Abstract:

A full-length in-vitro UCBSV infectious clone (GenBank accession KX753357) was generated by cloning
the complete genome of the virus in a backbone pYES2.1 vector. An SP6 promoter was introduced at the 5’
end of the UCBSV genome to allow for in-vitro transcription. The constructed infectious clone was
confirmed to infect tobacco and cassava plants as shown by the mosaic like symptoms observed in the
infectious clone infected plants. RNA was extracted from the symptomatic plants and used in RT-PCR. PCR
and sequencing results confirmed the infection.
The full-length infectious clone was then mutated in the Pl protein to study the function of this protein in
UCBSV. Initially, an RNA suppression assay confirmed that Pl protein in UCBSV was an RNA silencing
suppressor. A mutation in the Pl encoding sequence that changed LRRA motif to LAAA abolished
completely the silencing suppression activity of UCBSV-P1. However, the same mutation in the full-length
infectious clone did not affect the infectivity of the infectious clone but the viral titres were observed to be
higher in plants inoculated with the mutant infectious clone as compared to the wildtype infectious clone.
The Pl protein was then completely deleted from the UCBSV infectious clone and the Pl mutant was used
to infect tobacco. It was observed that the Pl mutant infectious clone was able to move systemically within
the plants although RT-PCR showed low levels of the virus in the plants implying that Pl may be possibly
be involved in genome amplification.
Cassava is one of the most important staple foods to most of the African populations with over 800 million
people depending on cassava as the main source of starch. However, low average yields of cassava are caused
by a number of factors including susceptibility to pests and diseases. Among the viral diseases, cassava
brown streak disease (CBSD), caused by Cassava brown streak virus and (CBSV) and Ugandan cassava
brown streak virus (UCBSV) family Potyviridae and genus Ipomovirus, is one of the most devastating.
Disease spread has been controlled by planting clean and healthy cuttings. Breeding for resistant varieties
has been undertaken, however, there is a need to screen the generated breeding lines for resistance to CSBD,
and this has necessitated a need for infectious clones.
The main aim of this PhD work was to construct a full-length infectious clone of UCBSV Kikombe isolate
and to study the function of the Pl (first potyviral protein) and HAM1 proteins in the UCBSV genome.
Infectious clones of RNA viruses provide a homogenous and reproducible source of viral infection for
effective screening of breeding lines.
In order to construct the full-length infectious clone, initially, the complete genome of Kikombe isolate
(UCBSV) was amplified in sections by reverse transcription polymerase chain reaction (RT-PCR) and
Sanger sequenced. The complete sequence (GenBank accession number KX753356) was found to consist
of 9070 nucleotides (excluding the poly-A tail) which translated into a polyprotein of 2902 amino acids.
HAM1 UCBSV infectious clone mutants were used to characterize the role of HAM 1 protein in UCBSV
genome. It was observed that plants infected with the HAM1 mutant infectious clone developed yellow
mosaic symptoms which were different from the mild mosaics observed with the wildtype UCBSV
infectious clone implying that HAM1 may be involved in symptom development.
In conclusion, this work has for the first time reported construction of an intron-less, in-vitro infectious clone
of the full length cDNA of the UCBSV. Attempts to construct in-vivo infectious clones failed and that was
probably caused by positions selected for insertions of introns and suggest the sequences of UCBSV when
placed under strong constitutive promoters are toxic to E. coli cells. Further studies are recommended to investigate appropriate positions for insertions of the introns for construction of in-vivo infectious clones.