20 Apr Applying genome editing to improve banana
Leena Tripathi (IITA-Kenya)
Banana is a major staple food crop feeding more than 500 million people in tropical and subtropical countries. Its production is seriously affected by several factors, specifically stresses such as declining soil fertility, narrow genetic diversity in germplasm, and inadequate availability of clean planting material among smallholder farmers. Diseases and pests are a major factor limiting yields worldwide. Production is mainly reduced by many bacterial, fungal, and viral pathogens, mainly Xanthomonas campestris pv. musacearum (Xcm) causing banana Xanthomonas wilt (BXW), Ralstonia solanacearum causing moko and bugtok disease, Ralstonia syzygii subsp. celebesensis causing blood disease, Pseudocercospora fijiensis, P. musae, and P. eumusae causing black Sigatoka, yellow Sigatoka, and leaf spot disease, respectively, Fusarium oxysporum f. sp. cubense causing fusarium wilt (commonly known as panama disease), and viruses such as banana bunchy top virus (BBTV) and banana streak virus (BSV), and pests like nematodes and weevils.
There is a huge yield gap in banana production in areas where several of these pathogens and pests occur together. Therefore, improved varieties need to be developed with multiple and durable resistance to pathogens and pests. Modern breeding tools such as genome editing can be applied to improve banana, complementing traditional breeding. CRISPR/Cas9 has emerged as a potent editing tool that can be used efﬁciently to induce targeted mutations in the genomes of plant species to produce improved varieties. This technology has been successfully applied in many organisms including several plant species.
At IITA, researchers have established a genome editing tool for banana using phytoene desaturase (PDS) gene as a marker as mutations disrupting PDS causing albinism and dwarﬁng of plants (Figure 1, Tripathi et al., 2019a). Establishment of CRISPR/Cas9 system has paved the way for applying genome editing for improving banana varieties resistant to diseases and pests and tolerant of abiotic stresses.
Recently, we have applied CRISPR/Cas9 based genome editing to knock out the endogenous banana streak virus (eBSV) integrated in the B genome of Musa spp., overcoming a major challenge in banana breeding. BSV is a plant pathogenic badnavirus of the family Caulimoviridae, affecting banana production. It was first identified in West Africa in 1958 and is now reported in most banana and plantain growing countries. Like other badnaviruses, BSV sequences integrate into the genome of Musa spp. BSV is a complex of different viruses belonging to the pararetroviruses (PRVs) and classified as endogenous pararetroviruses (EPRVs) when they are integrated into a host genome. The EPRVs of BSV in banana are known as endogenous BSV (eBSV). The eBSV sequences are integrated in the B genome derived from Musa balbisiana.
Many economically important subgroups of banana, such as plantain (AAB), an important staple food in Africa, contain at least one B genome. When the banana plants are stressed, the eBSV recombines to produce a functional episomal viral genome and infectious viral particles and as a result the plant develops disease symptoms. The major epidemics caused by BSV are not due to natural transmission through insect vectors or through use of infected planting materials, but rather due to activation of integrated virus under stress conditions such as unfavorable conditions, in vitro culture for propagation, and hybridization through conventional breeding. Consequently, BSV is considered a major constraint in banana breeding programs, restricting the use of the diploid progenitor M. balbisiana or its derivants carrying a B genome as parents for introgression of desirable agronomic traits. It also restricts germplasm movement of genotypes with the B genome worldwide due to this potential activation of eBSV into the episomal infectious form. Therefore, it is crucial to design a strategy to irreversibly silence the latent eBSV in the B genome. IITA scientists in collaboration with University of California, Davis, USA applied CRISPR/Cas9-based genome editing technology to inactivate eBSV strain Obino l’Ewai (eBSOLV) sequences in the host plantain genome (Tripathi et al. 2019b).
The CRISPR/Cas9 reagents targeting the virus genome were delivered to embryogenic cells of plantain cultivar ‘Gonja Manjaya’, a false horn plantain, and complete plants were regenerated. The regenerated plants were validated by PCR analysis and Sanger sequencing of the targeted sites. The genome-edited plants and wild type control plants, developed from the same embryogenic cell line, were found to be phenotypically similar with no growth abnormalities. The regenerated genome-edited events of ‘Gonja Manjaya’ showed mutations in the targeted sites with the potential to prevent proper transcription or/and translation into functional viral proteins. The genome-edited plants along with control nonedited plants were evaluated under the glasshouse for activation of eBSOLV into infectious virus under water stress conditions. Seventy-five percent (6/8) of the edited events remained asymptomatic compared to the nonedited control plants under water stress conditions, confirming inactivation of eBSV and reversal of its ability to be converted into infectious viral particles. Disease symptoms such as broken or continuous streaks of yellow, chlorotic, black or brown color on the leaf appeared in all the wild type control plants tested, as would be expected if there was activation of eBSOLV into episomal infectious BSOLV. However, six out of eight genome-edited events tested remained asymptomatic and the remaining two events showed only moderate symptoms (Fig. 2). The presence of the infectious episomal form of BSOLV was confirmed by PCR and qPCR analysis.
This is the first report to demonstrate the knockout of integrated endogenous DNA sequences of the pararetrovirus in a host plant genome. We have demonstrated that CRISPR/Cas9 based targeted mutagenesis can permanently inactivate endogenous eBSV and presents a promising model for inactivating other endogenous viral genomes. This study paves the way for editing banana germplasm with B genome(s) that can be used as one or both parents in the breeding programs. This is a particularly important strategy for both improving plantains and enabling global dissemination of the resulting hybrids with improved B genome.
- Tripathi L., Ntui V.O., Tripathi N. 2019. Application of genetic engineering and genome editing for developing climate smart bananas. Food and Energy Security https://doi.org/10.1002/fes3.16
- Tripathi N., Ntui V.O., Ron M., Muiruri S.K., Britt A., Tripathi L. 2019. CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding. Communications Biology 2. https://doi.org/10.1038/s42003-019-0288-7