Cassava (Manihot esculenta Crantz) is affected by two major viral diseases, namely Cassava brown streak disease (CBSD) and Cassava mosaic disease (CMD). Two of the most widely distributed begomoviruses in East Africa associated with CMD, are East African cassava virus- Uganda2 (EACMV-UG2) and African cassava mosaic virus (ACMV). Despite efforts of generating improved Tropical Manihot Series (TMS) by traditional breeding and using highly resistant geminivirus cassava landraces such as Tropical Manihot Esculenta1 (TME1) and Tropical Manihot Esculenta3 (TME3), more recently two circular single stranded (ss) satellite-like DNA molecules (episomal DNA-II and DNA-III) have been found to be associated with CMD and are able to break resistance to EACMV-UG2 and enhance virus symptoms. The nature of these satellite-like DNA molecules is unknown, and furthermore, the discovery of integration of partial copies of DNA molecules (DNA-II and III fragments), and evidence for transcription from cassava Expressed Sequence Tag (EST) database screening, has led to an even more perplexing disease complex. In the present study, we attempted to further explore the interaction between the satellite-like DNAs and their associated cassava-infecting begomoviruses by investigating the impact of these DNA molecules on disease development in TME3 (tolerant) and cv. 60444 (susceptible) cassava cultivars, and to also gather biological evidence for transcription of integrated genomic and episomal (putative predicted ORFs) sequences in the ACMV and EACMV-UG2-associated DNA-II and DNA-III. Biolistic inoculation of EACMV-UG2, ACMV, and in co-bombardment with DNA-II, DNA-III, DNA-II + DNA-III was successfully performed. CMD symptoms were developed earlier on cassava plants inoculated with ACMV + DNA-II, ACMV + DNA-III, ACMV + DNA-II + DNA-III and EACMV-UG2 + DNA-II, EACMV-UG2 + DNA-III, EACMV-UG2 + DNA-II + DNA-III molecules compared with cassava plants inoculated with begomoviruses alone. Additionally, CMD symptoms were more severe in cv.60444 compared to TME3 when inoculated with begomoviruses alone, or in combination with DNA-II, DNA-III and DNA-II + DNA-III molecules. DNA-II and III were able to break resistance to the highly CMD-tolerant cassava landrace, TME3, and enhance virus symptoms. In order to confirm EST-generated evidence for transcription of DNA-II and III fragments, cDNA was subjected to RT-PCR. RT-PCR of transcripts was successful for only three putative ORFs: ORF C4 of the antisense DNA-II strand, ORF V1 on sense DNA-II strand, and ORF C2 on antisense strand for DNA-III. Primers for transcripts amplified 250 bp and 220 bp for ORF C4 of DNA-II and ORF V1 of DNA-III, respectively. Transcribed ORFs were confirmed by sequencing, and the sequences were similar to the published sequences of Begomovirus associated DNA-II satellite and Begomovirus associated DNA-III satellite, respectively. These results showed that at least two putative ORFs for DNA-II and one (the largest ORF VI) DNA-III can be transcribed. Furthermore, surveys were undertaken in order to ascertain the distribution of episomal and integrated DNA-II and III in cassava germplasm from several countries, namely Tanzania, Uganda, Kenya and Rwanda. Results from this research successfully established genetic diversity and wide geographical distribution of integrated DNA-II and DNA-III molecules. Two primer pairs were designed from a central conserved sequence found in all the integrated DNA-II or III fragments identified from the cDNA libraries (EST database). These primers also amplified integrated sequences of expected size in cassava accessions and wild Manihot species which were similar to satellite-like sequence occurrences in the ESTs. Using designed primers, PCR amplification yielded integrated DNA-II and DNA-III products of ~895 bp and ~306 bp, respectively. Analysis of 363 field leaf samples detected the presence of DNA-II or DNA-III from Kenya (3.3% or 8.3%), Uganda (18% or 2.5%), Rwanda (6.5% or 19.6%) and Tanzania (5.7% or 11.9%) , results which were confirmed by analysis of the sequenced PCR amplicons. Detection of both DNA-II and DNA-III molecules on the samples collected was also found from Kenya (73%), Uganda (69.1%), Rwanda (50%) and Tanzania (69.3%). Interestingly integrated DNA-II and II copies were amplified from healthy, symptomless and infected cassava samples. DNA-II sequences did not vary significantly (93.3% - 99.8%) and were highly similar to the sequences of Begomovirus associated sat DNA-II (AY836366) and 99% with mentha leaf deformity disease associated satellite DNA-II, while DNA-III sequences and Begomovirus associated DNA-II satellite (AY833667). In conclusion, this study has provided useful information that contributes to a further understanding of the biological function of integrated and episomal DNA-II and III molecules in begomoviruses infected cassava plant. However the relationship, if any between episomal and integrated forms needs to be established in future, and investigation into whether the transcribed ORFs can produce functional proteins, needs to be undertaken. How DNA-II and III interact with EACMV-UG2 and ACMV in disease modulation remains to be explored, and the replication of episomal DNA-II and III by these associated begomoviruses needs to be confirmed if these DNA molecules are to truly show a satellite-like relationship. Furthermore, the findings in this study that partial and varied-sized integrated DNA-II and III fragments occur widely in healthy and infected cassava germplasm will enable researchers (plant virologists and breeders) working on cassava in Sub Saharan Africa (SSA) to explore this complex more deeply in order to develop durable management strategies for CMD.

