The role of small RNAs in susceptibility and tolerance to cassava mosaic disease

Rogans, Sarah Jane
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Cassava (Manihot esculenta, Crantz) is considered to be an important food security crop consumed by over a billion peoples globally, many who subsist on it. Cassava mosaic disease (CMD) is one of the main biotic and economically important constraints to cassava cultivation in sub-Saharan Africa. Geminiviruses are the casual agents of CMD and cause disease to many staple food and cash crops of great economic importance worldwide. There are currently 11 species of Begomoviruses that belong to the Geminiviridae family. South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite (DNA A and DNA B components) begomovirus belonging to the family Geminiviridae, and is one of the causal agents of cassava mosaic disease (CMD) endemic to southern Africa. Various strategies to control CMD are currently being investigated, one of which is cis-genics, which involves manipulation of endogenous host genes to combat viral pathogens. In order to achieve this, it is imperative to elucidate molecular mechanisms involved in host-virus interactions. Endogenous small RNAs (sRNAs), including microRNAs (miRNAs), have been found associated with gene regulatory mechanisms in response to virus infection. Amongst the non-coding host sRNAs targeting viruses are small interfering RNAs (siRNAs) associated with posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS), which are involved in the host RNA silencing pathway. The RNA silencing pathway is a highly conserved basal immunity pathway involved in host defence against plant viruses. The aim of this study was to identify siRNAs and miRNAs associated with gene regulatory mechanism in response to SACMV infection and to determine if they a play a role in the susceptible or recovery phenotype observed in SACMV tolerant cassava landrace TME3 or T200, respectively. Furthermore, virus-derived siRNA (vsRNA) populations targeting the DNA A and B components of SACMV were also investigated. MicroRNAs (miRNAs) are an important class of endogenous non-coding single-stranded small RNAs (21-24 nt in length), which serve as post-transcriptional negative regulators of gene expression in plants. Despite the economic importance of Manihot esculenta Crantz (cassava) only 153 putative cassava miRNAs (from multiple germplasm) are available to date in miRBase (V.21). Therefore, both conserved and novel miRNAs needed to be identified in cassava before we could determine what association they had with SACMV infection. In this part of the study, mature sequences of all known plant miRNAs were used as a query for homologous searches against cassava EST and GSS databases, and additional identification of novel and conserved miRNAs were gleaned from next generation sequencing (NGS) of two cassava landraces (T200 from southern Africa and TME3 from West Africa) at three different growth stages post explant transplantation and acclimatization. EST and GSS derived data revealed 259 and 32 conserved miRNAs in cassava, and one of the miRNA families (miR2118) from previous studies has not been reported in cassava. NGS data collectively displayed expression of 289 conserved miRNAs in leaf tissue, of which 230 had not been reported previously. Of the 289 conserved miRNAs identified in T200 and TME3, 208 were isomiRs. Thirty-nine novel cassava-specific miRNAs of low abundance, belonging to 29 families, were identified. Thirty-eight (98.6%) of the putative new miRNAs identified by NGS have not been previously reported in cassava. Several miRNA targets were identified in T200 and TME3, highlighting differential temporal miRNA expression between the two cassava landraces. This study contributes to the expanding knowledge base of the micronome of this important crop. MicroRNAs play a crucial role in stress response in plants, including biotic stress caused by viral infection. Viruses however can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of miRNAs. This study aimed to understand the regulation of miRNAs in tolerant (TME3) and susceptible (T200) cassava landraces infected with SACMV. Next-generation sequencing was used for analysing small RNA libraries from infected and mock-inoculated cassava leaf tissue collected at 12, 32 and 67 dpi (days post-inoculation). The total number of differentially expressed miRNAs (normalized against mock-inoculated samples) across all three time points was 204 and 209 miRNAs, in TME3 and T200 infected plants, respectively, but the patterns of log2fold changes in miRNA families over the