Genome-wide transcriptome analysis of cassava challenged with Ugandan cassava brown streak virus (UCBSV)

Cassava is staple to millions in Africa, yet cassava brown streak disease (CBSD) greatly threatens cassava production. This study reports the mechanism of cassava’s resistance to CBSD using Ugandan cassava brown streak virus (UCBSV), one of the two CBSD-causal virus species. In a green house, five varieties: Albert, Kiroba, Mkombozi, Namikonga and NDL06/132 were analysed for response to UCBSV at 20 time points. In the first experiment, Albert and Namikonga were compared. In the second experiment, all five varieties were studied. Plants were phentyped for foliar and root symptoms of CBSD, virus titre was measured using qRT-PCR while RNAseq and QuantStudio were used to determine gene expression. Virus infected plants of susceptible variety (Albert) developed clear leaf symptoms and necrotic storage roots. UCBSV-infected plants of Namikonga (resistant) showed minimal leaf symptoms and storage roots were non-necrotic. UCBSV titre was highest in susceptible variety (Albert), and very low in the resistant variety (Namikonga). More genes were differentially expressed in Namikonga (10,028) compared to Albert (688). In Namikonga, highest expression was recorded at 2 dag and 5 dag, when only two genes were differentially expressed in Albert. GO terms for phosphorelay signal transduction, ribosome and elongation factors were enriched in Namikonga and none in Albert. In another experiment, UCBSV-infected storage roots from Kiroba were non-necrotic. Defence genes identified in Kiroba were similar to Namikonga’s, except that in Kiroba, genes were highly expressed at 1 dag and 51 dag. One storage root of Mkombozi developed a necrotic spot. Mkombozi had no distinct pattern of defence-gene expression as seen in Kiroba and Namikonga. Varieties NDL06/132 and Albert were susceptible, and showed limited numbers of differentially expressed genes. Therefore, Kiroba and Namikonga are resistant to CBSD. The resistance mechanism involves up regulation of known defence proteins, and restricted multiplication of UCBSV in infected plants.Thesis (PhD)--University of Pretoria, 2019.Bill and Melinda Gates Foundation & University of PretoriaPlant SciencePhDUnrestricte

Candidate genes for field resistance to cassava brown streak disease revealed through the analysis of multiple data sources

Cassava (Manihot esculenta Crantz) is a food and industrial storage root crop with substantial potential to contribute to managing risk associated with climate change due to its inherent resilience and in providing a biodegradable option in manufacturing. In Africa, cassava production is challenged by two viral diseases, cassava brown streak disease (CBSD) and cassava mosaic disease. Here we detect quantitative trait loci (QTL) associated with CBSD in a biparental mapping population of a Tanzanian landrace, Nachinyaya and AR37-80, phenotyped in two locations over three years. The purpose was to use the information to ultimately facilitate either marker-assisted selection or adjust weightings in genomic selection to increase the efficiency of breeding. Results from this study were considered in relation to those from four other biparental populations, of similar genetic backgrounds, that were phenotyped and genotyped simultaneously. Further, we investigated the co-localization of QTL for CBSD resistance across populations and the genetic relationships of parents based on whole genome sequence information. Two QTL on chromosome 4 for resistance to CBSD foliar symptoms and one on each of chromosomes 11 and 18 for root necrosis were of interest. Of significance within the candidate genes underlying the QTL on chromosome 4 are Phenylalanine ammonia-lyase (PAL) and Cinnamoyl-CoA reductase (CCR) genes and three PEPR1-related kinases associated with the lignin pathway. In addition, a CCR gene was also underlying the root necrosis-resistant QTL on chromosome 11. Upregulation of key genes in the cassava lignification pathway from an earlier transcriptome study, including PAL and CCR, in a CBSD-resistant landrace compared to a susceptible landrace suggests a higher level of basal lignin deposition in the CBSD-resistant landrace. Earlier RNAscope®in situ hybridisation imaging experiments demonstrate that cassava brown streak virus (CBSV) is restricted to phloem vessels in CBSV-resistant varieties, and phloem unloading for replication in mesophyll cells is prevented. The results provide evidence for the involvement of the lignin pathway. In addition, five eukaryotic initiation factor (eIF) genes associated with plant virus resistance were found within the priority QTL regions

