Table 1.
Primers used for SACMV DNA-A and DNA-B virulence proteins and PacBio SMRT sequence of NLR and LRR-PK/PL genes.
Fig 1.
South African cassava mosaic virus (SACMV) genome structure and the effect of SACMV on cassava leaf symptoms.
(a) A bipartite ssDNA-A and ssDNA-B genomic structure of SACMV. AC1: replication-associated protein (Rep); AC2: transcriptional activator protein (TrAP); AC3: replication enhancer protein (REn); AC4: silencing suppressor protein); AV1: coat protein (CP); AV2: pre-coat protein; BC1: movement protein (MP) and BV1: nuclear shuttle protein (NSP). (b) Leaf symptoms induced by SACMV manifested by leaf curl and yellow mosaic (arrows). In susceptible T200 symptoms persist at 32 and 67 dpi, while in Ukulinga 9 symptoms appear after 32 dpi (delayed susceptible phenotype). TME3 symptoms manifested at 32 dpi (tolerant, recovery phenotype) but the plant recovered at 67 dpi. TMS98/0505 exhibits no symptoms at any given time point post-infection (resistant genotype). (c) Viral load in T200, Ukulinga 9, TME3 and TMS98/0505 at 12 (pre-symptomatic stage), 32 (symptomatic stage) and 67 (recovery stage) dpi determined as DNA copies of CP using qPCR. Data represent the mean of three independent biological replicates. Error bars represent SD. An asterisk indicates a statistically significant difference according to unpaired Student’s t-test (two-tailed), * p ≤ 0.05. GTPb was used as a housekeeping gene.
Fig 2.
Differentially-expressed NLR genes in susceptible T200 and tolerant TME3 genotypes infected with SACMV.
(a) Total number of RNA transcripts DE in T200 and TME3 infected with SACMV at 12, 32 and 67 dpi. (b–d) A heatmap of DE NLR genes in T200 and TME3 infected plants at 12, 32 and 67 dpi. NL = nucleotide-binding LRR; RNL = coiled-coil NLR; CNL = RPW8-like coiled-coil NLR; and TNL = Toll/Interleukin-1 receptor/resistance NLRs. (e) Total number of NLR transcripts DE in T200 and TME3 infected with SACMV at 12, 32 and 67 dpi. (f) Overlap between DE genes in T200 and TME3 induced by SACMV at 12, 32 and 67 dpi. Values indicated in the Venn diagram present the number of downregulated and upregulated genes in T200 and TME3 compared to the mock infection. Transcripts were considered DE if the log2 fold-change > 0.5 and P ≤ 0.05. (n = 3 to 4 leaves collected from individual plants and one measurement per time point was taken and considered an overall representation of 3 biologicals). Numerical values in the heatmap indicate expression level calculated as F-value. Genes labelled in red and blue show significant upregulation and downregulation respectively in response to SACMV infection, compared to mock infection.
Table 2.
Expression levels of SACMV DNA-A and DNA-B encoded virulence factors in TME3 and T200 at 12, 32 and 67dpi measured by RT-qPCR.
Fig 3.
Transcript levels of MAPK, NIK, NLR and SA-dependent gene pathways and responsive genes.
Heatmap represents log2-transformed fold change of PTI, ETI and SA transcripts DE in T200 and TME3 SACMV infected and mock leaves at (a) 12, (b) 32 and (c) 67 dpi. Transcripts were considered DE if the log2 fold-change > 0.5 and P ≤ 0.05. Numerical values in the heatmap indicate expression level calculated as P-value. Genes labelled in red and blue show significant upregulation and downregulation respectively. (n = 3 to 4 leaves collected from individual plants and one measurement per time point was taken and considered an overall representation of 3 biologicals). RT-qPCR assay for PTI (d) MAPK, (e) NIK, (f) ETI and (g) SA key and response genes. Data represent the mean of three independent biological replicates. Error bars represent SD. An asterisk indicates a statistically significant difference according to unpaired Student’s t-test (two-tailed), * p ≤ 0.05. GTPb was used as a housekeeping gene.
Fig 4.
Expression profile of WRKY-TFs and ROS accumulation in susceptible T200 and tolerant TME3 cassava genotypes.
Total number of all DE RNA WRKY-TF transcripts (a) and ROS accumulation in T200 and TME3 leaves infected with SACMV at 12, 32 and 67 dpi (b). WRKY-TFs were considered DE if the log2 fold-change > 0.5 and p ≤ 0.05. (n = 3 to 5 leaves from individual plants combined and considered an overall representation of least 3 biological replicates. ROS data is the mean and SE of four independent biological replicates; significant differences in p values were determined by one-way ANOVA).
Table 3.
Total count of HiFi reads indicating gene variation in 24 cassava genotypes.
Fig 5.
Hypothetical model of the molecular mechanism underlying the ability of SACMV to suppress the host RNA silencing mechanism (PTGS) and systemic acquired resistance (SAR).
In susceptible response, SACMV AC2 encodes the transcriptional activator protein (TrAP) and suppresses host PTGS in both susceptible response (a) and tolerance/recovery response (b). During the late infection stage, suppression of host PTGS by SACMV is alleviated and nuclear shuttle protein (NSP) encoded by BV1 to promote virus cell-to-cell movement and inhibition of PTI in susceptible T200 (susceptible response) but the expression of NSP is suppressed in the tolerant/recovery response (TME3) possibly by NIKs, TNLs and WRKYs function through elevation of ROS levels and activation of SAR. PTGS: posttranscriptional gene silencing; NSP: nuclear shuttle protein; NIK: NSP-interacting kinase 1; SAR: systemic acquired resistance; ROS: Reactive oxygen species; TNL: N-terminal Toll/interleukin-1 receptor (TIR) domains; PTI: pathogen-triggered immunity.