Browsing by Author "Patil, Basavaprabhu L."

Now showing 1 - 4 of 4
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    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.
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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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    Transmission studies with Cassava brown streak Uganda virus (Potyviridae: Ipomovirus) and its interaction with abiotic and biotic factors in Nicotiana benthamiana
    (Journal of Virological Methods, 2010) Ogwoka, Emmanuel; Patil, Basavaprabhu L.; Alicai, Titus; Fauqueta, Claude M.
    Cassava brown streak disease (CBSD), caused by two distinct species, Cassava brown streak Uganda virus (CBSUV) and Cassava brown streak virus (CBSV), is a major constraint to cassava (Manihot esculenta Crantz) production in Africa. Absence of infectious clones of CBSUV or CBSV and the lack of efficient means of mechanical transmission of CBSD has hampered laboratory studies of this disease. Mechanical transmission, achieved mainly by plant sap inoculation, is a widely used technique for characterizing plant viruses. Efficient sap transmission of CBSUV/CBSV to the common laboratory host Nicotiana benthamiana is essential for both basic and applied studies of the virus. We report here the development of an efficient protocol for sap transmission of CBSUV to N. benthamiana and N. debneyi. Several factors affecting transmission efficiency were identified such as the effects of buffer composition, antioxidants, inoculum concentration, plant age and temperature. Higher temperatures (30 ◦C) favored rapid symptom initiation compared to lower temperatures (21 ◦C) when sap prepared in phosphate buffer of pH 7.0 was applied on the leaves of N. benthamiana dusted with the abrasive (carborundum). We demonstrated the usefulness of the transmission method in transient evaluation of CBSUV[UG:Nam:04]-derived RNA interference constructs for CBSD resistance and also in studying the interaction of CBSUV[UG:Nam:04] with cassava mosaic geminiviruses, another important group of viruses infecting cassava.