OpenUCT :: Browsing by Author "Rafudeen, Suhail"

Browsing by Author "Rafudeen, Suhail"

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Open Access
A biological study of the cellular response to heat stress in the South African alga Gracilaria gracilis
(2012) Boom, Taryn; Coyne, Vernon; Rafudeen, Suhail
Gracilaria gracilis is a commercially important alga, previously harvested from the wild South African population in Saldanha Bay as a feed for marine organisms and as a source of commercially important agar. Since 1974 however, a number of sporadic population collapses has lead to the destruction of this once flourishing resource. After numerous failed attempts at re-establishing this industry, the need to develop an alternative farming strategy became evident. In order to devise such a solution, a better understanding of the tolerances and responses of this alga to the environmental parameters responsible for the downfall of the population is required. Although the exact reasons remain unclear, Jaffray et al., 1997 have reported that increased water temperature in Saldanha Bay may be a contributing factor as the population collapses have repeatedly occurred during summer months. Thus the effect of heat stress on G. gracilis has been selected for this study.
Open Access
Characterisation of galactinol synthase II (XvGolSII) from the resurrection plant Xerophyta viscosa (Baker)
(2013) Neumann, Alexis Joy; Rafudeen, Suhail; Farrant, Jill M
The monocotyledonous Xerophyta viscosa belongs to a unique group of angiosperms known as resurrection plants. These plants possess a number of unique characteristics which allow them to survive and cope for extended periods with extreme abiotic stresses such as dehydration and cold stress. It is therefore of great interest to understand and elucidate the various molecular mechanisms which are specifically regulated in response to abiotic stress by identifying genes and proteins which may contribute to abiotic stress tolerance. These genes could potentially be utilized in the development of crops with improved tolerance to abiotic stresses. The aim of this study was to preliminarily characterize XvGolSII, a galactinol synthase, which had been isolated from a X. viscosa cold stress cDNA library. In this study, the XvGolSII cDNA was sequenced and both the nucleic and amino acid sequence analysed through in silico analysis. The XvGolSII cDNA sequence was shown to be 1434 bp in length, with an open reading frame (ORF) of 1018 bp. This ORF encodes a 339 amino acid protein with a molecular weight of 38.7 kDa, containing a characteristic hydrophobic carboxyterminal pentapeptide, APSAA. Recombinant XvGolSII protein was successfully expressed in E. coli BL21 cells using the pET29b expression vector. The recombinant XvGolSII protein showed in vitro galactinol synthase activity via an activity assay using HPAEC-PAD, where it produced galactinol from the substrates myo-inositol and UDPgalactose. Subcellular localisation examination, using an XvGolSII-YFP fusion protein, indicated localisation to the cell membrane of onion epidermal cells. Quantitative real time PCR analysis showed XvGolSII to be significantly down-regulated during dehydration stress while mildly up-regulated during the early stages of cold stress, though the latter increase was not significant. Western blot analyses did not detect XvGolSII in total protein extracted from X. viscosa leaf tissue during dehydration and rehydration treatments, using polyclonal antibodies generated from the XvGolSII recombinant protein. This study successfully characterized XvGolSII at the molecular level and provides a basis for further investigation of the role of XvGolSII in abiotic stress tolerance.
