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Thesis (M.S.)--University of Tennessee, Knoxville, 2009. / Title from title page screen (viewed on Oct. 23, 2009). Thesis advisor: Reza Hajimorad. Vita. Includes bibliographical references.

1) Geminiviruses are a group of plant viruses which contain circular single stranded DNA molecules as their genomes and the capsid consists of two icosahedra fused together to form twinned or geminate particles. The largest genus in the family Geminiviridae is that of begomoviruses which are of two kinds; the monopartite begomoviruses which contain only one circular single stranded DNA molecule as their genome and the bipartite begomoviruses which contain two circular single stranded DNA molecules (designated DNA-A and DNA-B) as their genomes. In bipartite viruses, the two DNA molecules are enclosed in separate geminate capsids. 2) In bipartite begomoviruses, the DNA-A encodes the proteins essential for replication and encapsidation of the viral genome while the DNA-B encodes the proteins involved in movement. The DNA-B encodes two proteins: the BV1 or the nuclear shuttle protein (NSP) and BC1 or the cell-to-cell movement protein. Geminiviruses have DNA genomes which replicate inside the host cell nucleus. The NSP, which contains nuclear localization signal, brings the viral DNA from nucleus to the cytoplasm while the BC1 serves to take the viral genome to the cell periphery for movement to the neighbouring cell through the plasmodesmata. 3) The monopartite begomoviruses do not contain DNA-B (which, in bipartite begomoviruses, encodes the proteins involved in movement) and it has been suggested that some of the proteins encoded by DNA-A take up the movement function. Based on studies on TYLCV and CLCuV, a model has been proposed for the movement of monopartite begomoviruses according to which the coat protein (CP) of monopartite begomoviruses serves as the functional equivalent of the NSP of bipartite begomoviruses 4) The present thesis deals with the biochemical characterization of the C4 protein of the monopartite begomovirus CLCuKV-Dab. As stated in statement (3) above, the V2 and C4 proteins of monopartite begomoviruses have been implicated to be involved in cell-to¬cell movement of the viral genome. In TYLCV, both the proteins were shown to be localized to the cell periphery and could move from one cell to another through the plasmodesmata. Further, the V2 protein of CLCuKV-Dab was shown to interact with the coat protein and bind to single stranded DNA. The biochemical properties of the C4 protein needed to be elucidated in order to strengthen the proposal of its probable involvement in movement. 5) The objectives of the present study were: i) Bioinformatic analysis of the C4 protein of CLCuKV-Dab ii) Biochemical characterization of ATPase and pyrophosphatase activities of the C4 protein. iii) Studies on the effect of V2-C4 interaction on the enzymatic properties of C4. iv) Functional characterization of C4 in planta. 6) The FoldIndex© and PONDR analyses predicted the C4 protein of CLCuKV-Dab to be natively unfolded. Similarly, in PSIpred analysis, most of the C4 protein was predicted to be a random coil without any well-defined secondary structure. Further, the protein sequence was analyzed using the motifscan server. However, no motif for any specific function was predicted in the C4 Protein. 7) The C4 gene was initially cloned into pRSET-C vector and overexpressed as histidine tagged protein and the solubility of the protein was tested in various conditions including low temperature (18° C) after inducing the expression of the protein, buffers of various pH and different salt concentrations but the protein remained insoluble. Subsequently, the protein was purified under denaturing conditions and attempts were made to refold the protein but the protein precipitated during refolding. In order to get the C4 protein in soluble form, the C4 gene was subcloned into pGEX-5X2 vector and overexpressed as a GST-tagged fusion protein (GST-C4). Some of the GST-C4 protein was soluble which was purified by using GST-bind resin. The purified fusion protein was observed as a 37 kDa band on SDS-PAGE gel. The purified protein was accompanied by a degraded product of approximately 30 kDa size. Both the intact GST-C4 protein and the degraded product were detected in western blot analysis using anti-GST antibody. 