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.

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 / 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. / LG2017

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science in the School of Molecular and Cell Biology. Johannesburg 2017 / With growing populations and climate change associated drought predicted for the future, cassava can provide one solution for food security and a source of starch for industrial use and biofuels in South Africa, and other countries in the SADC region. One of the severe constraints on cassava production is cassava mosaic disease (CMD) caused by cassava infecting begomoviruse species, including African cassava mosaic virus (ACMV), South African cassava mosaic virus (SACMV) and East African cassava mosaic virus (EACMV). Cassava begomoviruses (CBVs) are responsible for significant yield loss of the starchy tubers. Since no chemical control of virus diseases of plants is possible, one approach to develop virus resistance is via biotechnology, through genetic engineering (GE) of cassava with hairpin RNA (hpRNA) silencing constructs that express small interfering RNAs targeting CBVs and preventing severe disease development. The aim of this project was to subject previously transformed five CMM6 cassava lines (cv. 60444 transformed with a non-mismatched Africa cassava mosaic virus-[Nigeria:Ogorocco;1990] (ACMV-[NG:Ogo:90])-derived hpRNA construct, six AMM2 (cv. 60444 transformed with a mismatched ACMV-[NG:Ogo:90]-derived hpRNA construct), six CMM8 cassava lines (cv.60444 transformed with a non-mismatched SACMV BC1-derived hpRNA construct) and seven AMM4 cassava lines (cv.604444 transformed with a mismatched SACMV BC1-derived hpRNA construct) to reproducible trials, and evaluate for response to virus challenge. The ACMV-[NG:Ogo:90] hpRNAi constructs target 4 overlapping virus open reading frames (ORFs) (AC1 replication associated protein/AC4 and AC2 transcriptional/AC3 replication enhancer), while the SACMV hpRNAi constructs target the cell-to cell movement BC1 ORF. Non mismatched constructs consist of a transformation cassette that has an intron separating the sense and antisense arms of the viral transgene whilst mismatched constructs have the sense arm of the viral transgene treated with bisulfite to induce base mutation. This mutated sense arm is then separated from the non mutated antisense arm by a small spacer. Furthermore, a 229 bp inverted repeat hpRNA construct (DM-AES) was designed to target ACMV-[NG:Ogo:90] 117 nt putative promoter region (2714-49 nt), a 91 nt overlapping sequence (1530-1620 nt) between ACMV-[NG:Ogo:90] AC1 3’ end and AC2 5’ end (AC1 3’/AC2 5’-ter) as well as being efficient against SACMV and EACMV due to the inclusion of a 21 nt conserved sequence (1970-1990) of AC1/Rep shared between ACMV, EACMV and SACMV. Cassava landrace T200 friable embryogenic callus (FEC) were transformed with this construct. The selected transgenic lines were infected with either ACMV-[NG:Ogo:90] (CMM6 and AMM2 transgenic lines) or SACMV (CMM8 and AMM4 transgenic lines) by agro-inoculation and monitored at 14, 36 and 56, 180 and 365 days post infection (dpi) for symptom development, plant growth and viral load. From the ACMV trials 3 lines (CMM6-2, CMM6-6 and line AMM2-52) showed significantly lower symptom scores and lower viral load at 36, 56 and 365 dpi, compared with viral challenged untransgenic cv.60444. This phenotype is described as tolerance, not resistance, as despite ameleriorated symptoms virus replication persists at lower levels. From the SACMV infectivity trials even though all CMM8 and AMM4 transgenic lines had lower symptom severities and viral loads compared with infected untransformed cv.60444, the results were not highly significant (p˃ 0.05). From this study, tolerance or reduction of viral load and symptoms was attributed to the accumulation of transgene-derived siRNAs prior to infection. However