Using viral infectious clones to study cassava brown streak disease in its native host Manihot esculenta and exploring the role of viral Ham1 proteins

Abstract

Abstract disease is caused by virus (CBSV) and Ugandan virus (UCBSV) and it is one of the most devastating viral diseases infecting cassava in sub-Saharan East Africa. This study explored some practices to mitigate the impact of the disease using molecular technologies, including the development of viral infectious clones suitable for cassava infection, functional characterization of viral proteins, and CRISPR-Cas9 editing of targets in Nicotiana benthamiana. The main aim of this study involved the biolistic delivery of CBSV infectious clones into cassava. Methods for biolistic delivery were optimised, revealing Plant Line 60444 to be a very sensitive cassava type. That the growth conditions of plant material suitable for infection were paramount. Moreover, it showed that chimeric clones harboring the Cassava Torrado-like Virus Ham1 domain showed loss of pathogenicity, suggesting that Ham1s were not readily interchangeable, even if they show similar enzymatic properties. The hydrolytic activity of various viral Ham1 proteins was demonstrated, exhibiting its ability to hydrolyze non-canonical nucleotides such as ITP and XTP. Expression and assay of Ham1 proteins from CBSV, UCBSV, Euphorbia ringspot virus , Arracacha Virus A and CsTLV revealed different substrate preferences, but typically with a preference for ribose over deoxyribose noncanonical nucleotides. The role of the Serine-Histidine-Arginine motif in substrate selection was also highlighted. CRISPR-Cas9 technology was successfully employed to edit the Ham1 gene in the model plant, Nicotiana benthamiana. Frameshift as well as non-frameshift mutations were found in T1 and T2 generations, with homozygous lines showing stable Mendelian inheritance. Functional assays indicated that the Ham1 knockout lines were less affected by CBSV, evidenced by less chlorosis and necrosis symptoms. Intriguingly, Ham1 disruption did not impact infection by Turnip mosaic virus, implying a virus-specific activity of Ham1. Phenotypic characterisation suggested the Ham1 mutants were more sensitive to salt and osmotic stress, emphasizing the role of Ham1 in stress physiology and its crosstalk with other pathways. This study enhances the tools to better understand CBSV pathogenesis, the interplay of viruses and hosts, and the multifunctional role of the Ham1 protein. It also highlights CRISPR-Cas9 as a precise method for generating edited plant varieties.