Browsing by Author "Ogwok, Emmanuel"

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    Comparative analysis of virus-derived small RNAs within cassava (Manihot esculenta Crantz) infected with cassava brown streak viruses
    (Virus research, 2016) Ogwok, Emmanuel; Ilyas, Muhammad; Alicai, Titus; Rey, Marie E.C.; Taylor, Nigel J.
    Infection of plant cells by viral pathogens triggers RNA silencing, an innate antiviral defense mechanism. In response to infection, small RNAs (sRNAs) are produced that associate with Argonaute (AGO)-containing silencing complexes which act to inactivate viral genomes by posttranscriptional gene silencing (PTGS). Deep sequencing was used to compare virus-derived small RNAs (vsRNAs) in cassava genotypes NASE 3, TME 204 and 60444 infected with the positive sense single-stranded RNA (+ssRNA) viruses Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), the causal agents of cassava brown streak disease (CBSD). An abundance of 21-24 nt vsRNAs was detected and mapped, covering the entire CBSV and UCBSV genomes. The 21 nt vsRNAs were most predominant, followed by the 22 nt class with a slight bias toward sense compared to antisense polarity, and a bias for adenine and uracil bases present at the 5’-terminus. Distribution and frequency of vsRNAs differed between cassava genotypes and viral genomes. In susceptible genotypes TME 204 and 60444, CBSV-derived sRNAs were seen in greater abundance than UCBSV-derived sRNAs. NASE 3, known to be resistant to UCBSV, accumulated negligible UCBSV-derived sRNAs but high populations of CBSV-derived sRNAs. Transcript levels of cassava homologues of AGO2, DCL2 and DCL4, which are central to the gene-silencing complex, were found to be differentially regulated in CBSV- and UCBSV-infected plants across genotypes, suggesting these proteins play a role in antiviral defense. Irrespective of genotype or viral pathogen, maximum populations of vsRNAs mapped to the cytoplasmic inclusion, P1 and P3 protein-encoding regions. Our results indicate disparity between CBSV and UCBSV host-virus interaction mechanisms, and provide insight into the role of virus-induced gene silencing as a mechanism of resistance to CBSD.
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    Efficient conditions for in vitro establishment and regeneration of disease-free Ugandan farmer-preferred cassava genotypes
    (African Journal of Biotechnology, 2021) Apio, Hellen B.; Alicai, Titus; Ogwok, Emmanuel
    Cassava (Manihot esculenta Crantz) is majorly devastated by two viral diseases, cassava brown streak disease (CBSD) and cassava mosaic disease (CMD), resulting in 100% yield loss. Being a clonal plant, nodal cuttings (NC) and shoot apical meristems (SAMs) are the best explants for production of disease free planting materials. In this study, NCs and SAMs were used to determine reliable indicators for successful in vitro establishment of cassava. Eight cassava genotypes were used for the study. Leaf samples were collected from 30 stakes of each of the eight genotypes planted in the screen house. The leaf samples were pooled and screened for presence and/or absence of CBSD and CMD by PCR using virus specific primers. Nodal cuttings were excised from screen house grown plants, surface sterilized to rid-off contaminants and established on Murashige and Skoog (MS) Medium. Using the sprouted stakes, 5-mm sized SAMs were excised, surface sterilized and reduced to 0.5-1 and 2-3 mm sizes. The SAMs were established on MS medium with varying concentrations of plant growth regulators (0.5, 1, 2) ml/L Benzylaminopurine (BAP) and (2, 4) ml/L Naphthalene acetic acid (NAA), Kinetin (K) and BAP respectively. PCR results revealed the pooled leaf samples were free of both CBSD and CMD for all genotypes. Establishment and regeneration of NCs was possible with MS medium for all genotypes. For the SAMs, the concentrations of (2, 4) ml/LBAP followed by 2 ml/LNAA facilitated their establishment and regeneration in comparison to KIN.SAMs of 2-3 mm sizes regenerated better than 0.5 - 1 mm size. Both NCs and SAMs of the different genotypes produced leaves, nodes, roots and there was an increase in plant length. These parameters are critical indicators for in vitro establishment and regeneration of cassava.
