Transcriptome profiling in susceptible model and natural host systems in response to South African cassava mosaic virus
Date
2014-02-07
Authors
Pierce, Erica Joanna
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Abstract
Geminiviruses causes diseases to many staple food and cash crops of great economic importance worldwide. Currently eight species of Begomoviruses belonging to the Geminivirus family exist, of which South African cassava mosaic virus SACMV-[ZA:99] is a member, and is known to cause cassava mosaic disease (CMD). Cassava (Manihot esculenta, Crantz) is considered to be an important food crop consumed in many tropical, sub-tropical and African countries, and is increasingly becoming well-known for its ethanol production on a global a scale. Various strategies to control CMD are currently being implemented, one of which is to elucidate mechanisms involved in host-virus interactions with the aim of identifying defence-related genes involved in the disease process. Many defence genes within the plant kingdom are evolutionary conserved, potentially providing methods of control not only to CMD but to other diseases as well. The research outlined in this thesis aimed to identify networks and pathways involved in disease susceptibility between the model plant host system, Arabidopsis thaliana and cassava T200 upon SACMV-[ZA:99] infection. Conclusions were also drawn from within host comparisons between susceptible cassava T200 and resistant cassava TME3 cultivars in order to explore if similarities, differences or common patterns of expression existed between genes governing resistance and susceptibility.
Before transcriptomic profiling studies were carried out, it was important to improve South African cassava mosaic virus (SACMV-[ZA:99]) and African cassava mosaic virus (ACMV-[NG:Ogo:90]) infection efficiencies in recalcitrant crop systems such as cassava. Susceptible cassava cultivars T200, TMS60444, and SM14334 were tested for these purposes following infection with three different Agrobacterium strains (C58C1; AGL1; LBA4404). Results demonstrated that an overall increase in infection efficiency was achieved for each genotype and virus tested, although with varying infectivity levels, suggesting that although an improved method was established, basal levels of susceptibility differed between genotypes and therefore it was not possible to achieve 100% infection efficiencies for agroinfection methods.
A 4 x 44k microarray whole genome study was then conducted to identify susceptible host genes involved in the interaction between the model plant system Arabidopsis thaliana and SACMV-[ZA:99]. An infectivity assay was carried out across three time points (14, 24, and 36 dpi), confirming that disease symptoms and virus infectivity levels correlated with an increase in differentially expressed transcripts across time points, with SACMV-[ZA:99] predominantly causing host-gene suppression. Many complex genes and pathways were disrupted and were shown to be involved in categories pertaining to stress and defence responses, phytohormone signalling pathways, cellular transport, metabolism and cell-cycle regulation strongly suggesting an attempt made by SACMV-[ZA:99] to affect homeostasis and antagonize host defence responses. This was the first geminivirus study identifying differentially expressed transcripts across 3 time points.
Next generation sequencing (NGS) using the ABI Solid platform was then carried out on SACMV-[ZA:99] – infected susceptible cassava T200 cultivar at 3 time points (12, 32, and 67 dpi), comparing infection responses to mock-inoculated healthy controls. Similarly to the Arabidopsis microarray study, findings from this analysis also revealed a shift from up-regulated to down-regulated genes across time points, once again reflecting virus-specific suppression on host genes suggesting SACMV-[ZA:99] specific alterations were induced in the host, regardless of the host (Arabidopsis and cassava T200) or platform (microarray and NGS) used. Genes identified pertaining particularly to the susceptible cassava T200 - SACMV-[ZA:99] interaction such as the disease resistance protein families (TIR-NBS-LRR), RPP1, RPM1, and NHO1 were showing down-regulation demonstrating that SACMV-[ZA:99] pathogenicity proteins may be causing this suppression leading to inactivation of basal immunity. Comparisons between tolerant cassava TME3 and susceptible T200 showed similarities and differences in responses between the cultivars. Many similarities such as cell wall precursor proteins and glutathione-S-transferases were up-regulated in both cultivars, which may be due to the host attempting to mount appropriate defences. Opposite patterns of expression was observed for genes in categories involved in transcription and phytohormone signalling such as WRKY‘s, NAC, JAZ, and ERF where suppression was evident in susceptible cassava T200, confirming the suppressive nature of SACMV-[ZA:99] to establish a replication-competent environment. Findings in this study contributed to the little that is known about geminivirus disease progression within a previously uncharacterised susceptible host such as cassava.