Abstract
Cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) are currently two major viral diseases that severely reduce cassava production in large areas of Sub-Saharan Africa. Natural resistance has so far only been reported for CMD in cassava. CBSD is caused by two virus species, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). A sequence of the CBSV coat protein (CP) highly conserved between the two virus species was used to demonstrate that a CBSV-CP hairpin construct sufficed to generate immunity against both viral species in the cassava model cultivar (cv. 60444). Most of the transgenic lines showed high levels of resistance under increasing viral loads using a stringent top-grafting method of inoculation. No viral replication was observed in the resistant transgenic lines and they remained free of typical CBSD root symptoms 7 month post-infection. To generate transgenic cassava lines combining resistance to both CBSD and CMD the hairpin construct was transferred to a CMD-resistant farmer-preferred Nigerian landrace TME 7 (Oko-Iyawo). An adapted protocol allowed the efficient Agrobacterium-based transformation of TME 7 and the regeneration of transgenic lines with high levels of CBSV-CP hairpin-derived small RNAs. All transgenic TME 7 lines were immune to both CBSV and UCBSV infections. Further evaluation of the transgenic TME 7 lines revealed that CBSD resistance was maintained when plants were co-inoculated with East African cassava mosaic virus (EACMV), a geminivirus causing CMD. The innovative combination of natural and engineered virus resistance in farmer-preferred landraces will be particularly important to reducing the increasing impact of cassava viral diseases in Africa.
Highlights
Cassava (Manihot esculenta Crantz) production in large areas of Africa is severely affected by two major viral diseases, cassava brown streak disease (CBSD) and cassava mosaic disease (CMD)
Genetic diversity has been best characterized for the coat protein (CP) sequence of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) [18]
We confirmed by in silico analysis that the UCBSV-CP sequence is a potential target of a large number of hairpin-derived small RNAs based on the free energy of small interfering RNAs (siRNAs)-target duplexes (Figure S2, Table S1)
Summary
Cassava (Manihot esculenta Crantz) production in large areas of Africa is severely affected by two major viral diseases, cassava brown streak disease (CBSD) and cassava mosaic disease (CMD). CBSD is caused by two virus species, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (picornalike (+) ssRNA viruses, genus Ipomovirus, family Potyviridae) [1,2]. CMD in Africa is caused by at least seven cassava mosaic geminivirus species (CMGs) [3]. The etiologic agents, symptoms, and impact on cassava production are disease-specific, these two cassava viral diseases can overlap in their distribution [4,5]. Despite evidence of synergism between the two viruses in Nicotiana benthamiana [6], field survey data do not support any interaction between the viruses [4]. It is notable that CBSD is prevalent in CMD-resistant varieties [2,5] that have been deployed in recent decades as part of CMD mitigation management strategies [7]
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