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    Eleven years of breeding efforts to combat cassava brown streak disease
    (Breeding Science, 2016) Sezi Kawuki, Robert; Kaweesi, Tadeo; Esuma, Williams; Pariyo, Anthony; Kayondo, Ismail Siraj; Ozimati, Alfred; Kyaligonza, Vincent; Abaca, Alex; Orone, Joseph; Tumuhimbise, Robooni; Nuwamanya, Ephraim; Abidrabo, Philip; Amuge, Teddy; Ogwok, Emmanuel; Okao, Geoffrey; Wagaba, Henry; Adiga, Gerald; Alicai, Titus; Omongo, Christopher; Bua, Anton; Ferguson, Morag; Kanju, Edward; Baguma, Yona
    Cassava (Manihot esculenta Crantz) production is currently under threat from cassava brown streak disease (CBSD), a disease that is among the seven most serious obstacles to world’s food security. Three issues are of significance for CBSD. Firstly, the virus associated with CBSD, has co-evolved with cassava outside its center of origin for at least 90 years. Secondly, that for the last 74 years, CBSD was only limited to the low lands. Thirdly, that most research has largely focused on CBSD epidemiology and virus diversity. Accordingly, this paper focuses on CBSD genetics and/or breeding and hence, presents empirical data generated in the past 11 years of cassava breeding in Uganda. Specifically, this paper provides: 1) empirical data on CBSD resistance screening efforts to identify sources of resistance and/or tolerance; 2) an update on CBSD resistance population development comprising of full-sibs, half-sibs and S1 families and their respective field performances; and 3) insights into chromosomal regions and genes involved in CBSD resistance based on genome wide association analysis. It is expected that this information will provide a foundation for harmonizing on-going CBSD breeding efforts and consequently, inform the future breeding interventions aimed at combating CBSD.
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    Expansion of the cassava brown streak pandemic in Uganda revealed by annual field survey data for 2004 to 2017
    (Scientific Data, 2019) Alicai, Titus; Szyniszewska, Anna M.; Omongo, Christopher A .; Abidrabo, Phillip; Okao-Okuja, Geoffrey; Baguma, Yona; Ogwok, Emmanuel; Kawuki, Robert; Esuma, Williams; Tairo, Fred; Bua, Anton; Legg, James P.; Stutt, Richard O. J. H.; Godding, David; Sseruwagi, Peter; Ndunguru, Joseph; Gilligan, Christopher A.
    Cassava brown streak disease (CBSD) is currently the most devastating cassava disease in eastern, central and southern Africa affecting a staple crop for over 700 million people on the continent. A major outbreak of CBSD in 2004 near Kampala rapidly spread across Uganda. In the following years, similar CBSD outbreaks were noted in countries across eastern and central Africa, and now the disease poses a threat to West Africa including Nigeria - the biggest cassava producer in the world. A comprehensive dataset with 7,627 locations, annually and consistently sampled between 2004 and 2017 was collated from historic paper and electronic records stored in Uganda. The survey comprises multiple variables including data for incidence and symptom severity of CBSD and abundance of the whitefly vector (Bemisia tabaci). This dataset provides a unique basis to characterize the epidemiology and dynamics of CBSD spread in order to inform disease surveillance and management. We also describe methods used to integrate and verify extensive field records for surveys typical of emerging epidemics in subsistence crops.
