Dataset Information

Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence.

ABSTRACT: Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive-sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, novel cap-binding protein-1 (nCBP-1), and nCBP-2. Protein-protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9-mediated genome editing was employed to generate ncbp-1, ncbp-2, and ncbp-1/ncbp-2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp-1/ncbp-2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild-type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.

SUBMITTER: Gomez MA

PROVIDER: S-EPMC6335076 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence.

Gomez Michael A MA Lin Z Daniel ZD Moll Theodore T Chauhan Raj Deepika RD Hayden Luke L Renninger Kelley K Beyene Getu G Taylor Nigel J NJ Carrington James C JC Staskawicz Brian J BJ Bart Rebecca S RS

Plant biotechnology journal 20181005 2

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive-sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eI ...[more]

PMID: 30019807

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Project description:Cassava (Manihot esculenta) is an important tropical subsistence crop that is severely affected by cassava brown streak disease (CBSD) in East Africa. The disease is caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Both have a (+)-sense single-stranded RNA genome with a 5' covalently-linked viral protein, which functionally resembles the cap structure of mRNA, binds to eukaryotic translation initiation factor 4E (eIF4E) or its analogues, and then enable the translation of viral genomic RNA in host cells. To characterize cassava eIF4Es and their potential role in CBSD tolerance and susceptibility, we cloned five eIF4E transcripts from cassava (accession TMS60444). Sequence analysis indicated that the cassava eIF4E family of proteins consisted of one eIF4E, two eIF(iso)4E, and two divergent copies of novel cap-binding proteins (nCBPs). Our data demonstrated experimentally the coding of these five genes as annotated in the published cassava genome and provided additional evidence for refined annotations. Illumina resequencing data of the five eIF4E genes were analyzed from 14 cassava lines tolerant or susceptible to CBSD. Abundant single nucleotide polymorphisms (SNP) and biallelic variations were observed in the eIF4E genes; however, most of the SNPs were located in the introns and non-coding regions of the exons. Association studies of non-synonymous SNPs revealed no significant association between any SNP of the five eIF4E genes and the tolerance or susceptibility to CBSD. However, two SNPs in two genes were weakly associated with the CBSD responses but had no direct causal-effect relationship. SNPs in an intergenic region upstream of eIF4E_me showed a surprising strong association with CBSD responses. Digital expression profile analysis showed differential expression of different eIF4E genes but no significant difference in gene expression was found between susceptible and tolerant cassava accessions despite the association of the intergenic SNPs with CBSD responses.

Project description:CRISPR/Cas9 has become a powerful genome-editing tool for introducing genetic changes into crop species. In order to develop capacity for CRISPR/Cas9 technology in the tropical staple cassava (Manihot esculenta), the Phytoene desaturase (MePDS) gene was targeted in two cultivars using constructs carrying gRNAs targeting two sequences within MePDS exon 13. After Agrobacterium-mediated delivery of CRISPR/Cas9 reagents into cassava cells, both constructs induced visible albino phenotypes within cotyledon-stage somatic embryos regenerating on selection medium and the plants regenerated therefrom. A total of 58 (cv. 60444) and 25 (cv. TME 204) plant lines were recovered, of which 38 plant lines (19 from each cultivar) were analyzed for mutagenesis. The frequency of plant lines showing albino phenotype was high, ranging from 90 to 100% in cv. TME 204. Observed albino phenotypes were comprised of full albinos devoid of green tissue and chimeras containing a mixture of white and green tissues. Sequence analysis revealed that 38/38 (100%) of the plant lines examined carried mutations at the targeted MePDS site, with insertions, deletions, and substitutions recorded. One putatively mono-allelic homozygous line (1/19) was found from cv. 60444, while 1 (1/19) and 4 (4/19) putatively bi-allelic homozygous lines were found in 60444 and TME204, respectively. The remaining plant lines, comprised mostly of the chimeras, were found to be putatively heterozygous. We observed minor (1 bp) nucleotide substitutions and or deletions upstream of the 5' and or downstream of the 3' targeted MePDS region. The data reported demonstrates that CRISPR/Cas9-mediated genome editing of cassava is highly efficient and relatively simple, generating multi-allelic mutations in both cultivars studied. Modification of MePDS described here generates visually detectable mutated events in a relatively short time frame of 6-8 weeks, and does not require sequencing to confirm editing at the target. It therefore provides a valuable platform to facilitate rapid assessment and optimization of CRISPR/Cas9 and other genome-editing technologies in cassava.

