RNAi-mediated resistance to diverse isolates belonging to two virus species involved in Cassava brown streak disease

Abstract

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.

Figure 1

Screening of Nicotiana benthamiana transgenic lines in the T₂ generation for resistance against CBSUV‐[UG:Nam:04] (Ugandan isolate of Cassava brown streak Uganda virus). (A) Phenotype of resistant line NT7 derived from the RNAi construct targeting the N‐terminus of CBSUV‐[UG:Nam:04]/CP, showing 100% protection (left) when compared with complete infection in control plants (right). Individual plants are enlarged and shown in the bottom panel. (B) Different levels of resistance obtained in N. benthamiana transgenic lines derived from the RNAi constructs targeting full‐length (FL; left), N‐terminal (NT; centre) and C‐terminal (CT; right) parts of CBSUV‐[UG:Nam:04]/CP in the T₂ generation after challenge with CBSUV‐[UG:Nam:04] at 30 days post‐inoculation (dpi).

Figure 2

Molecular analysis of Nicotiana benthamiana transgenic plant lines (line numbers indicated for each lane) derived from three RNAi constructs targeting different regions of CBSUV‐[UG:Nam:04] (Ugandan isolate of Cassava brown streak Uganda virus) coat protein (CP) [N‐terminus (NT), C‐terminus (CT) and full‐length (FL)] in the T₂ generation. (A) Accumulation of mRNAs and siRNA as detected by Northern blot hybridization before CBSUV‐[UG:Nam:04] challenge. (B) Accumulation of viral RNA and siRNA after challenge with CBSUV‐[UG:Nam:04] in the same transgenic lines. ‘−’ in (A) is a nontransgenic healthy plant; ‘+’ in (A) is a small RNA from transient expression by agroinfiltration of the FL‐CP construct; ‘+’ in (B) is a nontransgenic virus‐infected control.

Figure 3

Screening of Nicotiana benthamiana transgenic lines in the T₂ generation for resistance against six distinct Cassava brown streak Uganda virus (CBSUV) and Cassava brown streak virus (CBSV) isolates. (A) Phenotype of a resistant line FL17 derived from the RNAi construct targeting the 894 nts of CBSV‐UG[UG:Nam:04]/CP, showing 100% protection (left) when compared with complete infection in control plants (right) when challenged with CBSV‐[TZ:Zan:07]. (B, C) Different levels of protection obtained in N. benthamiana transgenic lines derived from RNAi constructs targeting full‐length (FL; left), C‐terminal (CT; centre) and N‐terminal (NT; right) parts of CBSV‐UG[UG:Nam:04]/CP in the T₂ generation after challenge with six different isolates of CBSUV and CBSV. (B) shows protection for CBSUV isolates (CBSUV‐[UG:Kab:07], CBSUV‐[TZ:Kib:07] and CBSUV‐[KE:Mwa:07]) and (C) shows protection for CBSV isolates (CBSV‐[MZ:Nam:07], CBSV‐[TZ:Zan:07] and CBSV‐[TZ:Nal:07]). Different levels of siRNA expressed by these lines positively correlate (R= 0.86) with their resistance levels, as shown in (D).

Figure 4

Reverse trancriptase‐polymerase chain reaction (RT‐PCR) for the detection of the coat protein (CP) of Cassava brown streak Uganda virus (CBSUV) (A) and Cassava brown streak virus (CBSV) (B) isolates. Specific primers were used to amplify 1101 nts of CP for the CBSUV isolates (CBSUV‐[UG:Kab:07], CBSUV‐[TZ:Kib:07] and CBSUV‐[KE:Mwa:07]) and 1134 nts of CP for the CBSV isolates (CBSV‐[TZ:Zan:07], CBSV‐[TZ:Nal:07] and CBSV‐[MZ:Nam:07]) from the total RNA extracts for both symptomatic (first three/two lanes for each transgenic plant line) and nonsymptomatic (next three/two lanes) transgenic plants after challenge of transgenic N. benthamiana with CBSUV‐[UG:Kab:07] and CBSV‐[TZ:Zan:07].