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    Real time portable genome sequencing for global food security
    (F1000 Research, 2018) Boykin, Laura; Ghalab, Ammar; Rossitto De Marchi, Bruno; Savill, Anders; Wainaina, James M.; Kinene, Tonny; Lamb, Stephen; Rodrigues, Myriam; Kehoe, Monica; Ndunguru, Joseph; Tairo, Fred; Sseruwagi, Peter; Kayuki, Charles; Mark, Deogratius; Erasto, Joel; Bachwenkizi, Hilda; Alicai, Titus; Okao-Okuja, Geoffrey; Abridrabo, Phillip; Ogwok, Emmanuel; Osingada, John Francis; Akono, Jimmy; Ateka, Elijah; Muga, Brenda; Kiarie, Samuel
    Crop losses due to viral diseases and pests are major constraints on food security and income for millions of households in sub-Saharan Africa (SSA). Such losses can be reduced if plant diseases and pests are correctly diagnosed and identified early. Currently, accurate diagnosis for definitive identification of plant viruses and their vectors in SSA mostly relies on standard PCR and next generation sequencing technologies (NGS). However, it can take up to 6 months before results generated using these approaches are available. The long time taken to detect or identify viruses impedes quick, within-season decision-making necessary for early action, crop protection advice and disease control measures by farmers. This ultimately compounds the magnitude of crop losses and food shortages suffered by farmers. The MinION portable pocket DNA sequencer was used, to our knowledge globally for the first time, to sequence whole plant virus genomes. We used this technology to identify the begomoviruses causing the devastating cassava mosaic virus, which is ravaging smallholder farmers’ crops in sub-Saharan Africa.
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    RNAi-derived field resistance to Cassava brown streak disease persists across the vegetative cropping cycle
    (GM Crops & Food, 2014) Odipio, John; Ogwok, Emmanuel; Taylor, Nigel J.; Halsey, Mark; Bua, Anton; Fauquet, Claude M.; Alicai, Titus
    A confined field trial was established to determine durability of RNAi-mediated resistance to Cassava brown streak disease (CBSD). Stem cuttings were obtained from field-grown cassava plants of cv 60444 transgenic for construct p718, consisting of an 894 bp inverted repeat sequence from the Ugandan Cassava brown streak virus (UCBSV) coat protein. Plants were established from three transgenic lines previously shown to provide complete resistance to UCBSV and differing levels of protection to the non-homologous virus species Cassava brown streak virus (CBSV), and grown for 11 months. CBSD symptoms were observed on shoots and storage roots of all non-transgenic cv 60444 control plants and transgenic lines p718–002 and p718–005, but not on p718–001. RT-PCR diagnostic showed tissues of plant lines p718–002 and p718–005 to be infected with CBSV, but free of UCBSV. All leaves and roots of p718–001 plants were confirmed to carry no detectable levels of either pathogen. Plants of cv 60444 in this field trial showed severe cassava mosaic disease symptoms, indicating that presence of replicating geminiviruses did not cause significant suppression of RNAi-mediated resistance to CBSD. Resistance to CBSD across a vegetative cropping cycle confirms earlier field data, and provides an important step in proof of concept for application of RNAi technology to control of CBSD under conditions encountered in farmers’ fields.
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    RNAi-mediated resistance to Cassava brown streak Uganda virus in transgenic cassava
    (Molecular Plant Pathology, 2011) Yadav, Jitender S.; Ogwok, Emmanuel; Wagaba, Henry; Patil, Basavaprabhu L.; Bagewadi, Basavaraj; Alicai, Titus; Gaitan-Solis, Eliana; Taylor, Nigel J.; Fauquet, Claude M.
    Cassava brown streak disease (CBSD), caused by Cassava brown streak Uganda virus (CBSUV) and Cassava brown streak virus (CBSV), is of new epidemic importance to cassava (Manihot esculenta Crantz) production in East Africa, and an emerging threat to the crop in Central and West Africa. This study demonstrates that at least one of these two ipomoviruses, CBSUV, can be efficiently controlled using RNA interference (RNAi) technology in cassava. An RNAi construct targeting the near full-length coat protein (FL-CP) of CBSUV was expressed constitutively as a hairpin construct in cassava. Transgenic cassava lines expressing small interfering RNAs (siRNAs) against this sequence showed 100% resistance to CBSUV across replicated graft inoculation experiments. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed the presence of CBSUV in leaves and some tuberous roots from challenged controls, but not in the same tissues from transgenic plants. This is the first demonstration of RNAi-mediated resistance to the ipomovirus CBSUV in cassava.
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    RNAi-mediated resistance to diverse isolates belonging to two virus species involved in Cassava brown streak disease
    (Molecular plant pathology, 2011) Patil, Basavaprabhu L.; Ogwok, Emmanuel; Wagaba, Henry; Mohammed, Ibrahim U.; Yadav, Jitender S.; Bagewadi, Basavaraj; Taylor, Nigel J.; Kreuze, Jan F.; Maruthi, M. N.; Alicai, Titus; Fauquet, Claude M.
