UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG

Postgraduates

Chezlyn Chetty, Ph.D (Post Doctoral Fellow)

Transformation and Regeneration of Cassava (Manihot esculenta Crantz) 

Cassava production in South Africa faces numerous constraints that limit the full potential of the crop. Cassava mosaic disease (CMD) caused by geminiviruses is the most important disease affecting cassava in the country. Deployment of disease-free planting material has helped to reduce CMD impact on cassava production but is not sufficient due to the rapid post-planting dissemination of the viruses by its natural vector (Bemisia tabaci).  Therefore the transgenic approach to improve cassava production has been employed. Cassava is generally considered to be recalcitrant to transformation, but we have developed a stable, reliable and efficient transformation and regeneration protocol for the production of transgenics. Selected cultivars have been transformed with CMV viral sequences. Expression of the introduced viral sequence interferes with virus replication in the plant via Post-Transcriptional Gene Silencing mechanism (PTGS). This plant defense system results in degradation of mRNA produced both by the transgene and the virus. Special focus has been given to South African preffered cassava cultivars such as T200, which is a high-starch yielding  cultivar.  The importance of this study can be viewed as progress to developing a consistent and robust cassava transformation platform in southern Africa for transfer of desirable traits to farmer-preferred or commercial varieties.  

 

Alice Tadzei Maredza, Ph.D (Post Doctoral Fellow) 

Interaction of cassava begomoviruses with satellites and their impact on virus virulence

In southern and east Africa, cassava mosaic disease (CMD), caused by cassava mosaic geminiviruses (CMGs), is one of the major factors limiting cassava production.  Intervention to control CMD is by introduction of improved Tropical Manihot Series (TMS) or by genetic engineering.  CMGs require both their genomic components (DNA-A and-B) to produce typical disease symptoms in cassava (Manihot esculenta). Recently, two novel ssDNA molecules, associated with DNA-A from a natural CMG (East African cassava mosaic virus) infection, have been discovered in East Africa.  When present in co-infections with geminiviruses, these satellite DNA (Sat DNA) molecules cause increased viral accumulation and novel, severe disease symptoms. In addition, the high resistance to geminiviruses in the landrace TME 3 (a central component of cassava improvement programs) can be broken by sat DNA II. It was shown by sequence determination and analysis that the Sat DNA molecules have no significant sequence similarity to their ~2.7 kb helper virus genomes. This project aims to study the interactions of these unusual sub-genomic DNA molecules with their associated helper viruses, in southern and east African countries, in order to understand the nature of interaction and to develop virus resistance strategies.   

 

Richard Mundembe, Ph.D (Post Doctoral Fellow)

Cassava transformation by Agrobacterium-mediated transformation and bombardment for resistance to SACMV

The objective of the project is to genetically engineer cassava for resistance to South African Cassava Mosaic Virus (SACMV). This will be achieved by making plasmid constructs carrying a duplicated 193 bp fragment of SACMV AC1/AC4 gene fragment that forms a hairpin structure, and to use the constructs to transform cassava FECs, from which transgenic cassava plants will be regenerated.  Two forms of the hairpin structures, mismatched and non-mismatched, and two methods of plant transformation, Agrobacterium-mediated transformation and biolistics (bombardment), will be used.  Our cultivars of choice are TMS 60444 and T200.  Transgenic plants generated by Agrobacterium-mediated transformation will be used to evaluate which form of the hairpin constructs result in higher levels of RNAi-mediated resistance in cassava.  The best performing form of the hairpin constructs will be then be bombarded into cassava FECs as minimal cassettes, a strategy enabling the exclusion or removal of selection markers and undesirable vector sequences.

 

Farhahna van Schalk (Doctoral Fellow)

Gene Expression studies towards the elucidation of host responses to South African cassava mosaic virus (SACMV) 

At present cassava functional genomics is still in its infancy with very little known about the underlying mechanisms involved in Cassava Mosaic Disease (CMD), particularly with respect to susceptible interactions.This research therefore aims to use a combination of high throughput genomics and Bioinformatics approaches to study host-pathogen interactions (cassava-South African cassava mosaic virus) in order to identify potential genes that can be used to develop CMD resistant cassava varieties for improved income to small-scale and commercial cassava farmers in South Africa.