Open Access
Characterisation of two desiccation-linked dehydrins from Xerophyta humilis
(2016) Fan, Cynthia; Farrant, Jill M; Rafudeen, Suhail
In response to abiotic stresses, organisms throughout the plant kingdom, as well as microorganisms and micro-animals such as nematodes or tardigrades, have been observed to express Late Embryogenesis Abundant (LEA) proteins as protective mechanisms. However, despite two decades of research, little is understood about their physiological functions and this has led to extensive nomenclature, with a large amount of redundancy. The primary reason for this lack of insight into LEA protein functions is their highly hydrophilic and intrinsically disordered nature. Intrinsically disordered proteins (IDPs) cannot be studied using conventional methods of structural analyses such as X-ray crystallography and, therefore, alternative techniques are required. A combination of transgenic and in vitro studies have also shown that LEA proteins are most likely to behave as molecular chaperones by binding water and ions, preventing macromolecular aggregation and protecting enzymatic activity during dehydration. This study characterized two dehydrins that were expressed during dehydration in the desiccation tolerant plant, Xerophyta humilis. From a transcriptome analyses on X. humilis, cDNA for the two dehydrins were obtained. These sequences were first analysed using various in silico tools in order to identify putative dehydrin-specific characteristics. Subsequently, these two dehydrins were cloned and expressed for production of recombinant dehydrin protein. These proteins were then analysed in terms of structural and functional characteristics. Structurally, through the use of circular dichroism in an in vitro system, both dehydrins demonstrated the shift towards being increasingly alpha-helical when placed in environments of decreasing water content. The role of these two dehydrins in stabilizing enzymes during dehydration was subsequently investigated using citrate synthase (CS) and lactate dehydrogenase (LDH). The preservation of enzyme activity was observed in both CS and LDH. This preservation of enzyme activity was further maintained by the presence of trehalose. Anti-aggregation roles were also investigated, however, neither dehydrin demonstrated significant ability to minimize the aggregation of LDH. This study hopes to establish a pipeline for characterizing LEA proteins using structural and functional assays in order to provide alternative means of LEA protein classification.
Open Access
Evaluation of southern African maize germplasm for phytoalexin accumulation following inoculation by Fusarium verticillioides
(2017) Veenstra, Amy; Murray, Shane L; Rafudeen, Suhail
Maize is a socially and economically important crop in Africa (and worldwide) that is severely affected by many fungal pathogens. The pathogen Fusarium verticillioides causes Fusarium ear rot in maize, a disease that greatly reduces quantity and quality of annual maize yields. The pathogen produces mycotoxins called fumonisins, which have been linked to adverse health effects in both humans and animals. Maize produces terpenoid phytoalexins, which are antimicrobial compounds that directly reduce the growth of many fungal pathogens including F. verticillioides. Two families of maize phytoalexins, termed kauralexins and zealexins, have been characterized. Key genes putatively involved in the biosynthetic pathway of these phytoalexins have been identified from the rice model and subsequent studies on maize. This research aimed to evaluate the correlation between phytoalexin accumulation and fungal growth in diverse southern African maize lines in response to F. verticillioides inoculation. Maize lines were inoculated with F. verticillioides using a seed soak inoculation method and grown in vitro for up to two weeks. The harvested tissue was analysed for fungal growth using quantitative PCR, putative phytoalexin biosynthetic gene expression using RT-qPCR and phytoalexin accumulation using gas-chromatography mass spectrometry. Furthermore, an endophyte growing in one of the maize lines was isolated and identified as Trichoderma asperellum. Trichoderma spp. are used as biocontrol agents against many fungal pathogens, although research on the specific antagonistic effect of T. asperellum on F. verticillioides is limited. Phytoalexin accumulation in maize containing endophytic T. asperellum was compared to maize inoculated with F. verticillioides. In vitro competition assays were performed to analyse the antagonistic effect of T. asprellum on F. verticillioides. Results from this study show that inoculation of maize lines with F. verticillioides induces the accumulation of total phytoalexins, and more specifically the accumulation of total kauralexins. Putative phytoalexin biosynthetic genes are also up-regulated in response to inoculation. Maize growing with a T. asperellum endophyte accumulated phytoalexins to the same levels as F. verticillioides, suggesting that T. asperellum induces a defence response that 'primes' the plant for further infection. In vitro competition assays between F. verticillioides and T. asperellum showed that T. asperellum significantly inhibits F. verticillioides growth. These results will aid in the identification of maize lines that can be bred with increased resistance to F. verticillioides with the goal to reduce F. verticillioides incidence in southern Africa. Furthermore, analysis of the efficacy of T. asperellum as an antagonist against F. verticillioides may provide another method for disease reduction in the field.