course of infection differed between the two landraces. A high number were significantly altered at 32 dpi when T200 and TME3 plants showed severe symptoms. Notably, in T200 69% and 28 (100%) of miRNA families were upregulated at 12 and 32 dpi, respectively. In contrast, TME3 showed an early pre-symptomatic response at 12 dpi where a high number (87%) of miRNAs showed a significant log2fold downregulation. Endogenous targets were predicted in the cassava genome for many of the identified miRNA families including transcription factors, disease resistance (R)-genes and transposable elements. Interestingly, some of the miRNA families (miR162, miR168 and miR403) that were significantly affected in both T200 and TME3 upon SACMV infection were shown to target proteins (DCL1, AGO1 and AGO2) that play important roles in the RNA silencing pathway. From results, we suggest that the early (12 dpi) miRNA response to SACMV in TME3 appears to involve PTGS-associated AGO1, DCL2 and a cohort of R genes belonging to the miR395 family which may prime the plant for tolerance and recovery downstream, while in T200, SACMV suppresses AGO1, AGO2 (at 32 and 67 dpi), and DCL2 (32 dpi) mediated RNA silencing, leading to severe persistent disease symptoms. This study provides insights into miRNA-mediated SACMV cassava interactions and may provide novel targets for control strategies aimed at developing CMD-resistance cassava varieties Endogenous small RNAs (sRNAs) associated with gene regulatory mechanisms respond to virus infection, and virus-derived small interfering RNAs (vsRNAs) have been implicated in recovery or symptom remission in some geminivirus-host interactions. Transcriptional gene silencing (TGS) (24 nt vsRNAs) and post transcriptional gene silencing (PTGS) (21-23 nt vsRNAs) have been associated with geminivirus intergenic (IR) and coding regions, respectively. In this Illumina deep sequencing study, we compared for the first time, the small RNA response to South African cassava mosaic virus (SACMV) of cassava landrace TME3 which shows a recovery and tolerant phenotype, and T200, a highly susceptible landrace. Interestingly, different patterns in the percentage of SACMV-induced normalized total endogenous sRNA reads were observed between T200 and TME3. Notably, in T200 there was a significant increase in 21 nt sRNAs during the early pre-symptomatic response (12 dpi) to SACMV compared to mock, while in TME3, the 22 nt size class increased significantly at 32 dpi. While vsRNAs of 21 to 24 nt size classes covered the entire SACMV DNA- A and DNA-B genome components in T200 and TME3, vsRNA population counts were significantly lower at 32 (symptomatic stage) and 67 dpi in tolerant TME3 compared with T200 (non-recovery). It is suggested that the high accumulation of primary vsRNAs, which correlated with high virus titres and severe symptoms in susceptible T200, may be due to failure to target SACMV-derived mRNA. In contrast, in TME3 low vsRNA counts may represent efficient PTGS of viral mRNA, leading to a depletion/sequestration of vsRNA populations, supporting a role for PTGS in tolerance/recovery in TME3. Notably, in TME3 at recovery (67 dpi) the percentage (expressed as a percentage of total vsRNA counts) of redundant and non-redundant (unique) 24 nt vsRNAs increased significantly. Since methylation of the SACMV genome was not detected by bisulfite sequencing, and vsRNA counts targeting the IR (where the promoters reside) were very low in both the tolerant or susceptible landraces, we conclude that 24 nt vsRNA-mediated RNA directed genome methylation does not play a central role in disease phenotype in these landraces, notwithstanding recognition for a possible role in histone modification in TME3. This work represents an important step toward understanding variable roles of sRNAs in different cassava genotype-geminivirus interactions. Also, by comparing the differences between a tolerant and susceptible host the aim is to achieve better understanding of the effect of pathogens on host sRNAome, an area that is deserving of me attention in plant systems. The expectation is that these findings presented in the PhD will contribute to the long-term goals of devising new methods of disease control against SACMV and understanding the complex interconnected mechanisms involved in virus-host interactome.
A dissertation presented by Sarah Jane Rogans to The Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Molecular and Cell Biology. 2016
Rogans, Sarah Jane (2016) The role of small RNAs in susceptibility and tolerance to cassava mosaic disease, University of Witwatersrand, Johannesburg,>