Browsing by Author "Taylor, Nigel J."
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Item Artificial microRNA-derived resistance to Cassava brown streak disease(Journal of virological methods, 2016) Wagaba, Henry; Patil, Basavaprabhu L.; Mukasa, Settumba; Alicai, Titus; Fauquet, Claude M.; Taylor, Nigel J.Artificial miRNAs (amiRNA) were generated targeting conserved sequences within the genomes of the two causal agents of Cassava brown streak disease (CBSD): Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Transient expression studies on ten amiRNAs targeting 21 nt conserved sequences of P1(CBSV and UCBSV), P3(CBSV and UCBSV), CI(UCBSV), NIb(CBSV and UCBSV), CP(UCBSV) and the un-translated region (3 -UTR) were tested in Nicotiana benthamiana. Four out of the ten amiRNAs expressed the corresponding amiRNA at high levels. Transgenic N. benthamiana plants were developed for the four amiRNAs targeting the P1 and NIb genes of CBSV and the P1 and CP genes of UCBSV and shown to accumulate miRNA products. Transgenic plants challenged with CBSV and UCBSV isolates showed resistance levels that ranged between ∼20–60% against CBSV and UCBSV and correlated with expression levels of the transgenically derived miRNAs. MicroRNAs targeting P1 and NIb of CBSV showed protection against CBSV and UCBSV, while amiRNAs targeting the P1 and CP of UCBSV showed protection against UCBSV but were less efficient against CBSV. These results indicate a potential application of amiRNAs for engineering resistance to CBSD-causing viruses in cassava.Item 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.Item Efficient transmission of Cassava brown streak disease viral pathogens by chip bud grafting(BMC Research, 2013) Wagaba, Henry; Beyene, Getu; Trembley, Cynthia; Alicai, Titus; Fauquet, Claude M.; Taylor, Nigel J.Techniques to study plant viral diseases under controlled growth conditions are required to fully understand their biology and investigate host resistance. Cassava brown streak disease (CBSD) presents a major threat to cassava production in East Africa. No infectious clones of the causal viruses, Cassava brown streak virus (CBSV) or Ugandan cassava brown streak virus (UCBSV) are available, and mechanical transmission to cassava is not effective. An improved method for transmission of the viruses, both singly and as co-infections has been developed using bud grafts. Findings: Axillary buds from CBSD symptomatic plants infected with virulent isolates of CBSV and UCBSV were excised and grafted onto 6–8 week old greenhouse-grown, disease-free cassava plants of cultivars Ebwanateraka, TME204 and 60444. Plants were assessed visually for development of CBSD symptoms and by RT-PCR for presence of the viruses in leaf and storage root tissues. Across replicated experiments, 70-100% of plants inoculated with CBSV developed CBSD leaf and stem symptoms 2–6 weeks after bud grafting. Infected plants showed typical, severe necrotic lesions in storage roots at harvest 12–14 weeks after graft inoculation. Sequential grafting of buds from plants infected with UCBSV followed 10–14 days later by buds carrying CBSV, onto the same test plant, resulted in 100% of the rootstocks becoming co-infected with both pathogens. This dual transmission rate was greater than that achieved by simultaneous grafting with UCBSV and CBSV (67%), or when grafting first with CBSV followed by UCBSV (17%). Conclusions: The bud grafting method described presents an improved tool for screening cassava germplasm for resistance to CBSD causal viruses, and for studying pathogenicity of this important disease. Bud grafting provides new opportunities compared to previously reported top and side grafting systems. Test plants can be inoculated as young, uniform plants of a size easily handled in a small greenhouse or large growth chamber and can be inoculated in a controlled manner with CBSV and UCBSV, either singly or together. Disease symptoms develop rapidly, allowing better studies of interactions between these viral pathogens, their movement within shoot and root systems, and how they induce their destructive disease symptoms.Item Field Level RNAi-Mediated Resistance to Cassava Brown Streak Disease across Multiple Cropping Cycles and Diverse East African Agro-Ecological Locations(Frontiers in plant science, 2017) Wagaba, Henry; Beyene, Getu; Aleu, Jude; Odipio, John; Okao-Okuja, Geoffrey; Deepika Chauhan, Raj; Munga, Theresia; Obiero, Hannington; Halsey, Mark E.; Ilyas, Muhammad; Raymond, Peter; Bua, Anton; Taylor, Nigel J.; Miano, Douglas; Alicai, TitusCassava brown streak disease (CBSD) presents a serious threat to cassava production in East and Central Africa. Currently, no cultivars with high levels