Accelerating News Issue 18

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.Bill and Melinda Gates Foundation [OPP1068522]; Natural Science Foundation of China [31461143016]; Genus plcOpen access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Primers used to amplify coding sequences of cassava eIF4E and its homologues.

<p>Primers used to amplify coding sequences of cassava eIF4E and its homologues.</p

Boxplot of expression levels of eIF4E genes in six cassava lines.

<p>The 454 sequencing data were obtained from six cassava accessions (AR 37–80, AR40-6, Mkombozi, Kiroba, Nachinyaya, and Namikonga) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181998#pone.0181998.ref029" target="_blank">29</a>]. Reads mapped to each gene were normalized to total read counts and lengths of each gene in kilobases (FPKM) and subsequently used as a measurement of expression levels. Statistical analyses and boxplotting were performed with MATLAB version R2017a.</p

Manhattan scatter plots of probabilities of 1358 SNPs in eIF4E genes and surrounding regions (+/- 5 kb) associated with CBSV responses.

<p>Informative SNPs within +/- 5 kb regions of each eIF4E genes were tested for their likelihood to be associated with the susceptible or resistant responses to CBSD. Negative log-transformed probability scores are plotted again the relative distances of each SNP in each chromosomes for each of the SNPs. Horizontal axis represents relative distances of each SNP from each other. SNPs from each eIF4E gene are color-coded. The orange and red lines indicate p-value thresholds of 0.01 and 0.005, respectively. Schematic of the gene model in each locus is depicted below the graph and color-coded. eIF4E gene in each locus in indicated by red rectangles. For each eIF4E gene, exons are represented by rectangle boxes while intros are indicated by lines.</p

Boxplot of eIF4E gene expression levels of CDSD-susceptible and tolerant cassava accessions.

<p>The 454 sequencing data were obtained from three CBSD-susceptible accessions (AR 37–80, AR40-6, and Mkombozi) and three CBSD-tolerant accessions (Kiroba, Nachinyaya, and Namikonga) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181998#pone.0181998.ref029" target="_blank">29</a>]. Reads mapped to each gene were normalized to total read counts and lengths of each gene in kilobases (FPKM) and subsequently used as a measurement of expression levels. Data from the susceptible and tolerant accessions were grouped together for statistical analysis. Each sample in the susceptible and tolerant group was considered as an independent sample. Statistical analyses and boxplotting were performed with MATLAB version R2017a.</p

RT-PCR amplification of five cassava eIF4E ORFs with gene-specific primers.

<p>Total RNA was extracted from TMS60444 cassava line. The first-strand cDNA was synthesized using SuperScript^™ III Reverse Transcriptase (Invitrogen) and PCR was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181998#pone.0181998.t001" target="_blank">Table 1</a>. Lane 1: DNA marker; Lane 2: 016601 CDS amplified with primers 016601F and 016601R; Lane 3: 016620 CDS amplified with primers 016620F and 016620R; Lane 4: 015501 CDS amplified with 015501F and 015501R, Lane 5: 013223 CDS amplified with primers 013223lF and 013223R, Lane 6, 013223 CDS amplified with primers 013223sF and 013223R; Lane 7: 013732 CDS amplified with 013732lF and 013732R; Lane 8: 013732 CDS amplified with 013732sF and 013732R; and Lane 9: negative water control.</p

Response of selected cassava accessions to CBSV/UCBSV infection^*.

<p>Response of selected cassava accessions to CBSV/UCBSV infection^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181998#t002fn001" target="_blank">*</a>}.</p