there was no observable correlation between levels (semi-qauntitative northern blots) of siRNAs and tolerance or susceptible phenotypes. Tuber yield evaluation of the three tolerant lines (CMM6-2, CMM6-6 and line AMM2-52) showed that the tuber fresh and dry weight at 365 dpi was not affected by the viral presence. These are promising lines for larger greenhouse and field trials. A comparison between the two different constructs showed that the two tolerant CMM6 lines-2 and 6 appeared to perform better (viral load) compared with AMM2 tolerant line-52 with regards to levels of viral amplification. The mismatched construct in AMM4 lines and the nonmismatched construct in CMM8 lines induced the same viral and symptom severity score (sss) reduction. Transformation of T200 FECs with the DM-AES construct was unsuccessful due to the age (more than six months old) of the FECs. FECs are more likely to lose their regeneration and totipotent nature with age. We therefore propose the use of fresh T200 FECs in future transformation studies to test the DM-AES construct. / MT2017

Cassava (Manihot esculenta Crantz) is affected by two major viral diseases, namely Cassava brown streak disease (CBSD) and Cassava mosaic disease (CMD). Accurate and efficient detection and identification of plant viruses are fundamental aspects of virus diagnosis leading to sustainable disease management. In the present study I describe two techniques, the first based on a single tube duplex and multiplex polymerase chain reaction (m-PCR), developed for simultaneous detection of African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV) and East African cassava mosaic Malawi virus (EACMMV), and second, a technique based on Restriction Fragment Length Polymorphism (RFLP) analysis of Reverse Transcribed (RT) -PCR amplified Cassava brown streak viruses species, Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV). In this work, the single tube duplex and multiplex PCR for simultaneous detection of the four cassava mosaic begomoviruses (CMBs) was developed successfully. Four primer pairs were designed from published DNA-A component sequences targeting specific amplification of the four cassava mosaic begomoviruses (CMBs). Evaluation of the primers sensitivity in serially diluted virus samples revealed that the new primers amplified their target virus to a dilution of 10-4 and 10-3 for uniplex and multiplex PCR respectively. Developed multiplex assay enabled specific amplification of the viruses in producing 950, 503, 435 and 260 base pairs (bp) for ACMV, EACMMV, EACMCV and EACMZV respectively in single and mixed infections of CBSVs. Analysis of 172 field samples from Kenya, Malawi, Mozambique, Rwanda, Tanzania and Zambia detected both single and mixed infections, results which were proved by analysis of the sequenced amplicons. Second, a technique based on 2 Restriction Fragment Length Polymorphism (RFLP) analysis of RT-PCR amplified cassava brown streak viruses, Cassava brown streak virus (CBSV) and cassava brown streak Uganda virus (CBSUV), was performed. A degenerate primer amplifying 785 bp of the coat protein gene (CP) of CBSV and CBSUV was also designed. Two restriction endonucleases, HindIII and EcoR1 (identified by a software package, Vector NTI® Express v1.0 from Life Technologies/Invitrogen), which produce different fragments upon digestion of RT-PCR amplicons from CBSV and CBSUV, were used to distinguish the two viruses RFLP analysis using EcoRI has no site in CBSV producing one fragment (785 bp), two fragments (525 bp and 224 bp) for CBSUV and three fragments (785, 525 and 224 bp) for the mixed infections. On the other hand, HindIII has no site in CBSUV producing one fragment (785 bp), three fragments (437 bp, 267 bp and 81 bp) were produced for CBSV, and four fragments (785, 437, 267 and 81 bp) for CBSV and CBSUV mixed infections. In both multiplex and RFLP analyses, results from the sequenced PCR/RT-PCR amplicons agreed with