Open Access
The functional analysis of XhLEA3-2 - a LEA_4 from the resurrection plant, Xerophyta humilis
(2017) Dennis, Timothy James; Farrant, Jill M; Rafudeen, Suhail
Climate change is a pressing reality in the current era. Changing environmental conditions and limited water availability are associated with the loss of arable land in areas where farming has traditionally thrived. Thus, linked to climate change, is the risk of a global food shortage. Resurrection plants are phenomenal in that they are able to survive extended periods of drought in a state of anhydrobiosis and then resume full metabolism upon rehydration. These plants serve as models to scientists and genetic engineers who hope to replicate, to a degree, the 'resurrection phenomenon' in drought sensitive crop species. The ability of resurrection plants to survive drought needs to be studied on a molecular level if it is to be implemented in transgenic crops. Currently, the molecular mechanisms of desiccation tolerance are only somewhat understood, and considerable investigation is still required. Xerophyta humilis is a monocotyledonous resurrection plant in which one of the responses to extreme water loss is the upregulation of several Late Embryogenesis Abundant (LEA) genes. The protein products of these genes, called LEA proteins, are known to be correlated with abiotic stress tolerance in plants, invertebrates and microorganisms. However, the precise molecular mode(s) of action of LEA proteins are still poorly understood. In this study, a group LEA_4, LEA protein, which we have termed XhLEA3-2, shown to be transcriptionally upregulated during desiccation of the resurrection plant X. humilis, has been characterized. A bioinformatic, predictive analysis was performed to detect any LEA-like characteristics of XhLEA3-2. Recombinant XhLEA3-2 was produced in Escherichia coli, purified, and used to generate XhLEA3-2 specific antibodies for expression analyses. The ability of XhLEA3-2 to function as a molecular chaperone was assessed using a lactate dehydrogenase (LDH) enzyme stability assay. Transgenic expression of XhLEA3-2 in E. coli and tobacco was also investigated. In summary, this thesis demonstrates that XhLEA3-2: has typical LEA protein properties according to bioinformatic analyses, has two close homologs in X. viscosa, is present in dry X. humilis leaf tissue, has homologs present in dry X. viscosa leaf tissue, has some molecular chaperone activity, can protect E. coli from desiccation but not from osmotic stress, and can be transiently expressed in tobacco.
Open Access
Genetic diversity in tef [Eragrostis tef (Zucc.) Trotter]
(2015) Assefa, Kebebew; Cannarozzi, Gina; Girma, Dejene; Kamies, Rizqah; Chanyalew, Solomon; Plaza-Wüthrich, Sonia; Blösch, Regula; Rindisbacher, Abiel; Rafudeen, Suhail; Tadele, Zerihun
Tef [Eragrostis tef (Zucc.) Trotter] is a cereal crop resilient to adverse climatic and soil conditions, and possessing desirable storage properties. Although tef provides high quality food and grows under marginal conditions unsuitable for other cereals, it is considered to be an orphan crop because it has benefited little from genetic improvement. Hence, unlike other cereals such as maize and wheat, the productivity of tef is extremely low. In spite of the low productivity, tef is widely cultivated by over six million small-scale farmers in Ethiopia where it is annually grown on more than three million hectares of land, accounting for over 30% of the total cereal acreage. Tef, a tetraploid with 40 chromosomes (2n = 4x = 40), belongs to the family Poaceae and, together with finger millet (Eleusine coracana Gaerth.), to the subfamily Chloridoideae. It was originated and domesticated in Ethiopia. There are about 350 Eragrostis species of which E. tef is the only species cultivated for human consumption. At the present time, the gene bank in Ethiopia holds over five thousand tef accessions collected from geographical regions diverse in terms of climate and elevation. These germplasm accessions appear to have huge variability with regard to key agronomic and nutritional traits. In order to properly utilize the variability in developing new tef cultivars, various techniques have been implemented to catalog the extent and unravel the patterns of genetic diversity. In this review, we show some recent initiatives investigating the diversity of tef using genomics, transcriptomics and proteomics and discuss the prospect of these efforts in providing molecular resources that can aid modern tef breeding.
