Use of infectious clones to gain insights into Cassava brown streak virus gene function

  • Katherine Tomlinson

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

Cassava is the second most important food staple in terms of per capita calories consumed in Africa and offers unique climate change adaptation opportunities. Unfortunately, production in sub- Saharan Africa is currently constrained by cassava mosaic disease (CMD) and cassava brown streak disease (CBSD), which together are estimated to cause annual losses worth US$1 billion and adversely affect food security across the entire region. Compared with CMD, relatively little is currently understood about CBSD infections and there are no cassava varieties with high levels of CBSD resistance available to farmers. CBSD is caused by at least two potyviral species: Cassava brown streak virus and Ugandan cassava brown streak virus, collectively referred to as U/CBSVs. Little is known about U/CBSV gene function; to date only the silencing suppressor activity of the UCBSV P1 protein has been reported. Viral infectious clones are highly valuable tools that can be used to screen for viral resistance and to gain fundamental insights into viral infections. Unfortunately, until recently the construction of U/CBSV ICs has been circumvented by sequence instability during propagation in Escherichia coli.
In 2014 -16, two U/CBSV ICs were constructed at the University of Bristol, as part of a larger project related to this work. The main aim of this study was to test the tractability of these ICs and use them to characterise U/CBSV gene functions.
The UCBSV IC exhibited high levels of sequence instability during propagation in all tested E. coli strains. In contrast, the CBSV IC which contains introns, demonstrated sequence stability during propagation in the E. coli strains ccdB and TOP10. This permitted the insertion of GFP at two genome positions. N. benthamiana infections with CBSV_GFP ICs enabled visualisation of viral movement in planta.
U/CBSV genomes encode Ham1-like peptides with conserved ITPase pyrophosphohydrolase motifs, which have only been reported in Euphorbia ringspot virus. Eu/prokaryotic ITPase proteins selectively hydrolyze non-canonical nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby reduce mutation rates. In vitro pyrophosphohydrolase assays, demonstrated that U/CBSV Ham1 proteins have significantly higher activities with non-canonical nucleotides compared with canonical nucleotides. However, no significant differences were found in the number of viral variants during N. benthamiana infections with full-length CBSV IC and a CBSV IC containing Ham1 deletion, indicating that CBSV Ham1 may not reduce viral mutation rates.
Additionally, mutation of the CBSV IC demonstrated that a Ham1 pyrophosphohydrolase motif is associated with the development of severe necrosis during N. benthamiana infections and a chimera IC, consisting of the CBSV genome with UCBSV coat protein replacement caused systemic N. benthamiana infections; raising the possibility for transcapsidation to occur during mixed U/CBSV infections in the field. These insights into CBSV gene function demonstrates the utility of U/CBSV ICs and should aid future studies into these important viruses.
Date of Award23 Jan 2019
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorGary D Foster (Supervisor) & Andy M Bailey (Supervisor)

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