induced genome editing (VIGE) based on barley stripe mosaic virus (BSMV) in Cas9-transgenic barley. Somatic and heritable editing in the ALBOSTRIANS gene (CMF7) resulting in albino/variegated chloroplast-defective barley mutants is shown. In addition, somatic editing in meiosis-related candidate genes in barley encoding ASY1 (an axis-localized HORMA domain protein), MUS81 (a DNA structure-selective endonuclease), and ZYP1 (a transverse filament protein of the synaptonemal complex) was achieved. Hence, the presented VIGE approach using BSMV enables rapid somatic and also heritable targeted gene editing in barley.

SUBMITTER: Tamilselvan-Nattar-Amutha S

PROVIDER: S-EPMC10315673 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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

Tamilselvan-Nattar-Amutha Suriya S Hiekel Stefan S Hartmann Franziska F Lorenz Jana J Dabhi Riddhi Vijay RV Dreissig Steven S Hensel Goetz G Kumlehn Jochen J Heckmann Stefan S

Frontiers in plant science 20230619

Genome editing strategies in barley (Hordeum vulgare L.) typically rely on Agrobacterium-mediated genetic transformation for the delivery of required genetic reagents involving tissue culture techniques. These approaches are genotype-dependent, time-consuming, and labor-intensive, which hampers rapid genome editing in barley. More recently, plant RNA viruses have been engineered to transiently express short guide RNAs facilitating CRISPR/Cas9-based targeted genome editing in plants ...[more]

PMID: 37404527

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Project description:Barley stripe mosaic virus (BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used Agrobacterium tumefaciens-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.

Dataset Information

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

Publications

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

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