of resistance to CBSD are available to farmers. Transgenic RNAi technology was employed to combat CBSD by fusing coat protein (CP) sequences from Ugandan cassava brown streak virus (UCBSV) and Cassava brown streak virus (CBSV) to create an inverted repeat construct (p5001) driven by the constitutive Cassava vein mosaic virus promoter. Twenty-five plant lines of cultivar TME 204 expressing varying levels of small interfering RNAs (siRNAs) were established in confined field trials (CFTs) in Uganda and Kenya. Within an initial CFT at Namulonge, Uganda, non-transgenic TME 204 plants developed foliar and storage root CBSD incidences at 96–100% by 12 months after planting. In contrast, 16 of the 25 p5001 transgenic lines showed no foliar symptoms and had less than 8% of their storage roots symptomatic for CBSD. A direct positive correlation was seen between levels of resistance to CBSD and expression of transgenic CP-derived siRNAs. A subsequent CFT was established at Namulonge using stem cuttings from the initial trial. All transgenic lines established remained asymptomatic for CBSD, while 98% of the non-transgenic TME 204 stake-derived plants developed storage roots symptomatic for CBSD. Similarly, very high levels of resistance to CBSD were demonstrated by TME 204 p5001 RNAi lines grown within a CFT over a full cropping cycle at Mtwapa, coastal Kenya. Sequence analysis of CBSD causal viruses present at the trial sites showed that the transgenic lines were exposed to both CBSV and UCBSV, and that the sequenced isolates shared >90% CP identity with transgenic CP sequences expressed by the p5001 inverted repeat expression cassette. These results demonstrate very high levels of field resistance to CBSD conferred by the p5001 RNAi construct at diverse agro-ecological locations, and across the vegetative cropping cycle.Item 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, TitusA 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.Item 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.Item 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 virusItem Successful application of FTA® Classic Card technology and use of bacteriophage 29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes(Journal of virological methods, 2007) Owor, Betty E.; Shepherd, Dionne N.; Taylor, Nigel J.; Edema, Richard; Monjane, Ad´erito L.; Thomson, Jennifer A.; Martin, Darren P.; Varsani, ArvindLeaf samples from 155 maize streak virus (MSV)-infected maize plants were collected from 155 farmers’ fields in 23 districts in Uganda in May/June 2005 by leaf-pressing infected samples onto FTA® Classic Cards. Viral DNA was successfully extracted from cards stored at room temperature for 9 months. The diversity of 127 MSV isolates was analysed by PCR-generated RFLPs. Six representative isolates having different RFLP patterns and causing either severe, moderate or mild disease symptoms, were chosen for amplification from FTA cards by bacteriophage 29 DNA polymerase using the TempliPhiTM system. Full-length genomes were inserted into a cloning vector using a unique restriction enzyme site, and sequenced. The 1.3-kb PCR product amplified directly from FTA-eluted DNA and used for RFLP analysis was also cloned and sequenced. Comparison of cloned whole genome sequences with those of the original PCR products indicated that the correct virus genome had been cloned and that no errors were introduced by the 29 polymerase. This is the first successful large-scale application of FTA card technology to the field, and illustrates the ease with which large numbers of infected samples can be collected and stored for downstream molecular applications such as diversity analysis and cloning of potentially new virus genomes. © 2006 Elsevier B.V. All rights reserved.Item Transgenic overexpression of endogenous FLOWERING LOCUS T-like gene MeFT1 produces early flowering in cassava(PLoS ONE, 2020) Odipio, John; Getu, Beyene; Chauhan, R. D.; Alicai, Titus; Bart, Rebecca; Nusinow, Dmitri A.; Taylor, Nigel J.Endogenous FLOWERING LOCUS T homolog MeFT1 was transgenically overexpressed under control of a strong constitutive promoter in cassava cultivar 60444 to determine its role in regulation of flowering and as a potential tool to accelerate cassava breeding. Early profuse flowering was recorded in-vitro in all ten transgenic plant lines recovered, causing eight lines to die within 21 days of culture. The two surviving transgenic plant lines flowered early and profusely commencing as soon as 14 days after establishment in soil in the greenhouse. Both transgenic lines sustained early flowering across the vegetative propagation cycle, with first flowering recorded 30–50 days after planting stakes compared