8) Because C4 has been implicated to be involved in movement of monopartite begomoviruses and movement is an energy requiring process, it was of interest to determine if GST-C4 possesses ATPase activity. The purified GST-C4 protein was incubated with γ-[32P]-ATP, the product of the reaction was separated by thin layer chromatography and the chromatography plate was analyzed by phosphorimager. The hydrolysis of ATP by GST-C4 and the release of inorganic phosphate was clearly observed, suggesting that GST-C4 might possess ATPase activity. 9) The reaction conditions for the ATPase activity of GST-C4 were standardized. The activity increased linearly upto 2.60μM of the protein. The optimum temperature and pH for the ATPase activity were found to be 30 C and 6.0 respectively. The activity was inhibited by EDTA, suggesting that it is dependent on divalent metal ions. The activity was stimulated by Mg+2, Mn+2 and Zn+2 but inhibited by Ca+2ions. Further, in the time course experiment, it was observed that the ATPase activity increased linearly upto one hour. 10) The Km, Vmax and kcat for the ATPase activity of GST-C4 were found to be 51.72 ± 2.5 µM, 7.2 ± 0.54 nmoles/min/mg of the protein and 0.27 min-1 respectively. Some of the other virally encoded ATPases have been found to exhibit kcat similar to that found for GST-C4 but it is much lower than those of most of the prokaryotic and eukaryotic ATPases (as mentioned in Table 3.3, page 100, chapter 3). Further, the presence of the degraded product did not affect the kinetic constants as described in chapter 3, pages 95¬-98. It is possible that the enzymatic activity might increase upon interaction with some ligand. 11) In the absence of any putative ATP binding motifs, systematic deletions from N-and C-termini were made to delineate the regions of C4 important for the ATPase activity. GST-N∆15-C4 and GST-N∆30-C4 exhibited approximately 70 % reduction in the ATPase activity while all the C-terminal deletion mutants (GST-C∆10-C4, GST-C∆20¬C4 and GST-C∆30-C4) retained the activity similar to the full length GST-C4 protein. This suggested that the N-terminal region of C4 may contain the residues important for the ATPase activity of GST-C4. 12) In the N-terminal region of C4, there is a sequence CSSSSR which closely resembles the sequence present at the active site of phosphotyrosine phosphatases (CXXXXXR). However, GST-C4 did not catalyze the hydrolysis of p-Nitrophenyl phosphate, a substrate analogue commonly used to assay phosphotyrosine phosphatase activity. It was of interest to determine if the cysteine and arginine in this sequence are important for the ATPase activity of GST-C4. GST-R13A-C4 exhibited an approximately two fold reduction in Vmax suggesting that R13 in C4 may be catalytically important for the ATPase activity of GST-C4. On the other hand, the C8A mutation did not affect the ATPase activity of GST-C4. 13) The GST-C4 protein was tested for its ability to hydrolyze several other phosphate containing compounds as mentioned chapter 2, pages 53-55. Among these compounds, GST-C4 catalyzed the hydrolysis of sodium pyrophosphate, that is, GST-C4 exhibited an inorganic pyrophosphatase activity. 14) The reaction conditions for the inorganic pyrophosphatase activity of GST-C4 were initially standardized. The pyrophosphatase activity of GST-C4 increased linearly upto 3.38 µM of the protein. The optimum temperature and pH for the pyrophosphatase activity were found to be 37° C and 7.0 respectively. The pyrophosphatase activity was inhibited by EDTA, suggesting that it is dependent on divalent metal ions. The activity was most efficiently stimulated by Mg+2, although it was also stimulated by Mn+2and Zn+2but inhibited by Ca+2ions. Thus, the pyrophosphatase activity of GST-C4 resembles the family I inorganic pyrophosphatases in metal ion requirements. Further, the pyrophosphatase activity increased linearly upto 1 hour 30 minutes. 