    Cassava brown streak disease (CBSD) is emerging as one of the most important viral diseases of cassava (Manihot esculenta) and is considered today as the biggest threat to cassava cultivation in East Africa. The disease is caused by isolates of at least two phylogenetically distinct species of single-stranded RNA viruses belonging to the family Potyviridae, genus Ipomovirus. The two species are present predominantly in the coastal lowland [Cassava brown streak virus (CBSV); Tanzania and Mozambique] and highland [Cassava brown streak Uganda virus (CBSUV); Lake Victoria Basin, Uganda, Kenya and Malawi] in East Africa. In this study, we demonstrate that CBSD can be efficiently controlled using RNA interference (RNAi). Three RNAi constructs targeting the highland species were generated, consisting of the full-length (FL; 894 nucleotides), 397-nucleotide N-terminal and 491- nucleotide C-terminal portions of the coat protein (CP) gene of a Ugandan isolate of CBSUV (CBSUV-[UG:Nam:04]), and expressed constitutively in Nicotiana benthamiana. After challenge with CBSUV-[UG:Nam:04], plants homozygous for FL-CP showed the highest resistance, followed by the N-terminal and C-terminal lines with similar resistance. In the case of FL, approximately 85% of the transgenic plant lines produced were completely resistant. Some transgenic lines were also challenged with six distinct isolates representing both species: CBSV and CBSUV. In addition to nearly complete resistance to the homologous virus, two FL plant lines showed 100% resistance and two C-terminal lines expressed 50–100% resistance, whereas the N-terminal lines succumbed to the nonhomologous CBSV isolates. Northern blotting revealed a positive correlation between the level of transgene-specific small interfering RNAs detected in transgenic plants and the level of virus resistance.This is the first demonstration of RNAi-mediated resistance to CBSD and protection across very distant isolates (more than 25% in nucleotide sequence) belonging to two different species: Cassava brown streak virus and Cassava brown streak Uganda virus
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    Transgenic RNA interference (RNAi)-derived field resistance to cassava brown streak disease
    (Molecular plant pathology, 2012) Ogwok, Emmanuel; Odipio, John; Halsey, Mark; Gaitán-Solís, Eliana; Bua, Anton; Taylor, Nigel J.; Fauquet, Claude M.; Alicai, Titus
    Cassava brown streak disease (CBSD), caused by the Ipomoviruses Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV), is considered to be an imminent threat to food security in tropical Africa. Cassava plants were transgenically modified to generate small interfering RNAs (siRNAs) from truncated full-length (894-bp) and N-terminal (402-bp) portions of the UCBSV coat protein (DCP) sequence. Seven siRNA-producing lines from each gene construct were tested under confined field trials at Namulonge, Uganda. All nontransgenic control plants (n = 60) developed CBSD symptoms on aerial tissues by 6 months after planting, whereas plants transgenic for the full-length DCP sequence showed a 3-month delay in disease development, with 98% of clonal replicates within line 718-001 remaining symptom free over the 11-month trial. Reverse transcriptase-polymerase chain reaction (RT-PCR) diagnostics indicated the presence of UCBSV within the leaves of 57% of the nontransgenic controls, but in only two of 413 plants tested (0.5%) across the 14 transgenic lines. All transgenic plants showing CBSD were PCR positive for the presence of CBSV, except for line 781-001, in which 93% of plants were confirmed to be free of both pathogens. At harvest, 90% of storage roots from nontransgenic plants were severely affected by CBSD-induced necrosis. However, transgenic lines 718- 005 and 718-001 showed significant suppression of disease, with 95% of roots from the latter line remaining free from necrosis and RT-PCR negative for the presence of both viral pathogens. Crossprotection against CBSV by siRNAs generated from the full-length UCBSV DCP confirms a previous report in tobacco.The information presented provides proof of principle for the control of CBSD by RNA interference-mediated technology, and progress towards the potential control of this damaging disease.