Assessing the role of DNA methylation in response of cassava to South African cassava mosaic virus

Abstract

Manihot esculenta Crantz (cassava) is a root crop vegetatively propagated by stem cuttings and serves as a dietary staple for poverty alleviation in tropical America, Asia and Africa. The tuber crop feeds hundreds of millions worldwide and has been positioned as the third most staple carbohydrate source in Africa. As with all crops, cassava is faced with challenges such as the geminivirus South African cassava mosaic virus (SACMV) which leads to cassava mosaic disease (CMD) that results in substantial losses to production and consequently having a negative impact on the economy. Cassava landraces T200 and TME3 display susceptibility and tolerance to SACMV, respectively. Furthermore, TME3 displays a recovery phenotype. Plant hosts have developed strategies to defend themselves against invading geminiviruses by employing epigenetic modifications via DNA methylation. The first aim of this study was to compare the SACMV viral load and global methylation status of T200 and TME3 during symptomatic (32 dpi) and recovery (in TME3; 67 dpi) phases of SACMV infection using relative qPCR, ELISA and Liquid Chromatography Mass Spectrometry (LCMS/MS), respectively. Infectivity results revealed that T200 plants had higher viral load than TME3 plants at 32 and 67 dpi. Global DNA methylation was higher in AGL1 control T200 plants at 32 and 67 dpi compared to TME3, suggesting that hypermethylation may be a hallmark of this landrace. This correlates with the global transcriptome results where a significantly higher number of genes are downregulated at 32 and 67 dpi in T200 compared to TME3. Global plant DNA methylation was significantly decreased in SACMV-inoculated T200 but marginally inTME3 at 32 and 67 dpi compared to AGL1-inoculated controls. This result suggests that SACMV targets/supresses methylation related genes/pathways to induce genes required for systemic spread and DNA replication. It is therefore concluded that hypomethylation is related to susceptibility. The second objective of the study was aimed at quantifying the expression levels of MET1, CMT3, AGO4, SAM and AGO5 in T200 and TME3 at 32 and 67 dpi. Relative qPCR revealed that SACMV infection led to decreased expression of MET1, CMT3, AGO4 and SAM in T200 and TME3 plants at 32 and 67 dpi compared to AGL-1, which correlated to hypomethylation observed in infected cassava. Interesting, the expression levels of AGO5 only increased at 67 dpi in TME3 plants at 67 dpi, suggesting a role in transcriptional gene silencing (TGS) known to be associated with recovery to geminiviruses in several plant hosts. In summary, our results demonstrate that SACMV causes a decrease in global DNA methylation in T200 and TME3 plants by decreasing MET1, CMT3, AGO4, and SAM expression leading to a decrease in de novo methylation. In conclusion, further analysis on the gene-specific methylation profile between T200 and TME3 landraces requires further study.