Open Access
An investigation of Fusarium verticillioides infection in maize using physiological and molecular approaches
(2017) Lambarey, Humaira; Rafudeen, Suhail; Murray, Shane
Abstract Maize (Zea mays L.) is an important staple food crop in sub-Saharan Africa providing food security to millions of people. Fusarium verticillioides is an important fungal pathogen that infects maize and causes 'Fusarium Ear Rot' which decreases maize kernel yield and quality. In addition, the fungus produces mycotoxins which contaminate the kernel and upon ingestion have negative health consequences for both people and livestock. To this date, there is still no African maize line completely resistant to infection by F. verticillioides. In this study, an African maize line, Zea mays CML144, was infected with F. verticillioides using a soak-seed inoculation method and grown for two weeks under controlled conditions. Analysis of the morphological characteristics showed that compared to the control (mock-infected) maize, infected maize seedlings displayed signs of stunting with leaves shorter & thinner while roots were shorter and displayed visible signs of rotting. Control and infected maize plants were also characterised physiologically and biochemically. Electrolyte leakage experiments were conducted on the meristem regions of the plants after week one and two of infection and showed that leakage increased over time in both control and infected samples with no significant difference observed between the two groups. Biochemical characterisation by analysing superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) antioxidant enzymes showed an increase after the two weeks of infection, indicating a defense response by the plants in response to infection by the fungal pathogen. RNA-sequencing, the main aim of this study was conducted on control and infected plants after two weeks of infection to identify differentially expressed genes (DEGs) involved in F. verticillioides infection. The Illumina NextSeq 500 platform was used to sequence the transcriptome and quantify changes in gene expression. Analysis of the RNA-seq data using the Tuxedo suite of protocols revealed significant DEGs that were both up- and down-regulated in the infected samples compared to the control. Data analysis was conducted using the DNA subway online bioinformatics tool and these results were compared to those obtained using a separate analysis which also incorporated the Tuxedo suite of protocols. Bioinformatic analysis on the RNA-seq DEGs were performed using the agriGO analysis tool which revealed three significant Gene Ontology (GO) terms for both the up- and down-regulated genes, respectively, with the 'response to stimulus' GO-term (within the down-regulated genes) being of specific interest. Other GO-terms included response to chemical stimulus, carbohydrate metabolic process and ion bonding, which also played a role in the defense response when plants were infected by the fungal pathogen. Quantitative Real-Time PCR was performed on five DEGs that were either up- or down-regulated in response to F. verticillioides infection to validate RNA-seq data as well as the GO-analysis results. Quantitative Real-Time PCR was also used as a pre-validation (before RNA-seq) on shrunken-1, a down-regulated gene found in a previously conducted study. We observed that in response to infection by F. verticillioides, expression of shrunken-1 was down-regulated, however, this was not shown to be significant (p>0.05). The results in the current study and the identification of the genes in Zea mays CML144 responding to fungal infection will aid in the goal to develop a maize line completely resistant to F. verticillioides in Africa and in particular South Africa. This would provide improved food security and minimise health risks to the population in the long term. To our knowledge, this is the first study investigating F. verticillioides infection in the African maize line Zea mays CML144 using the soak-seed inoculation method.
Open Access
Investigation of the Gracilaria gracilis proteome response to nitrogen limitation
(2012) Naidoo, René Kathleen; Coyne, Vernon; Rafudeen, Suhail
In the past, commercial quantities of G. gracilis were harvested from Saldanha Bay until population collapses of the natural resource necessitated the need for research into alternative aquaculture programs for G. gracilis cultivation. One of the reasons for the G. gracilis population collapse was attributed to adverse conditions during summer which led to thermal stratification of the water column and subsequent nutrient limiting conditions. Inorganic nitrogen has been identified as the major nutrient factor limiting growth and production of G. gracilis populations in South Africa. Although the physiological mechanisms implemented by G. gracilis which allow adaption to low nitrogen environments have been investigated, not much is known about the molecular mechanisms which underlie these adaptions. Thus, it is necessary to elucidate the molecular basis of these adaptions in G. gracilis to complement the existing physiological data.