 

Natasha Abraham (MSc Student; Submitted March 2012)
Characterization of defective interfering molecules associated with South African cassava mosaic virus

Several animal and plant viruses are associated with small subgenomic particles. These subgenomic viral particles have been found to be present naturally at low levels in geminivirus-infected plants. These components are related to and dependent on their parents or “helper virus” for replication. Defective interfering (DI) molecules are a kind of subviral DNA. DIs are associated with viruses from the families Geminiviridae, Nanoviridae and Caulimoviridae. Sequence analyses of DIs indicate that they form due to deletions duplication, inversion or rearrangements of the viral genome and its satellites or as a result of insertion of foreign DNA (non-viral DNA sequences). DIs are associated with a delay in the onset and attenuation of symptoms. They have an inhibitory effect on the helper virus as they compete for essential host and viral factors and thus are known as defective interfering molecules. DIs have been associated with symptom amelioration. It is believed that symptom amelioration occurs through the DIs directly interacting and competing with the viral and host components and hence decreasing the helper virus load post infection. In a preliminary study conducted by Choge et al. (2000), on naturally SACMV-infected cassava; a putative DI DNA molecule was identified. It was characterized and was found to be a deletion of DNA-B with the entire BV1 ORF deleted and the C terminal of BC1 ORF partially deleted. This leads to the expression of a truncated protein. In a further study conducted by Kuhn (2003), putative defective interfering molecules were isolated from field cassava infected with SACMV and infectious head-to-tail dimer of the putative DIs was constructed. The aim of this project is to : 
1.Establish effect of defective interfering molecules on symptom development/amelioration on model plant system using Nicotiana benthamiana agroinfected with South African cassava mosaic virus.
2.To test, defective interfering molecules, for protection against South African cassava mosaic virus in its natural host cassava.
3.Challenge transgenic tobacco, containing tandem repeats of DI sequence, resistant to SACMV infection

 

Debbie Carmichael (MSc Student; To Submit in March 2012)

Developing a sensitive, high-throughput tool for rapid detection of agronomically important seed-borne pathogens of tomato
The inability to adequately detect infected material within imported tomato seed lots in the past, has let to outbreaks and epidemics of devastating diseases in tomato fields, resulting in significant losses in yield and income. To reduce the risk of introducing such detrimental pathogens into tomato populations, countries importing seed have implemented specific phytosanitary requirements which only permit trade if seed-lots are certified for the absence of pathogens that post a potential threat. In order to abide by these regulations, exporting companies require a tool for screening parent plants and seed-lots, that can successfully detect a range of significant tomato pathogens with accuracy and reliability. Current phytodiagnostic tools are severely limited by the number of pathogens that may be detected in a single assay, making the analysis of seed-lots for a range of pathogens laborious, time-consuming and costly. Most available techniques also demonstrate insufficient specificity and sensitivity to detect pathogens at the standard required for seed quality testing.

The introduction of microarrays as phytodiagnostic tools has offered a potential solution to these encountered problems. Microarrays consist of a solid support substrate to which thousands of oligonucleotide probes are hybridised. These probes are designed to be specifically complimentary to target nucleic acid and are therefore able to detect complimentary sequences in a sample visualised by the emission of a hybridisation signal. In phytodiagnostics, probes are specifically designed against targeted pathogen sequences, and due to the high-throughput nature of microarrays, numerous probes may be included on a single array allowing for a range of pathogens to be detected in a single assay, thereby reducing time and labour. Due to the highly specific nature of the designed probes, microarrays also demonstrate high sensitivity and specificity making the detection of pathogens in a sample accurate and reliable. The recent development of the padlock-probe technology has further improved the resolution of microarrays through ability to discriminate between true matches and false positives or background signals. With improved detection limits reported for a range of pathogens from crude samples, microarrays represent a promising tool for seed quality testing in tomato trade.

The aim of the proposed study is thus to develop a sensitive, high-throughput of phytodiagnostic tool based on microarray technology, which is able to rapidly detect and quantify agronomically important seed-borne pathogens of tomato with high accuracy and reliability.


