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

C R A Duff-Farrier,H Bunawan,K R Tomlinson,T Alicai,A M Bailey,A M James,J L Pablo-Rodriguez,G D Foster,S E Seal,S Nanyiti,D R Mbanzibwa

doi:10.1007/s12033-018-0139-7

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.

Highlights

Cassava brown steak disease (CBSD) is the most important disease threatening cassava (Manihot esculenta) production in Africa [1, 2]
Restriction digestion, PCR and sequence analysis confirmed the integrity of the Ugandan cassava brown streak virus (UCBSV) ‘Kikombe’ infectious clones (ICs) sequence (NC_KX753357.1) with no obvious rearrangements
To infect indicator plants, capped in vitro transcripts were generated from the UCBSV ‘Kikombe’ IC and mechanically inoculated onto N. benthamiana and N. clevelandii

Summary

Introduction

Cassava brown steak disease (CBSD) is the most important disease threatening cassava (Manihot esculenta) production in Africa [1, 2]. CBSD is caused by at least two viral species: CBSV and UCBSV, which belong to the Ipomovirus genus of the Potyviridae family, hereafter referred to as U/ CBSVs [3, 4]. The disease was first reported in the 1930s in coastal regions of Tanzania [6] and until recently CBSD was largely restricted to lowland coastal areas of East Africa. In recent years CBSD has spread rapidly through Eastern and Central Africa, increasing in both geographical range and altitudinal distribution [1, 2]. CBSD is the leading cause of cassava losses in East Africa and its on-going spread threatens the food security of subsistence farmers across sub-Saharan Africa [1]

Methods

Results

Conclusion