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CRISPR/Cas9 Editing of Endogenous banana Streak virus in the B Genome of Musa Spp. Overcomes a Major Challenge in Banana Breeding

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CRISPR/Cas9 Editing of Endogenous banana Streak virus in the B Genome of Musa Spp. Overcomes a Major Challenge in Banana Breeding

Jaindra N Tripathi et al. Commun Biol.

Abstract

Presence of the integrated endogenous banana streak virus (eBSV) in the B genome of plantain (AAB) is a major challenge for breeding and dissemination of hybrids. As the eBSV activates into infectious viral particles under stress, the progenitor Musa balbisiana and its derivants, having at least one B genome, cannot be used as parents for crop improvement. Here, we report a strategy to inactivate the eBSV by editing the virus sequences. The regenerated genome-edited events of Gonja Manjaya showed mutations in the targeted sites with the potential to prevent proper transcription or/and translational into functional viral proteins. Seventy-five percent of the edited events remained asymptomatic in comparison to the non-edited control plants under water stress conditions, confirming inactivation of eBSV into infectious viral particles. This study paves the way for the improvement of B genome germplasm and its use in breeding programs to produce hybrids that can be globally disseminated.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Schematic representation of banana streak virus (BSV) strain Obino l’Ewai (BSOLV) and molecular analysis of Gonja Manjaya plants. a Episomal BSOLV showing three open-reading frames (ORF) and gene annotation in ORF3 (GenBank accession KJ013506). The colors purple, light blue, brown, and black indicate ORF1, ORF2, ORF3, and the intragenic region of BSOLV, respectively; P1–P4 show the position of primer pairs; S1–S3 indicate target sites for gRNA1, gRNA2, and gRNA3, respectively. b Endogenous BSV strain Obino l’Ewai (eBSOLV) integrated in banana genome (adapted from ref. ). The duplication of BSV sequences, either complete or partial in the same or opposite direction, within the integrated eBSOLV are shown in the diagram. Green indicates banana genome; SBP indicates position of probe used for Southern hybridization; P1–P4 represent PCR primer pair locations. c PCR amplification to confirm the presence of BSOLV and/or eBSOLV in Gonja Manjaya plantlets. 1‒4, in vitro mother plantlets of Gonja Manjaya; PCR products using P1–P3 primers are labeled on the top of the gel picture; M molecular marker. d PCR analysis to confirm the absence of episomal BSOLV in in vitro mother plantlets of Gonja Manjaya used for genome editing. PC1 and PC2, symptomatic field plants of plantain Agbagba; PC3, symptomatic Gonja Manjaya plant from glasshouse under stress condition; CW, asymptomatic in vitro plantlet of Cavendish Williams as negative control; NTC, no template control. e Southern blot hybridization to confirm the presence of eBSOLV in Gonja Manjaya plantlets. Genomic DNA for Southern blot was digested with HindIII. The same four individual Gonja Manjaya plants (1‒4) were used for PCR analyses and Southern hybridization
Fig. 2
Fig. 2
Regeneration of genome-edited events by delivering CRISPR/Cas9 construct through Agrobacterium-mediated transformation of cell suspension of Gonja Manjaya. a Schematic representation of T-DNA region of binary vector pMR215-BSV1 showing the positions of three gRNAs. LB, left border; RB, right border; PAM, protospacer adjacent motif. Sequences of the gRNAs are also indicated. b and c Germination of Agro-infected embryos in selective medium containing kanamycin. d Well-rooted plantlets in proliferation medium. e Genome-edited Gonja Manjaya plants in glasshouse. f Non-edited wild type control Gonja Manjaya plants in glasshouse
Fig. 3
Fig. 3