sequence identities of the respective published virus species. Experience from using developed multiplex and RFLP techniques show that time was saved and amount of reagents used were reduced. RFLPs confirmed the presence of CBSV and CBSUV in RT-PCR amplicons without requirement for sequencing. Additionally, modified protocols from Dellaporta et al. (1983) and Chang et al. (1993), were used to extract DNA and RNA respectively from dry and fresh cassava leaves with comparable results. I also demonstrated a method of collecting and preserving cassava leaf samples to retain their integrity during storage for a period of over one month. The two diagnostic tools can be used routinely in germplasm indexing, disease surveillance, and disease monitoring programs 3 Problem Statement and Rationale In east and southern Africa, cassava (Manihot esculenta Crantz) is one of the leading crops in terms of production and has become an important source of income to households and small-scale farmers. However, the production across the region is greatly affected by Cassava mosaic disease (CMD) and Cassava brown streak disease (CBSD). Reports from different authors (Gibson. 1996; Ogbe et al., 1996; Legg et al., 1999; Fondong et al., 2000; Bisimwa et al., 2012) have reported the occurrence of CMD in different countries in the SSA. In Tanzania, CMD has been reported from many locations. Comprehensive characterization by Ndunguru et al. (2005) showed seven cassava mosaic geminiviruses species occur in Tanzania. Mbanzibwa et al. (2009a) reported prevalence of two potyvirus species causing CBSD in the Lake Victoria basin and along the coastal belt of Indian Ocean. A countrywide survey of all major cassava-growing areas in Kenya by Bull et al. (2006) reported presence of six CMG species with novel begomoviruses and a new recombinant strain of EACMV, demonstrating increasing diversity and geographical distribution of CMGs. Similarly, recent reemergence of CBSD has been reported in many districts in Uganda (Alicai et al., 2007) as well as from Malawi (Winter et al., 2010), Kenya (Mware et al., 2009) and Rwanda (Shirima et al., 2012). No reports of occurrence of CBSD have been reported from Zambia. With the current development of more robust diagnostic tools such as RT-PCR and real-time PCR, the diagnosis of CMD and CBSD has also improved in many cassava- producing countries. Similarly, the challenges to obtain more sensitive broad-spectrum cost-effective diagnostic tools also increase. This is evident following discovery of more 4 virus species causing CMD and CBSD (Mbanzibwa et al., 2009a and Winter et al., 2010) which can easily be overlooked. In the field the co-infections of many CMBs and CBSVs is common. Therefore, it will require several tools to detect the multiple infections using the diagnostic tools currently available. Thus, development of efficient and affordable diagnostic tools for simultaneous detection and identification of CMBs and CBSVs is vital and will have a significant impact on development and implementation of cassava virus disease management. Diagnostics will be used for disease monitoring in cassava multiplication plots production and distribution of disease- free cassava planting materials. Therefore, this research make use of the available sequence information in the database for both CMBs and CBSVs to develop sensitive tools for the simultaneous detection of four species of cassava begomoviruses namely: African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV) and East African cassava Mosaic Zanzibar Virus (EACMZV) using multiplex PCR. Also identification and differentiation of two species of Cassava brown streak viruses namely Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV) by RT-PCR/RFLP approach. This study generated knowledge and new tools that will enhance the diagnosis of both CMD and CBSD. The tools will facilitate deployment of virus-indexed cassava planting materials within the region.