15) The Km, Vmax and Kcat for the pyrophosphatase activity of GST-C4 were found to be 0.76 ± 0.04 mM, 141.16 ± 20 nmoles/min/mg of the protein and 5.2 minrespectively. The kcat for the pyrophosphatase activity was approximately 20 fold higher than that for the ATPase activity (0.27 min-1). 16) GST-N∆15-C4 and GST-N∆30-C4 exhibited >70 % reduction in the pyrophosphatase activity, a finding similar to that for the ATPase activity. On the other hand, while GST-C∆10-C4 retained the activity similar to the full length GST-C4 protein, GST-C∆20-C4 and GST-C∆30-C4 exhibited 20 % and 60 % reduction in the pyrophosphatase activity, respectively, as compared to the full length GST-C4 protein. This suggested that the C-terminal region of C4 may also contain the residues important for the pyrophosphatase activity of GST-C4. However, the C-terminal deletion mutants retained the ATPase activity similar to the full length protein. 17) The pyrophosphatase activity of GST-C4 was stimulated more than three fold by several reducing agents. The C4 protein contains only one cysteine (at position 8 in the C4 sequence). This was the first clue that the cysteine may be important for the pyrophosphatase activity of the GST-C4 protein. Further, the pyrophosphatase activity of GST-C4 did not exhibit preference for a particular kind of reducing agent like that of the pyrophosphatase activity in Streptococcus faecalis. 18) GST-C8A-C4 exhibited more than two fold reduction in Vmax, suggesting that the C8 may be catalytically important for the pyrophosphatase activity of GST-C4. On the other hand, the R13A mutation did not affect the pyrophosphatase activity of the GST-C4 protein. Thus, it is possible that during catalysis, the cysteine thiolate of C4 makes a 19) The pyrophosphatase activity of GST-C4 was inhibited by vanadate and fluoride. Vanadate was found to be a competitive inhibitor with Ki 0.33 mM while fluoride was a non-competitive inhibitor with Ki 2.82 mM. A comparative account of the two enzymatic activities of GST-C4 is presented in table 6.1

Three distinct plant viruses, transmitted by the tobacco whitefly Bemisia tabaci Genn., were associated with diseased food or fiber crops grown in the southwestern deserts of Arizona. The cotton leaf crumple virus (CLCV), thought to affect only cotton Gossypium (L.) spp., is now known to infect other malvaceous plants and members of the Convolvulaceae and Leguminosae. Results of an experimental host range study suggest that potential virus-vector reservoirs may exist in cotton growing regions which include both weeds and cultivated plants. Geminivirus-like (GVL) particles of ∼18 x 30 nm were isolated for the first time from CLCV-infected bean, Phaseolus vulgaris (L.), 'Red Kidney', a plant which was a better purification host than cotton. Studies of CLCV-vector relationships indicated that the acquisition- and inoculation-access times, latent period and length of retention by whitefly vectors were similar to those of the original isolate reported in California in 1954. When growth chamber temperatures of 26, 32, and 37C were used in virus-vector studies, optimal acquisition and transmission occurred at 32C while temperatures of 37C were lethal to whitefly adults. Two additional virus-like agents were isolated from single and mixed infections of lettuce or melons, respectively. The virus-like agent from lettuce infected primarily members of the Chenopodiaceae, Compositae and Cucurbitaceae, and was whitefly but not mechanically transmissible. Long flexuous closterovirus-like rods of ∼10 x 1400-2000 nm were visualized in extracts prepared from plants inoculated with the lettuce isolate. The isolate was similar to the lettuce infectious yellow virus (LIYV) based upon host range, transmission characteristics and unique particle morphology. Both long flexuous rods like those associated with the lettuce isolate and GVL particles of 18 x 30 nm were associated with diseased melons. The host range of the GVL agent was confined to the Cucurbitaceae and Leguminosae and the agent was separated from the mixed infection by mechanical transmission to a non-LIYV host. The GVL-agent was distinct from previously described cucurbit viruses including the squash leaf curl virus, based upon host range and transmission characteristics and was tentatively designated as the watermelon curly mottle virus (WCMV).