Open Access
Molecular and bio-analytical characterisation as a means to understand genetic diversity within Kenyan Aspergillus flavus strains
(2018) Mitema, Alfred Ochieng; Rafudeen, Suhail; Okoth, Sheila
Toxigenic Aspergillus species produce mycotoxins that are carcinogenic, hepatotoxic and teratogenic immunosuppressing agents in both human and animals. Kenya frequently experiences outbreaks of aflatoxicosis with the worst occurring in 2004, which resulted in 125 deaths. This study sought to find possible reasons for frequent aflatoxicosis outbreaks in Kenya by isolating Aspergillus flavus strains from maize kernels sampled from different climatic regions of Kenya. Using diagonal transect random sampling, maize kernels were collected from Makueni, Homa Bay, Nandi, and Kisumu regions. The genetic diversity and variation among the isolates was examined by characterising the strains according to morphology, phenotype, vegetative compatible groups and molecular systematics. Selected atoxigenic and aflatoxigenic A. flavus isolates were also further analysed for aflatoxin production potential using quantitative real-time PCR and various bioanalytical techniques. The influence of the maize lines grown in Kisumu, Homa Bay, Nandi and Makueni region on A. flavus infection and aflatoxin production was also examined and served as the basis for an in vitro biocontrol assay. Out of 37 isolates identified, nitrate non-utilizing auxotroph’s complementation test revealed 20 vegetative compatibility groups. These groups were further designated using the prefix ʻʻKVCGʼʼ, where ʻʻKʼʼ represented Kenya and consequently assigned numbers 1 to 20 based on our findings. KVCG14 and KVCG15 had highest distribution frequency (n = 13; 10.8 %). The distribution of the L, S and S/L- morphotypes across the regions were 57 % (n = 21); 7 % (n = 3) and 36 % (n = 13) respectively. The phylogenetic analysis exhibited high diversity of A. flavus isolates from Makueni. ITS1 and ITS2 markers did not reveal significant information within intraspecies speciation of A. flavus. Furthermore, a unique isolate (KSM015) was identified that had characteristics of S-morphotype, but produced both aflatoxins B and G. Coconut agar medium (CAM) assay, TLC, HPLC and LCMS/MS analyses confirmed the presence or absence of aflatoxins in selected toxigenic and atoxigenic isolates. qPCR analysis revealed aflP, aflS, aflR and aflO transcripts as the most upregulated genes across the tested isolates whereas false detection of aflD gene transcript was observed in both induced and uninduced A. flavus isolates. Diversity Index (H) analyses ranged from 0.11 (Nandi samples) to 0.32 (Kisumu samples). Heterokaryon compatibility ranged from 33 % (for the Makueni samples, n = 3) to 67 % (Nandi samples, n = 6). The KDV1 maize line was more sensitive to A. flavus infection in comparison to GAF4. We also tested the biocontrol of atoxigenic isolates to inhibit toxin production by aflatoxigenic strains on infected maize kernels. It was shown that the atoxigenic strain (KSMO12) could inhibit the aflatoxigenic strain (KSM014) depending on the atoxigenic concentration during infection. To our knowledge, this is the first reported study for A. flavus genetic diversity, variation and distribution in Nandi, Homa Bay and Kisumu regions in comparison to and could assist researchers in the selection of biocontrol strategies to mitigate aflatoxin contamination, especially in Makueni and neighbouring regions.