Project description:Cassava brown streak disease (CBSD) is a severe virus disease of cassava and prevalent in the eastern regions of Africa. The disease is characterized by distinct vein chlorosis and streak symptoms on leaves and stems and necrosis of storage roots. This necrosis can encompass large areas of the root, rendering it inedible so that the entire cassava harvest can be lost. African cassava varieties are susceptible to either of the two viruses causing the disease, cassava brown streak virus (CBSV) and Uganda cassava brown streak virus, and while there are less sensitive varieties, all cassava eventually succumb to the disease. The lack of CBSD resistance in African cassava varieties prompted this search for new sources of virus resistance in the diversity of South American cassava germplasm held in the collection at International Center for Tropical Agriculture, Columbia. Our search for CBSD resistance in South American cassava germplasm accessions revealed that most of the 238 South American cassava lines infected with CBSV established systemic virus infections with moderate to severe disease symptoms on leaves and stems. Fifteen cassava accessions did not become virus infected, remained free of symptoms, and CBSV was undetected by qRT-PCR. When tuberous roots of those lines were examined, necrotic tissue was found in eight lines and CBSV was detected. The remaining seven cassava accessions remained clear of symptoms on all tissues and organs and were virus free. A broad spectrum of virus resistance also including other virus isolates was confirmed for the breeding lines DSC167 and DSC118. While detailed infection experiments with other cassava lines selected for resistance are still ongoing, this indicates that the resistance identified may also hold against a broader diversity of CBSVs. Taken together, we present the results of a comprehensive study on CBSV resistance and susceptibility in cassava germplasm accessions from South America. The virus resistance in cassava germplasm identified provides compelling evidence for the invaluable contribution of germplasm collections to supply the genetic resources for the improvement of our crops.

Project description:Cassava mosaic geminiviruses (CMGs) and cassava brown streak viruses (CBSVs) cause the highest yield losses in cassava production in Africa. In particular, cassava brown streak disease (CBSD) is and continues to be a significant constraint to optimal cassava production in Eastern and Southern Africa. While CBSD has not been reported in West Africa, its recent rapid spread and damage to cassava productivity in Eastern, and Southern Africa is alarming. The aim of this study was to evaluate Nigerian cassava genotypes in order to determine their responses to CBSD, in the event that it invades Nigeria, the world's largest cassava producer. The study gathered information on whether useful CBSD resistance alleles are present in the elite Nigerian cassava accessions. A total of 1,980 full-sib cassava seedlings from 106 families were assessed in the field at the seedling stage for a year. A subset of 569 clones were selected and assessed for another year at the clonal stage in Namulonge, central Uganda, a known hotspot for CBSD screening. Results indicated that foliar and root incidences and severities varied significantly (p ≤ 0.01, p ≤ 0.001) except for CBSD foliar incidence at 6 months (CBSD6i ). Highest and lowest plot-based heritability estimates for CBSD were registered for CBSD root severity (CBSD rs ) (0.71) and CBSD6i (0.5). Positive and highly significant correlations were noted between CBSD root incidence (CBSD ri ) and CBSD rs (r = 0.90***). Significant positive correlations were also noted between CBSD foliar severity at 3 months (CBSD3s ) and CBSD foliar incidence at 6 months (CBSD6i ) (r = 0.77***), CBSD3s and CBSD rs (r = 0.35***). Fresh root weight (Fresh RW ) negatively correlated with CBSD ri and CBSD rs , respectively (r = -0.21*** and r = -0.22***). Similarly, CBSD3s correlated negatively with cassava mosaic disease severity at 3 (CMD3s ) and 6 months (CMD6s ), respectively (r = -0.25*** and r = -0.21***). Fifteen clones were selected using a non-weighted summation selection index for further screening. In conclusion, results revealed that the elite Nigerian accessions exhibited significant susceptibility to CBSD within 2 years of evaluation period. It is expected that this information will aid future breeding decisions for the improvement of CBSD resistance among the Nigerian cassava varieties.

Dataset Information

Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence.

Publications

Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence.

Similar Datasets

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