Cassava brown streak disease (CBSD) caused by two distinct ssRNA virus species (CBSV and UCBSV of genus Ipomovirus, family Potyviridae) and transmitted by whitefly (Bemisia tabaci), is a major constraint to cassava production in Africa, including Mozambique. In this research, two studies were conducted. First, in order to monitor the incidence, severity and geographical distribution of cassava brown streak disease and associated viruses in Mozambique, field surveys were performed in six cassava major growing provinces. A total of one hundred and fifteen fields and one hundred and forty six fields were surveyed in 2010 and 2012, respectively. The disease was only found in three of six provinces namely Zambezia, Nampula and Cabo Delgado. The CBSD incidence was highest (61.3% and 82.2% in 2010 and 2012, respectively) in Zambezia and lowest (23.6% and 35.1% in 2010 and 2012, respectively) in Cabo Delgado, with cultivars such as Cadri and Robero showing the highest susceptibility to CBSD, while Likonde and Amwalikampiche had relatively low CBSD incidence, illustrating some tolerance to the disease. The results, when compared to previous surveys conducted in 1999 and 2003, demonstrated that the disease is increasing, and replanting new fields with disease-affected cuttings could be responsible for the spread. The second aim of the study was to investigate the genetic diversity of Cassava brown streak viruses, based on analysis of partial sequences of the coat protein gene, in Mozambique. Collections of CBSD-symptomatic leaves were done between June 2010 and June 2012. Diagnostic RT-PCR, using specific primers to screen for the two species, revealed for the first time the presence of Uganda cassava brown streak virus (UCBSV) in Mozambique. UCBSV was found in mixed infections with CBSV, and only confined to a single province of Zambézia, while CBSV species were widely distributed. The phylogenetic analysis revealed two subgroups within CBSV, which were 6.7% divergent in nucleotide sequence. The heterogeneity observed among CBSV isolates in Mozambique suggests that in the future studies more sampling is needed to characterize strains and variants. Addtionally, sequencing of the full CP sequence of CBSaVs isolates is required, which may reveal even more diversity. Infectivity assays of cassava brown streak viruses (CBSV and UCBSV) were established using the host indicator plant Nicotiana benthamiana. Plant sap was extracted from infected cassava leaves and inoculated into N.benthamiana plants. CBSD-like symptoms were observed, and RT-PCR revealed the presence of CBSV in all samples, except for one which was co-infected with UCBSV and CBSV. This study provided further evidence that CBSaVs are efficiently transmitted to N.benthamiana. There is scanty information on alternative hosts, therefore more research is needed to identify other potential hosts of CBSaVs in order to develop an effective strategy to control CBSD.

The limited specificity, sensitivity and multiplex capacity of detection techniques currently available for important seed-borne pathogens of tomato is a significant risk for the global tomato trade and production industry. These pathogens can be associated with seed at low concentrations but, due to their highly virulent nature, these low levels can be sufficient to infect germinating seedlings and spread to neighbouring plants and fields, potentially causing epidemics and economic losses. In this study, detection techniques currently available for phytodiagnostics were evaluated for the capacity to accurately detect and identify five agronomically important seed-borne pathogens of tomato: Pepino mosaic virus (PepMV), Tomato mosaic virus (ToMV), Clavibacter michiganensis subsp. michiganensis (Cmm), Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato. A prototype diagnostic microarray was also designed in an attempt to develop a tool that could simultaneously detect these five seed-borne pathogens from a single sample. Viral detection based on serological techniques was rapid, accurate and reliable but only detected a single pathogen per assay and required supplementary bioassays to indicate the viability of detected viral pathogens. Selective media plating for bacterial detection demonstrated unreliable recovery of targeted bacteria from infected seed and leaf samples and required supplementary tests to validate the identity of presumptive positives. Assays were lengthy, laborious and sometimes too ambiguous for accurate diagnosis of bacterial pathogens. Nucleic acid-based technologies demonstrated improved sensitivity and specificity for detection of targets from pure culture, leaf and seed extracts, compared to conventional and serological methods, yet also required supplementary bioassays or media assays to validate the viability of detected pathogens. Amplification efficiency however, was affected by the presence of PCR inhibitors and despite positive detection, variable banding intensity in electrophoretic analysis of amplified products necessitated the use of reference cultures to validate diagnosis. The developed microarray incorporated 152 pathogen-specific and control probes to facilitate diagnosis and taxonomic classification of detected pathogens. The array was challenged with pure culture extracts of the five target pathogens, selected related and non-target, unrelated pathogens of tomato. Positive detection of each of the pathogens was demonstrated but the production of hybridisation signals was highly variable and extremely sensitive to minor technical differences. Each of the five pathogens were successfully detected in combination proving that different classes of seed-borne pathogens could be detected from a single sample using the developed microarray. This prototype microarray has good potential for phytodiagnostic screening of the five targeted pathogens, and further validation, optimisation and extension for testing tomato seed samples may facilitate incorporation of this array into standard diagnostic protocols.