A dissertation submitted to the Faculty of Science of the University of Witwatersrand, Johannesburg, in full fulfilment of the requirements for the degree of Master of Science, 2019 / Cassava, Manihot esculenta Crantz, is categorized as a food security crop, producing large starchy tubers that are gaining interest from both international and local agro-processing industries for products such as bioethanol, textiles, and food additives. However, cassava is currently under threat from a group of begomoviruses that cause cassava mosaic disease (CMD) in all countries in sub-Saharan Africa where cassava is cultivated. CMD can result in up to 100% crop loss. South African cassava mosaic virus (SACMV) is particularly a threat to the growing cassava industry in southern Africa. Despite extensive breeding programs over the past 70 years to develop CMD-resistant farmer-preferred cassava landraces, total resistance has not been achieved. Furthermore, the high mutational rates of begomoviruses, and mixed infections in the field, have exacerbated the problem. TME3 is a West African landrace that displays tolerance to begomoviruses, including SACMV. Infection of TME3 by SACMV leads to recovery, hallmarked by low virus loads and milder symptoms compared to a susceptible southern African landrace T200. The molecular processes that govern tolerance in crops, including cassava, are not well understood. However, systemic immune responses, which are controlled by hormoneresponsive transcription factors (TFs), are required by the plant to successfully combat an invading pathogen. Two different branches of systemic immunity have been described, namely systemic acquired resistance (SAR), facilitated by salicylic acid (SA) signalling, and induced systemic resistance (ISR), which is induced through jasmonic acid (JA) and ethylene (ET) signalling in the presence of beneficial rhizobacteria. In 2014, Allie et al. compared global transcriptomic responses occurring in TME3 and the T200 during early 12 days’ post inoculation (dpi), middle (32 dpi) and late (67 dpi) stages of SACMV infection. In order to give greater context to transcriptomic data, which is inheritably large and complex, network analysis may be implemented. By placing the differentially expressed (DE) gene homologs/orthologs identified from the cassava transcriptome datasets into protein-protein networks, functions of SACMV-responsive genes, interacting partners, and potential hubs, can be derived. Cassava gene functions are based on the model crop Arabidopsis thaliana, as despite the sequencing of the cassava genome, the annotations are incomplete. The aim of this study was to identify potential candidate TFs, and their associated hormones and other network partners, that confer either tolerance (TME3) or susceptibility (T200) to SACMV. / TL (2020)

Bemisia tabaci is the vector of cassava mosaic begomoviruses and cassava brown streak viruses which are main production constraints to cassava in sub-Saharan Africa. Current vector dynamics involved in the spread of both viruses in the region was established through comparison of the mitochondria cytochrome oxidase I DNA. Two distinct species were obtained: sub-Saharan Africa clade 1 (SSA1), comprising of two sub-clades (I & II), and a South West Indian Ocean Islands (SWIO) species. SSA1 sub-clade I whiteflies were widely distributed in East Africa. SSA1 sub-clade II whiteflies predominated the coast regions of Kenya, southern & coast regions of Tanzania and widespread in Uganda. SWIO whiteflies occurred in the coastal region of Kenya. This study also revealed that SSA1 sub-clade I haplotypes performed significantly better than SSA1 sub-clade II haplotypes with respect to mean number of eggs laid, developing instars and hatched adults on healthy, African cassava mosaic virus-[Tanzania:2001 ] and East African cassava mosaic Kenya virus-infected plants. There was no boost in whitefly numbers by the CMB-infected plants. The fecundity and development differences observed between SSA1 sub-clade I and II haplotypes have major epidemiology implications on the CMGs in the region

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2013. / Unable to load abstract.

A thesis submitted 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. Johannesburg, 2015. / With rising global food prices, growing populations, climate change and future demand for tuber crops for feed and potential energy source, cassava is well positioned to meet the needs of many countries in the SADC region, including South Africa. However a major constraint to cassava cultivation is cassava infecting begomoviruses (CBVs), including African cassava mosaic virus (ACMV) and South African cassava mosaic virus (SACMV). ACMV and SACMV belong to the family Geminiviridae, comprising of circular single-stranded bipartite. Symptoms associated with CBVs infection include yellow and/or green mosaic, leaf deformation, leaf curling and stunted plant growth. Since no chemical control of virus diseases of plants is possible, one approach to develop virus resistance is via biotechnology, through genetic engineering (GE) of cassava to express hairpin RNA (hpRNA) silencing constructs against CBV. However cassava is recalcitrant and difficult to transform and regenerate. The aim of this study was to produce hpRNA/inverted repeat (IR) hpRNA constructs targeting ACMV AC1/4:AC2/3 open reading frames (ORF) and hpRNA targeting SACMV BC1 ORF to engineer hpRNA expressing transgenic cassava resistant to ACMV and SACMV. Furthermore, the approach was to stack two ACMV contiguous overlapping reading frames (AC1/4) and (AC2/3) in an attempt to improve resistance to CBV. However IR sequences are prone to unfavourable tight secondary structure formation known as cruciform structures. To circumvent this, one set of constructs (mutated sense-arm: mismatch constructs) were designed to contain sodium bisulfite deamination-induced mutations in the hairpin sense-arm making it less complementary to the antisense arm and therefore enhancing IR stability and cruciform junction formation. MM2hp (mismatch construct targeting ACMV AC1/4:AC2/3) and MM4hp (mismatch construct targeting SACMV BC1) were generated. The second construct set, non-mismatch: gateway, was designed based on the most currently used Gateway construct system. Gateway constructs contained an intron positioned between the IR fragments. MM6hp (non-mismatch construct targeting ACMV AC1/4:AC2/3) and MM6hp (non-mismatch construct targeting SACMV BC1) were generated. Similar to the deamination-induced mutations, the intron assisted with IR stability. ACMV- or SACMV-derived hpRNA constructs were transformed into model cassava cultivar cv.60444. Additionally, since few farmer-preferred cultivars or landraces have been transformed for resistance, South African high starch landrace T200 was also transformed with the hpRNA constructs. Agrobacterium-mediated transformation of friable embryogenic callus (FEC) was used and plants regenerated. Several transgenic cv.60444 and T200 lines were regenerated. Cassava landraces are generally less amenable to transformation however were able to report 79 % and 76 % for model cv.60444 and landrace T200, respectively. T200 transformation efficiency reported in this study is 43% higher than previously reported. This is also the first report of South African cassava landrace T200 transformation with ACMV and SACMV-derived hpRNA constructs. Transgenic lines were selected and infected with ACMV and SACMV infectious virus clones. Lines were then monitored at 12, 32 and 67 days post infection (dpi) for symptom development, plant growth and SACMV and ACMV viral load. At 67 dpi, a more significant difference between transgenic lines and untransformed infected cv.60444 was observed. At 67 dpi, 69 % and 75% of ACMV AC1/4:AC2/3 and SACMV BC1 transgenic lines, respectively, showed lower symptoms and reduced viral load compared to control susceptible wild-type cv.60444, but comparable to virus-challenged non-transgenic tolerant landrace control TME3. Notably, a lack of correlation between viral load and symptoms was not always observed. Plant to plant variation was observed between individual transgenic lines generated from each construct (MM2hp; MM4hp; MM6hp and MM8hp) transformation events (A-MM2, A-MM4, C-MM6 and C-MM8). However, overall a positive correlation between symptoms and viral load was observed for virus challenge trials of transgenic lines generated from A-MM4, C-MM6 and C-MM8 transformation events, this overall positive correlation was observed at all 3 dpi (12, 32 and 67 dpi). A number of ACMV and SACMV tolerant transgenic lines were obtained for both mismatch and non-mismatch hpRNA expressing transgenic lines, where virus replication persisted, but symptoms were lower at 67 dpi compared to non-transgenic plants. CBV tolerance levels observed in transgenic lines expressing mismatch technology hpRNA was not significantly different to CBV tolerance levels observed in transgenic lines expressing non-mismatch hpRNA. Expression of ACMV and SACMV- derived constructs generated tolerant cassava lines, where tolerance is defined as plants displaying virus replication but lower to no symptoms. In addition to this, a recovery phenotype was observed in five MM2hp (ACMV AC1/4:AC2/4)- derived hp expressing transgenic lines at 365 dpi, where recovery is defined as no to mild symptoms after an initial period of symptoms, and a reduction in or no viral load. In five MM4hp (SACMV BC1)-derived hpRNA expressing transgenic lines, complete recovery was observed at 365 dpi; no symptoms and no detectable virus. From this study we propose that expression of CBV- derived hpRNA targeting ACMV AC1/4:AC2/4 and SACMV BC1 in CBV susceptible cv.60444 enhances cv.60444 ACMV and SACMV tolerance. Mismatch (mutated sense-arm) construct technology offered tolerance levels comparable to the more conventional and more expensive non-mismatch (Gateway) technology. We therefore also propose that the use of mismatch hpRNA technology in cassava genetic engineering can be used as an alternative approach to transgenic crop production. Promising transgenic lines, showing moderate SACMV and ACMV resistance, were identified and these will be used in further trials as they could be considered favourable to farmers.