to 90 days for non-transgenic controls. Transgenic plant lines completed five flowering cycles within 200 days in the greenhouse as opposed to twice flowering event in the controls. Constitutive overexpression of MeFT1 generated fully mature male and female flowers and produced a bushy phenotype due to significantly increased flowering-induced branching. Flower induction by MeFT1 overexpression was not graft-transmissible and negatively affected storage root development. Accelerated flowering in transgenic plants was associated with significantly increased mRNA levels of MeFT1 and the three floral meristem identity genes MeAP1, MeLFY and MeSOC1 in shoot apical tissues. These findings imply that MeFT1 encodes flower induction and triggers flowering by recruiting downstream floral meristem identity genes.Item 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, TitusCassava 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.Item The VIRCA Project: Virus resistant cassava for Africa(GM Crops & Food, 2012) Taylor, Nigel J.; Halsey, Mark; Gaitán-Solís, Eliana; Anderson, Paul; Gichuki, Simon; Miano, Douglas; Bua, Anton; Alicai, Titus; Fauquet, Claude M.The VIRCA (Virus Resistant Cassava for Africa) project is a collaborative program between the Donald Danforth Plant Science Center, USA the National Crops Resources Research Institute, Uganda and the Kenya Agricultural Research Institute, Kenya. VIRCA is structured to include all aspects of the intellectual property, technology, regulatory, biosafety, quality control, communication and distribution components required for a GM crop development and delivery process. VIRCA's goal is to improve cassava for resistance to the viral diseases cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) using pathogen-derived RNAi technology, and to field test, obtain regulatory approval for and deliver these products to small landholder farmers. During Phase I of the project, proof of concept was achieved by production and testing of virus resistant plants under greenhouse and confined field trials in East Africa. In VIRCA Phase II, two farmer-preferred varieties will be modified for resistance to CBSD and CMD, and lead events identified after molecular and field screening. In addition to delivery of royalty-free improved planting materials for farmers, VIRCA capacity building activities are enhancing indigenous capability for crop biotechnology in East Africa.Item A Virus-Derived Stacked RNAi Construct Confers Robust Resistance to Cassava Brown Streak Disease(Frontiers in Plant Science, 2017) Beyene, Getu; Deepika Chauhan, Raj; Ilyas, Muhammad; Wagaba, Henry; Fauquet, Claude M.; Miano, Douglas; Alicai, Titus; Taylor, Nigel J.Cassava brown streak disease (CBSD) threatens food and economic security for smallholder farmers throughout East and Central Africa, and poses a threat to cassava production in West Africa. CBSD is caused by two whitefly-transmitted virus species: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (Genus: Ipomovirus, Family Potyviridae). Although varying levels of tolerance have been achieved through conventional breeding, to date, effective resistance to CBSD within East African cassava germplasm has not been identified. RNAi technology was utilized to integrate CBSD resistance into the Ugandan farmer-preferred cassava cultivar TME 204. Transgenic plant lines were generated expressing an inverted repeat construct (p5001) derived from coat-protein (CP) sequences of CBSV and UCBSV fused in tandem. Northern blots using probes specific for each CP sequence were performed to characterize 169 independent transgenic lines for accumulation of CP-derived siRNAs. Transgenic plant lines accumulating low, medium and high levels of siRNAs were bud graft challenged with the virulent CBSV Naliendele isolate alone or in combination with UCBSV. Resistance to CBSD in the greenhouse directly correlated to levels of CPderived siRNAs as determined by visual assessment of leaf and storage root symptoms, and RT-PCR diagnosis for presence of the pathogens. Low expressing lines were found to be susceptible to CBSV and UCBSV, while medium to high accumulating plant lines were resistant to both virus species. Absence of detectable virus in the best performing p5001 transgenic lines was further confirmed by back-inoculation via sap or graft challenge to CBSD susceptible Nicotiana benthamiana and cassava cultivar 60444, respectively. Data presented shows robust resistance of transgenic p5001 TME 204 lines to both CBSV and UCBSV under greenhouse conditions. Levels of resistance correlated directly with levels of transgene derived siRNA expression such that the latter can be used as predictor of resistance to CBSD