Solanum lycopersicon (the cultivated tomato) is a commodity of great economic importance in South Africa (SA) as well as worldwide. A destructive viral disease known as Tomato curly stunt virus, ToCSV-[ZA:Ond:98], belonging to the genus Begomovirus has negatively impacted on tomato production in SA. This has brought about the need to develop resistant cultivars to ToCSV. Since all cultivated tomato cultivars are susceptible to ToCSV, resistance genes against the virus found in wild tomato plant species have been introgressed into the cultivated tomato by plant breeding techniques. Wild relatives of tomato were adapted to many pathogens (including viruses) as well as stresses from the surrounding environment. During breeding for improved fruit quality and increased yield, the gene networks giving rise to many biotic and abiotic stress resistances have been lost leaving the domesticated tomato extremely susceptible. Plant breeders have reconstituted some of the gene networks into the cultivated tomato that provide tolerance to stresses including viruses. They have achieved this by the help of marker-assisted selection (MAS), where the associated marker is used as an indirect selection criterion. This is an important process in commercial breeding programs as it allows for a speedy selection of selected traits in the development of tomato hybrids. The defence response to abiotic stresses in plants includes the expression of heat shock proteins (HSPs) that function as stress response proteins, molecular chaperones and proteases which repair or degrade damaged proteins. The objective of this study was to elucidate the type of resistance mechanism of a tomato inbred line (TAM), to ToCSV. Since TYLCV-IL shows 77% nucleotide identity with ToCSV, molecular markers already established for the detection of resistance genes for TYLCV-IL were used to screen TAM. The inbred line, TAM, was screened for the absence of any of the known resistant genes to TYLCV-IL using molecular markers already established for the screening of TYCLV-IL resistance genes. TAM was crossed with susceptible cultivar, Rooikhaki, to produce F1 hybrids. These F1 hybrids were selfed to produce an F2 population. Infection trials using ToCSV were conducted using TAM inbred line, F1 hybrids and the F2 population. Since TAM did not have any of the known resistance genes to TYLCV-IL, a possible novel resistance source to ToCSV was speculated. A clue to the resistant mechanism against ToCSV resistance in TAM was indicated by the segregation patterns of the F2 population after inoculation with ToCSV. The results suggest that the resistance is under the control of partially dominant resistant genes. The level of resistance of commercial South African tomato cultivars (Tyler and Tovi-star) against TYLCV-IL was investigated. The heat shock protein (HSP) profiles of these two SA lines including susceptible cultivar, Rooikhaki, were treated with abiotic stresses (salt and heat) and results were compared with a similar study conducted with TYCLV-IL resistant and susceptible tomato cultivars. Heat shock protein 70 accumulation patterns were similar in that HSP70 was more stable in the resistant cultivars throughout the application when abiotic stresses were applied to the SA resistant and susceptible tomato cultivars as compared to Israel resistant and susceptible breeding lines. A relation between infection severity and the pattern of HSP expression was found. A higher level of HSP 70 in resistant tomato plants could contribute to a lower symptom severity phenotype.