Aobakwe Matshidiso (MSc Student)

A comparative evaluation of computational tools to assemble a cassava transcriptome

Next generation sequencing technologies, referred to as RNA-seq or NGS, provide massive improvements over existing technologies in terms of quality and cost. Also, previously known information does not have to be inferred onto current datasets. NGS technologies can perform both de novo sequencing and re-sequencing, and can discover unique molecules in a complex mixture, and quantify them at any given point. Due to their short length, NGS reads need to be assembled efficiently and accurately for one to extract biological information from the assembled transcriptome. Due to NGS’s efficacy and depth of coverage, the richness of the biological information needs to be captured without considerable time delays.

Analysis Next Generation Sequencing has some challenges, including repeats and the length of the reads. Numerous tools exist to facilitate the analysis of the said data, but many have different features, treat data differently and are optimized differently. Aobakwe’s research project aims to comparatively evaluate the performance and utility of several short read assembly tools, to determine the competitive advantages and shortcomings of each.  Another object of the study is to obtain knowledge regarding the assembly and alignment processes, and how these impact on further downstream processes like annotation. This will then be used to build an improved transcriptome that is better assembled and well-aligned.

 

Maabo Moralo (Doctoral Fellow)

Designing silencing hairpin RNA constructs against African cassava mosaic virus and East African cassava mosaic virus

Cassava production in Africa has for long been devastated by virus diseases. Genetic engineering approaches such as pathogen-derived resistance provides an alternative to develop virus resistant cassava. Pathogen-derived resistance is a genetic engineering strategy that involves the use of virus-derived genes to interfere with a specific step during virus replication or movement.  With regard to pathogen-derived strategies, several approaches have been taken for cassava begomoviruses CBVs), and these include antisense and sense constructs, constructs encoding intron-spliced RNAs and constructs encoding hairpin RNA structures (IR) which fold to produce dsRNA and hence induce siRNAs.  These siRNAs target incoming viral transcripts and reduce infection. This research involves designing silencing hairpin RNA constructs, genetic engineering of cassava for virus resistance using the constructs, followed by testing of RNA hairpin structures for efficient RNA silencing induction and CBV resistance. RNA silencing could provide a significant new strategy that offers a combination of specificity and the exploitation of a plant cellular pathway that has evolved naturally to combat disease, to fight cassava mosaic disease in cassava. 

 

Sarah Rogans (MSc Student)

Project Title:

 

I Mwaba (Doctoral Fellow)

Project Title: 

(Started PhD in 2012)

 

Part-time and International Students

E Pierce (Doctoral Fellow)

Project Title: Mining genes that confer resistance to Cassava Mosaic Disease and Whitefly for increased productivity in Southern Africa


O Ndomba  (Doctoral Fellow)

Project Title: Influence of satellite DNA molecules on severity of Cassava Begomoviruses and the breaking down of resistance to cassava mosaic disease in Tanzania

(Degree awarded, thesis corrections underway)


L Esterhuizen (Doctoral Fellow, Part time) University of Johannesburg

Project Title: Epidemiology and molecular characterization of Tomato curly stunt virus in southern Africa


M Rauwane (Doctoral Fellow, In abeyance June 2011)


V Nuaila (MSc Student) 

Project Title: Epidemiology of Tomato Curly Stunt Virus (ToCSV) in Mozambique

(To submit in April 2012)

 

K Dias (MSc Student)

Project Title: Evaluation of resistance to Tomato Curly Stunt Virus (ToCSV) in tomato

(MSc degree awarded, corrections underway)


J Amisse (MSc Student)

Project Title: Molecular Characterization of Cassava Brown Streak Viruses in Mozambique


C Kitona (MSc Student)

Project Title: Development and evaluation of efficient diagnostic tools for cassava mosaic and cassava brown streak diseases 


H Mugerwa (MSc Student)

Project Title: Regional Cassava Virus Diagnostic Project (Genetic variability of cassava Bemisia tabaci and its effects on the spread of cassava mosaic geminiviruses in east and southern Africa)


H Mollel (MSc Student)

Project Title: Interaction and impact of cassava begomoviruses and their associated satellites