A time series transcriptome analysis of cassava (Manihot esculenta Crantz) varieties challenged with Ugandan cassava brown streak virus

Abstract

A time-course transcriptome analysis of two cassava varieties that are either resistant or susceptible to cassava brown streak disease (CBSD) was conducted using RNASeq, after graft inoculation with Ugandan cassava brown streak virus (UCBSV). From approximately 1.92 billion short reads, the largest number of differentially expressed genes (DEGs) was obtained in the resistant (Namikonga) variety at 2 days after grafting (dag) (3887 DEGs) and 5 dag (4911 DEGs). At the same time points, several defense response genes (encoding LRR-containing, NBARC-containing, pathogenesis-related, late embryogenesis abundant, selected transcription factors, chaperones, and heat shock proteins) were highly expressed in Namikonga. Also, defense-related GO terms of 'translational elongation', 'translation factor activity', 'ribosomal subunit' and 'phosphorelay signal transduction', were overrepresented in Namikonga at these time points. More reads corresponding to UCBSV sequences were recovered from the susceptible variety (Albert) (733 and 1660 read counts per million (cpm)) at 45 dag and 54 dag compared to Namikonga (10 and 117 cpm respectively). These findings suggest that Namikonga's resistance involves restriction of multiplication of UCBSV within the host. These findings can be used with other sources of evidence to identify candidate genes and biomarkers that would contribute substantially to knowledge-based resistance breeding.

Figure 1

Leaf and root symptoms of UCBSV-inoculated versus mock-inoculated Albert and Namikonga plants during the experiment. (A) Albert, UCBSV inoculated, 8 dag; (B) Albert, mock inoculated, 8 dag; (C) Namikonga, UCBSV inoculated, 8 dag; (D) Namikonga, mock inoculated, 8 dag; (E) Albert, UCBSV inoculated, 54 dag; (F) Namikonga, UCBSV inoculated, 54 dag; (G) Albert root, UCBSV inoculated, 3 mag; (H) Namikonga root, UCBSV inoculated, 3 mag; (I) Albert root, UCBSV inoculated, 12 mag; (J) Namikonga root, UCBSV inoculated, 12 mag.

Figure 2

Diagnostic RT-PCR for UCBSV in the inoculum source NDL06/132, Albert and Namikonga plants at 3 mag. RNA from cassava plants from the UCBSV-inoculation experiment were amplified using the UCBSV diagnostic primers CBSDDF2/CBSDDR, and the RT-PCR products were separated by agarose gel electrophoresis (2%). Lanes 1–3: UCBSV inoculum source (variety NDL06/132); lanes 4–6: Albert, mock grafted with virus-negative scions of NLD06/132; lanes 7–9: Albert, UCBSV inoculated; lanes 10–12: Namikonga, mock grafted with virus-negative scions of NLD06/132; lanes 13–15: Namikonga, UCBSV inoculated and M: 1 Kb+ ladder.

Figure 3

Number of UCBSV reads per million RNAseq reads retrieved from unmapped RNAseq reads of UCBSV inoculated (I) and control (C) samples from Albert (A) and Namikonga (N) varieties. In both varieties, UCBSV reads were only detected at 45 dag and 54 dag.

Figure 4

Number of DEGs between UCBSV-inoculated and mock-inoculated cassava varieties Albert and Namikonga at 6 hag, 1 dag, 2 dag, 5 dag, 8 dag, 45 dag and 54 dag. The DEGs were identified using the DESeq method.

Figure 5

Over-represented GO terms for up-regulated genes in Namikonga at 2 dag and 5 dag. A = Over-represented GO terms of the category “biological process” in the Namikonga up-regulated genes at 5 dag; B = Over-represented GO terms of the category “molecular function” in the Namikonga up-regulated genes at 5 dag; C = Over-represented GO terms of the category “cellular component” in the Namikonga up-regulated genes at 5 dag; D = Over-represented GO terms of the category “biological process” in the Namikonga up-regulated genes at 2 dag; E = Over-represented GO terms of the category “molecular function” in the Namikonga up-regulated genes at 2 dag. Results are presented for over-represented GO terms with adjusted p-values in the 95% confidence interval.

Figure 6

Selected DEGs that were expressed at significantly different levels in susceptible (Albert) and resistant (Namikonga) varieties at different time points (RNAseq data). Gene families include A and B = Leucine-rich repeat (LRR)-containing proteins, C and D = NBARC-containing proteins, E and F = Pathogenesis-related (PR) proteins, G and H = Late embryogenesis abundant (LEA) proteins, I and J = WRKY DNA, K and L = Heat shock proteins, M and N = Chaperones and O and P = Transcription factors.

Figure 7

Model of cassava responses to UCBSV based on transcriptomics. DEGs = differentially expressed genes; avg = average; dag = days after grafting and mag = months after grafting.