Photocopy of typescript. / Digitized by Kansas Correctional Industries

Cassava (Manihot esculenta) is a major food crop in sub-Saharan Africa, where cassava is mostly used as a subsistence crop. In southern Africa, cassava production is affected by cassava mosaic disease (CMD). The impact of CMD on cassava yield has a devastating impact on the economy in southern Africa as it is also an important industrial crop. South African cassava mosaic virus (SACMV) is a distinct geminivirus known to cause CMD in Southern Africa. SACMV is a bipartite virus where the two components DNA-A (2800bp) and DNA-B (2760bp) code for important proteins needed for viral replication, movement and transmission. Defective interfering molecules are a type of subgenomic molecule associated with geminiviruses. DIs are dependent on the helper virus for replication. DIs are known to lead to symptom amelioration in plants infected with its cognate helper virus as a result of interfering with the helper virus replication in the plants. It is believed that they interfere with the replication, by competing for limited host and viral factors needed for replication by the helper virus. They also have a size-advantage to be selected over the helper virus, allowing for increased DI proliferation. A putative defective interfering (DI) molecule, isolated from a naturally infected field cassava, has been associated with SACMV. This DI molecule is derived from the DNA-B component of SACMV and is 1389bp in size, approximately half the size of DNA-B. In this study, the objectives were to investigate the effect of DI on SACMV replication and symptom development in a model host – N. benthamiana and cassava, the natural host of SACMV, in order to determine whether the DI was indeed an interfering molecule. Viral load (DNA-A and DNA-B) were determined using quantitative real-time PCR, and symptoms were scored according to a symptom severity index (1-5). The results from this study show that the DI did influence viral titres in N. benthamiana and cassava when infected with SACMV. However, the impact of DI on reducing viral replication and symptom attenuation was different in the two plant systems. In cassava, the symptom attenuation was more pronounced compared with N. benthamiana, which correlates to the respective viral titres, thus highlighting the differences in viral-host factor interactions during viral replication between a model host (Nicotiana benthamiana) and a natural host (cassava). It was also observed that the DI had an impact on viral replication and symptom attenuation, when it was present with SACMV- from onset of infection, but titres of DNA A and DNA B showed a cyclic pattern of increases and decreases during the infection process. A surprising observation made from this study was that either the presence of DI or reduced titres of DNA-B, due to interference by the DI, had a direct effect on the ability of the helper virus SACMV to develop a specific chlorotic symptom phenotype in infected leaves, suggesting that DNA B plays an important role in symptom development. Additionally, transgenic N. benthamiana transformed with a DI insert, which only replicates in the presence of the SACMV, was tested as a resistance strategy. Again, quantitative realtime PCR was used to determine viral load and symptoms were scored according to the symptom severity index. Unexpectedly, ours results showed that neither decreased viral load nor attenuated symptoms were observed when transgenic N. benthamiana was infected with SAMV, deeming the use of DI as a transgenic resistance approach non-viable. This suggests that although DI does decrease viral titres and lead to symptom attenuation during natural systemic infections or experimental inoculations, these DIs don’t hold potential in combating SACMV and CMD in a transgenic system.

Corrigendum inserted at the back. Includes bibliographical references (leaves 133-158). Ch. 1. General introduction -- ch. 2. General materials and methods -- ch. 3. Biological characterisation of Australian PSbMV isolates -- ch. 4. Developing nucleic acid based diagnostics for PSbMV -- ch. 5. Detection of PSbMV isolates by RT-PCR and RFLP analysis -- ch. 6. Developing an internal control for PSbMV RT-PCR -- ch. 7. Molecular analysis of the PSbMV VPG -- ch. 8. PSbMV sequence and phylogenetic analysis -- ch. 9. General discussion Sixteen pea seed borne mosaic virus (PSbMV) isolates were collected between 1995 and 1998. These isolates were biologically distinct yet serologically indistinguishable. The conclusion is that PSbMV is widespread and occurs at a low incidence in Australia. Reports sequence information on new isolates of PSbMV which has allowed genomic regions to be identified which distinguish PSbMV pathotypes and isolates; and, to the development of PSbMV nucleic acid hybridisation and RT-PCR assays.