Sequence analysis of genome-edited plants of Gonja Manjaya to detect mutations in S1 and S2 target sites. Nucleotides in brown represent the target sites and blue nucleotides indicate protospacer adjacent motif (PAM). Dotted lines (…) represent deletions, nucleotides in red indicate insertion, and green nucleotides indicate replacement. BSOLV, sequence of BSV strain Obino l’Ewai; WT, sequences same as wild type non-edited control Gonja Manjaya; 1–100, 20 independent transgenic events. The mutations in S1, S2, and S1/S2, number of indels as deletions (−), insertions (+) or replacements (r) and percentage of clones of transgenic event showing same mutations are indicated on the right side panel. The PCR products amplified using primer P1 (see Fig. 1b) were purified, cloned, and 10 individual clones per event were analyzed for mutations at target sites S1 and S2 by Sanger sequencing
Fig. 4
Fig. 4
Sequence analysis of genome-edited plants of Gonja Manjaya to detect targeted mutations in S3 target site. Nucleotides in brown represent the target site and blue nucleotides indicate PAM. Dotted lines (…) represent deletions, nucleotides in red indicate insertion. BSOLV, sequence of BSV strain Obino l’Ewai; WT, wild type non-edited control Gonja Manjaya; 1‒100, independent transgenic events. The number of indels as deletions (−) or insertions (+) and percentage of clones of transgenic event showing same mutations are indicated on the right side of the figure. The PCR products amplified using primer set P3 (see Fig. 1b) were purified, cloned, and 10 individual clones per event were analyzed for mutations at target site S3 by Sanger sequencing
Fig. 5
Fig. 5
Evaluation of genome-edited and wild type non-edited control plants of Gonja Manjaya for induction of BSV symptoms under water stress conditions. Two-month-old plants were subjected to water stress for 2 weeks. Disease symptoms as chlorosis or yellow streaks were recorded at the end of the stress period and pictures were taken. a Pictures of asymptomatic genome-edited plants (17, 76, and 81), symptomatic-edited plants (66 and 97), and wild type control plants (WT). b PCR diagnostic to detect activation of episomal BSOLV in genome edited and control plants under water stress conditions. c qPCR analysis to detect episomal BSOLV in genome edited and control plants under water stress conditions. PC1‒2, symptomatic plant of Agbagba from field as positive control; WT, wild type non-edited control Gonja Manjaya plant under stress conditions; 17, 66, 76, 81, 97, edited plants under stress conditions; CW, asymptomatic in vitro plantlet of Cavendish Williams as negative control; NTC no template control. For PCR and qPCR, leaf samples from three symptomatic wild type non-edited control plants (WT) of Gonja Manjaya were pooled for DNA extraction. Similarly, the leaves from three replicates for symptomatic and asymptomatic-edited events were pooled for PCR and qPCR. CT values were presented as means and standard error of six technical replicates from two independent experiments

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References

    1. Cheng C, Lockhart BEL, Olszewski NE. The ORF I and II proteins of Commelina yellow mottle virus are virion-associated. Virology. 1996;223:263–271. doi: 10.1006/viro.1996.0478. - DOI - PubMed
    1. Leclerc D, et al. The open reading frame III product of cauliflower mosaic virus forms a tetramer through a N-terminal coiled coil. J. Biol. Chem. 1998;273:29015–29021. doi: 10.1074/jbc.273.44.29015. - DOI - PubMed
    1. Fargette D, et al. Molecular ecology and emergence of tropical plant viruses. Annu. Rev. Phytopathol. 2006;44:235–260. doi: 10.1146/annurev.phyto.44.120705.104644. - DOI - PubMed
    1. Harper G, Osuji JO, Heslop-Harrison JS, Hull R. Integration of banana streak virus infection into the Musa genome: molecular and cytogenetic evidence. Virology. 1999;255:207–213. doi: 10.1006/viro.1998.9581. - DOI - PubMed
    1. Chabannes M, et al. Three infectious viral species lying in wait in the banana genome. J. Virol. 2013;87:8624–8637. doi: 10.1128/JVI.00899-13. - DOI - PMC - PubMed

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