Strategies for the Construction of Cassava Brown Streak Disease Viral Infectious Clones

Abstract

Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli . Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate ‘Kikombe’, such an approach failed to deliver full-length clones for CBSV isolates ‘Nampula’ or ‘Tanza’, necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex. Electronic supplementary material The online version of this article (10.1007/s12033-018-0139-7) contains supplementary material, which is available to authorized users.

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

“…To investigate in greater depth whether the CBSV Ham1 protein specifically reduces the CBSV mutation rate, the complete Ham1 sequence was deleted from the CBSV_Tanza IC (Duff‐Farrier et al , ) to produce the CBSV_Tanza Ham1 knockout IC (CBSV_HKO). N. benthamiana plants were then infected with either CBSV_Tanza or CBSV_HKO.…”

“…51045/197610/2 and handled according to Brewer et al (). IC manipulations were performed through homologous yeast recombination according to the protocol outlined in Duff‐Farrier et al (, ). Briefly, the CBSV_Tanza IC (NCBI: MG570022) was linearized with the restriction enzyme Bam H1 (Thermo Fisher Scientific).…”

Screening for Resistance in Farmer-Preferred Cassava Cultivars from Ghana to a Mixed Infection of CBSV and UCBSV

“…However, both methods have lower infection rates compared to the top grafting method, which usually achieves 100% infection rates [ 27 , 31 , 32 ]. Although the recent construction of an infectious UCBSV clone opens new opportunities to develop robust inoculation methods [ 33 ], graft inoculation methods have so far been most effective for the transmission of CBSV and UCBSV to non-infected cassava plants [ 20 , 27 , 31 ].…”

Application of Reverse Genetics in Functional Genomics of Potyvirus

“…Since then, infectious clones for many other plant RNA viruses were successfully obtained as either in vivo or in vitro trancripts. For in vitro strategy, the viral cDNAs are cloned under bacteriophage promoters such as T7, T3, or Sp6 followed by the generation of in vitro transcripts [ 22 , 23 ]. On the other hand, in vivo infectious transcripts are driven by Cauliflower mosaic virus 35S promoter in a binary vector.…”