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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.

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.

SUBMITTER: Tripathi JN

PROVIDER: S-EPMC6355771 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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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.

Tripathi Jaindra N JN Ntui Valentine O VO Ron Mily M Muiruri Samwel K SK Britt Anne A Tripathi Leena L

Communications biology 20190131

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 ...[more]

PMID: 30729184

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Project description:BackgroundBananas (Musa spp.) are a globally significant crop and are severely afflicted by diseases for which there are no effective chemical controls. Banana microbiomes may provide novel solutions to these constraints but are difficult to manage due to their high diversity and variability between locations. Hence 'common core' taxa, which are a subset of the microbiome that frequent all, or most, individuals of a host species, represent logical targets for the development of microbiome management approaches. Here, we first performed a pot experiment to characterise the effects of two factors that are likely to differ between farms (viz. edaphic conditions and host genotype) on bacterial diversity in bulk soil and seven plant compartments. From this experiment, we created shortlisted core 'candidates' that were then refined using a survey of 52 field-grown Musa spp. We confirmed the importance of the core through network analysis and by comparing the sequences of our core taxa with those reported in 22 previous studies.ResultsDiversity was found to differ between plant compartments and soils, but not genotypes. Therefore, we identified populations that were frequent across most plants irrespective of the soil in which they were grown. This led to the selection of 36 'common core' bacteria, that represented 65-95% of the dominant taxa in field-grown plants and were identified as highly interconnected 'hubs' using network analysis - a characteristic shown to be indicative of microbes that influence host fitness in studies of other plants. Lastly, we demonstrated that the core taxa are closely related to banana-associated bacteria observed on five other continents.ConclusionsOur study provides a robust list of common core bacterial taxa for Musa spp. Further research may now focus on how changes in the frequencies and activities of these most persistent taxa influence host fitness. Notably, for several of our core taxa, highly similar populations have already been isolated in previous studies and may be amenable to such experimentation. This contribution should help to accelerate the development of effective Musa spp. microbiome management practices.

Project description:BackgroundIn plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana.ResultsUsing embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6-8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target sequences (26-nt and 19-nt).ConclusionsRNAi-induced silencing was achieved in banana, both at transient and stable level, resulting in significant reduction of gene expression and enzyme activity. The success of silencing was dependent on the targeted region of the target gene. The successful generation of transgenic ECS for second transformation with (an)other construct(s) can be of value for functional genomics research in banana.

Dataset Information

CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding.

Publications

CRISPR/Cas9 editing of endogenous <i>banana streak virus</i> in the B genome of <i>Musa</i> spp. overcomes a major challenge in banana breeding.

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