Cassava Manihot esculenta Crantz (Euphorbiaceae), is a source of food for more than 700 million people in developing countries and is cultivated in estimated global area of 18.6 million hectares with total annual production of 238 million tonnes. Diseases however, take a substantial toll of yield, with CMD being the most important disease and major constraint for cassava production in Tanzania and Africa. The disease causes an estimated loss of over US$ 14 million per annum. A study was undertaken in 2006/2007 to investigate the influence of satellite DNA molecules on severity of cassava begomoviruses and the breakdown of resistance to cassava mosaic disease (CMD) in Tanzania. The goal was to appraise the nature of resistance to CMD in indigenous and improved cassava cultivars in the presence of resistance-breaking satellites. Three specific aims were earmarked: to identify and characterize cassava mosaic virus isolates and satellite DNA molecules in major cassava growing areas of Tanzania; to screen cassava cultivars for resistance to begomoviruses in presence and absence of the satellite DNA molecules; and to determine the nature of interaction between begomovirus DNAs and Satellite DNA molecules in Nicotiana benthamiana. To achieve these aims, a survey was done in the major cassava growing areas of Tanzania to investigate occurrence of cassava mosaic begomoviruses and associated satellites namely, SatDNA-II and SatDNA-III. Stems from plants showing CMD symptoms were collected from field. The stems were re-planted in screenhouse to study more about the symptoms. Symptomatic leaves from sprouting cuttings were collected for DNA extraction to be used in two downstream assays - amplification of EACMV, ACMV, SatDNA-II and SatDNA-III by polymerase chain reaction (PCR) and sequencing. In another experiment to evaluate cassava cultivars for resistance to CMD in presence of satellites, stem cuttings of the classical CMD-resistant cultivars were planted in greenhouse. Infectious clones of EACMV-TZ and EACMV-UG2 comprising both DNA-A and DNA-B components of bipartite begomoviruses (EACMV-TZ and EACMV-UG2) as well as infectious clones of SatDNA-II and SatDNA-III were bombarded onto the greenhouse cassava plants using a gene gun. Emerging disease symptoms on inoculated plants were scored using standard procedure. Total nucleic acid extraction from the inoculated plants was done and PCR was performed to amplify AC1 and βC1 genes as well as full length SatDNA-II and SatDNA-III. Southern blot analysis was performed to determine the presence of AC1, βC1, SatDNA-II and SatDNA-III on the DNA. In order to study interaction between cassava mosaic begomovirus (EACMV-TZ) and satellites, infectious clones of EACMV-TZ (DNA-A and DNA-B) and that of SatDNA-II and SatDNA-III were used. The clones (DNAs) were used to infect Nicotiana benthamiana by abrasion. Inoculated plants were covered with a plastic dome and placed in insect-free growth chamber for symptom development, which were scored on a standard scale of 1 to 5. Total DNA was extracted from the N. benthamiana leaves and used for Southern blot analysis. Results from the field survey showed that disease incidences varied from 60 to 90% in the Lake Victoria Zone and from 10 to 90% in the Eastern Zone. Cultivar Lyongo had the highest disease symptom severity in the Lake Victoria Zone while in the Eastern zone plants with high severity levels were from cvs Maiza and Tabora. In the screenhouse, some sprouted cuttings remained healthy up to 16 days after planting (DAP) and others recovered from the disease. Reversion was also observed in some cultivars. Using PCR, East African cassava mosaic Tanzania virus (EACMV-TZ) was amplified from 72.8% of tested samples while African cassava mosaic virus (ACMV) was amplified from 4.3%. Five percent of plants had dual infection of the two viruses. While ACMV was detected in samples collected from Lake Victoria, EACMV-TZ was mostly found on samples from the Eastern zone. Sequencing showed the presence of two new virus isolates: EACMV-TZ [TZ113] and EACMV-TZ [TZ108]. Seventy five percent of plants, which showed reversion of symptoms, contained SatDNA-II. It was found that full length SatDNA-II occurred in both zones, while SatDNA-III was exclusive to the Lake Zone. Multiple DNA bands were noted in PCR agarose gels, more so in SatDNA-II than SatDNA-III. For SatDNA-II, the multiple bands were more evident for samples collected from Eastern zone than for those from the Lake Zone. Using primers based on expressed sequence tags (EST-primers) for SatDNA-II (895 bp) and SatDNA-III (306 bp), genome integrated forms of the satellites were amplified from 68% and 71.17% of samples, respectively. Thirty percent of the samples showed co-infection of the satellites. While EST-primers for detection of the