Open Access
Molecular characterisation of the "LEAome" in the resurrection plant Xerophyta humilis (Baker)
(2015) Waters, Robyn; Farrant, Jill M; Rafudeen, Suhail; Peton, Nashied
Studies on resurrection plants and other anhydrobiotic organisms, have shown that Late Embryogenesis Abundant (LEA) proteins are expressed upon the onset of desiccation and are therefore inferred to be associated with the desiccation tolerance response. To date, despite some 25 years of research on these proteins, there is still very little understanding of the physiological function(s) of the majority of LEAs. This is because they lack tertiary structure in the hydrated state, making assigning of physiological roles difficult. This MSc study was undertaken to investigate the gene expression of a set of 21 putative LEAs during dehydration and subsequent rehydration stress, in the resurrection plant Xerophyta humilis (Baker). Recombinant proteins were expressed for 3 of the LEA genes from this set in order to perform structural studies and to ascertain their LEA status. These studies were conducted with the purpose of shedding light on the role of LEAs in desiccation tolerance, to add to the ever-growing transcriptomic and proteomic data, and to the current knowledge of these enigmatic proteins. Quantitative real-time gene expression (qPCR) analysis was conducted on the set of 21 full length X. humilis cDNA clone nucleotide sequences, with similarities to late embryogenesis mRNA sequences, derived from a study conducted by Collett et al., (2004). Expression analysis was conducted in both leaves and roots, across a dehydration and rehydration profile of X. humilis. Of this total group of 21 full length cDNA clones, three LEAs; XhLEA2-3 and XhLEA2-6 (two putative Group 2 LEA genes) and XhLEA3-5 (a putative Group 3 LEA gene), were chosen for cloning and expression studies. cDNAs of these XhLEAs were cloned into a modified bacterial expression vector and recombinant protein expression was attempted in E. coli.
Open Access
Molecular characterisation of the lateral organ boundaries gene (XvLOB) from the resurrection plant Xerophyta viscosa
(2011) Sattar, Shakiera; Rafudeen, Suhail
The aim of this study is to preliminary characterise XvLOB, a LOB domain containing gene, which has been isolated from a dehydration stress library in X. viscosa. In this study the gene was isolated and cloned, the DNA and amino acid sequences were analysed by in silico analysis, the recombinant XvLOB protein expressed, subcellular localisation examined and both the transcript and protein expression levels in response to dehydration was investigated.
Open Access
Molecular characterisation of XvRG6 and XvRF17 genes isolated from the resurrection plant Xeropyta viscosa
(2007) Felix, Mba Medie; Rafudeen, Suhail; Farrant, Jill M
A number of genes up-regulated in response to abiotic stress have been isolated from X. viscosa by various methods, one of which includes differential screening of cDNA libraries. Of these genes, a large number are undescribed, with no identity to known plant genes. Two such genes, XvRG6 and XvRF17, isolated from a X. viscosa cDNA library have been partially characterised. Southern blot analysis confirmed the presence of both XvRG6 and XvRF17 genes in the X. viscosa genome. In silico analyses predicted XvRG6 to be proline-rich protein, since it possesses many of the features common to PRP such as a signal peptide, proline repeats, a cysteine residue and possible phosphorylation sites. It also possesses a tyrosine residue present in some of the repeats, and this residue is belivied to play a role in protecting the plant against environmental stresses. The XvRF17 protein was predicted by in silico analyses to belong to the family of metallothioneins, a family of metal-binding proteins. Anaylis of the XvRG6 mRNA transcript showed that the gene was not dehydration stress inducible, but was induced by ethylene, endogenous ABA, SA and JA application. In contrast, the XvRF17 mRNA transcript was shown by RT-PCR to be induced by dehydration stress, endogenous ABA, SA and JA application but not by ethylene treatment. Interestingly, western blot analyses revealed that XvRG6 protein levels increased only during dehydration and not during any other imposed stress. This study has shown that both XvRG6 and XvRF17 and responsive to stress. Since there are no known orthologues of these genes, their respective roles in X. viscosa remains speculative.
Open Access
Molecular characterisation of XvVTC2, a gene coding for a GDP-L-galactose phosphorylase from Xerophyta viscosa
(2010) Bresler, Andries Petrus; Rafudeen, Suhail
Climate change is predicted to have a negative impact on world food security in the next 40 years. Resurrection plants can withstand highly variable and harsh climatic conditions. This makes them ideal candidates to elucidate possible mechanisms which can be used to adapt crop plants to tolerate variable climatic conditions associated with climate change.

Browsing by Author "Rafudeen, Suhail"

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