integrated forms of SatDNA-III produced single bands on gels, those of SatDNA-II still produced additional bands, most noteworthy being the closely spaced „double bands‟. Upon sequencing, the satellite DNA isolates showed similarity with sequences deposited in the genebank and bearing accession numbers AY836366 and AY836367 for SatDNA-II and SatDNA-III isolates, respectively. Alignment reports (Clustal W) revealed presence of GC-rich regions, TATA protein binding motifs (TATAAAT) and CAAT boxes as well as poly (A) signal. GC-rich regions in SatDNA-II were mostly trinucleotides (CGC) and hexanucleotides (CCGCCG) while in SatDNA-III the regions were trinucleotides (CGC) and pentanucleotides (CCGCC). Following biolistic inoculation, five-week scoring for the symptoms showed that plants from cvs AR37-92, CR27-24 and AR16-3 remained symptomless while plants from cv T200 were symptomatic. PCR amplification of βC1 gene five weeks post inoculation (wpi) gave PCR products in 19.6% of the samples while AC1 was amplified from only two plants. Full-length SatDNA-II was amplified from 70% of DNA samples, mostly from plants in which a begomovirus was co-inoculated with SatDNA-II. Amplification of full-length SatDNA-III from bombarded plants was unsuccessful. Amplification of integrated fragments of SatDNA-II from bombarded plants using EST-primers gave a PCR product in 93.7% of the samples. PCR amplification of the fragments from DNAs extracted from plants of cvs AR17-5 and CR27-24 previously inoculated with EACMV-TZ + SatDNA-II and EACMV-UG2 + SatDNA-III, respectively, gave closely spaced bands on 13% of the DNA samples. Amplification of integrated forms of SatDNA-III gave bands in 52.4% of samples. Probing for full-length SatDNA-II, SatDNA-III and AC1 from DNAs extracted from plants pre-inoculated with these DNAs using DIG- labeled probes gave hybridization signals in 60%, 83% and 68% of the samples, respectively. Further analysis of the signals in the context of screening suggested that cvs AR37-92 and AR37-96 were highly resistant to CMD while cv AR40-10 was susceptible. In the interaction experiment, Nicotiana benthamiana plants inoculated with an infectious clone of EACMV-TZ developed moderate CMD symptoms 7 days post inoculation (dpi) with symptoms consisting of leaf distortion and moderate stunting of plants. There were also plants which recovered from the symptoms by 35 dpi. Plants inoculated with EACMV-TZ + SatDNA-II produced similar symptoms with N. benthamiana plants developing symptoms 7 dpi that became severe by 14 dpi and without recovery even after 35 dpi. Very severe symptoms were also observed when N. benthamiana plants were inoculated with EACMV-TZ + SatDNA-II + SatDNA-III. Plants inoculated with SatDNA-II or SatDNA-III alone remained asymptomatic even after 35 dpi. Southern blot analysis showed clear increase in DNA accumulation when EACMV-TZ was inoculated together with both SatDNA-II and SatDNA-III as compared to when EACMV-TZ was inoculated alone or with SatDNA-II only and probed with EACMV-TZ. In conclusion, symptom recovery and reversion of symptoms in screenhouse plants is associated with virus resistance. There is a wide occurrence of satellites (SatDNA-II and SatDNA-III) across the sampled regions consistent with distribution of their helper cassava begomoviruses. The satellites are of a wider occurrence and diversity in Eastern zone than elsewhere in the country. The occurrence of SatDNA-III was not confined to the Lake zone as previously thought. There is evidence for satellite sequence integration into host plant genome, a further indication that the satellites are wider spread in cassava germplasm than earlier conceptualized. In few instances, both SatDNA-II and SatDNA-III isolates co-existed in the same plant though its effect on symptom enhancement could not be immediately established. The observed recovery in screening studies is thought to result from resistance introduced in the plant materials involved. Since labeled probes for satellites that were used in hybridization had been prepared from satellite sequences considered to be integrated, the hybridization signals did not depend on whether the leaf samples were picked from symptomatic or asymptomatic plants. From the study, three observations clearly suggest that SatDNA-II and SatDNA-III are biologically functional and that their effects on host plants are distinctly different. The study has demonstrated enhanced cassava begomovirus symptoms in N. benthamiana in the presence of satellite DNA molecules. This is the first detailed study undertaken to highlight the occurrence and role played by satellite DNA molecules in breaking the resistance to CMD of cassava cultivars grown in Tanzania. Keywords: Cassava mosaic disease, Cassava mosaic begomoviruses, Satellite DNA molecules, Tanzania.