Summary Cassava brown streak disease (CBSD) is a leading cause of cassava losses in East and Central Africa, and is currently having a severe impact on food security. The disease is caused by two viruses within the Potyviridae family: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), which both encode atypical Ham1 proteins with highly conserved inosine triphosphate (ITP) pyrophosphohydrolase (ITPase) domains. ITPase proteins are widely encoded by plant, animal, and archaea. They selectively hydrolyse mutagenic nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby function to reduce mutation rates. It has previously been hypothesized that U/CBSVs encode Ham1 proteins with ITPase activity to reduce viral mutation rates during infection. In this study, we investigate the potential roles of U/CBSV Ham1 proteins. We show that both CBSV and UCBSV Ham1 proteins have ITPase activities through in vitro enzyme assays. Deep‐sequencing experiments found no evidence of the U/CBSV Ham1 proteins providing mutagenic protection during infections of Nicotiana hosts. Manipulations of the CBSV_Tanza infectious clone were performed, including a Ham1 deletion, ITPase point mutations, and UCBSV Ham1 chimera. Unlike severely necrotic wild‐type CBSV_Tanza infections, infections of Nicotiana benthamiana with the manipulated CBSV infectious clones do not develop necrosis, indicating that that the CBSV Ham1 is a necrosis determinant. We propose that the presence of U/CBSV Ham1 proteins with highly conserved ITPase motifs indicates that they serve highly selectable functions during infections of cassava and may represent a euphorbia host adaptation that could be targeted in antiviral strategies.
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.
Rice (Oryza spp; 2n=24.) production in Uganda and Africa in general, is seriously threatened by the Rice yellow mottle virus disease (RYMVD), a disease caused by Rice yellow mottle virus (RYMV) within the genus Sobemovirus; family Sobemoviridae. This study investigated the existence and distribution of resistance-breaking RYMV pathotype in the three major lowland rice catchment areas in Uganda. Four known rice accessions resistant to Rice yellow mottle virus (RYMV) namely; Gigante, Tog5672, Tog5674 and Tog5681, carrying resistant allele’s rymv1-2, rymv1-4 & RYMV3, rymv1-5 and rymv1-3, respectively, were tested for their response to different RYMV isolates. The isolates were collected from three major lowland rice catchment areas of Doho, Kibimba, and Olweny in Uganda. Out of 100 leaf samples collected from the field and assayed for RYMV and confirmed to be positive using RT-PCR, 83 isolates induced symptoms on IR64- the RYMV susceptible line. Seventy-seven (92.8%) isolates were able to overcome resistance in at least one of the four differential rice accessions, as confirmed by the presence of RYMV symptoms; while 6 (7.2%) isolates were asymptomatic. Variation in time (days) for symptom development post-inoculation (dpi) and AUDPC were observed. Symptoms appeared within 5-7 days on IR64; while it took on average 11, 18, 36, and 18 days to appear on Gigante, Tog5672, Tog5674 and Tog5681, respectively. The highest AUDPC was observed on IR64 (254.7); while the lowest was observed on Tog5681 (74.1). Two major patho-groups were observed; those that broke down resistance in Gigante only (25.3%) and Gigante & Tog5672 (33.7%). Five isolates from Doho (Budaka & Bugiri districts) and Kibimba (Butaleja district) catchment areas broke down RYMV resistance in three accessions i.e. (Tog5681, Gigante & Tog5672) and (Tog5674, Gigante & Tog5672), respectively. Resistance breaking isolates were confirmed in all the three sampled catchment zones, however, Doho and Kibimba had some unique isolates that broke down resistance in accessions carrying resistance allele rymv 1-3 and rymv1-5 in addition to rymv1-2. Results from this study showed that RYMV isolates in Uganda can break down resistance conferred by the rymv1-2 resistance gene allele. However, accessions Tog5681 and Tog5674 seem to hold stable RYMV resistance and, thus are recommended for RYMV breeding.
Rice yellow mottle virus disease, caused by Rice yellow mottle virus (RYMV), is the most important disease of lowland rice in Uganda. However, little is known, about its genetic diversity in Uganda and relationships with other strains elsewhere across Africa. A new degenerate primer pair that targets amplification of the entire RYMV coat protein gene (ca. 738 bp) was designed to aid virus variability analysis using RT-PCR and Sanger sequencing. A total of 112 rice leaf samples from plants with RYMV mottling symptoms were collected during the year 2022 in 35 lowland rice fields within Uganda. The RYMV RT-PCR results were 100% positive, and all 112 PCR products were sequenced. BLASTN analysis revealed that all isolates were closely related (93-98%) to those previously studied originating from Kenya, Tanzania, and Madagascar. Despite high purifying selection pressure, diversity analysis on 81 out of 112 RYMV CP sequences revealed a very low diversity index of 3% and 1.0% at the nucleotide and amino acid levels, respectively. Except for glutamine, amino acid profile analysis revealed that all 81 Ugandan isolates shared the primary 19 amino acids based on RYMV coat protein region examined. Except for one isolate (UG68) from eastern Uganda that clustered alone, phylogeny analysis revealed two major clades. The Ugandan RYMV isolates were phylogenetically related to those from Democratic Republic of Congo, Madagascar, and Malawi, but not to RYMV isolates in West Africa. Thus, the RYMV isolates in this study are related to serotype 4, a strain common in eastern and southern Africa. RYMV serotype 4 originated in Tanzania, where evolutionary forces of mutation have resulted in the emergence and spread of new variants. Furthermore, mutations are evident within the coat protein gene of the Ugandan isolates, which may be attributed to changing RYMV pathosystems as a result of rice production intensification in Uganda. Overall, the diversity of RYMV was limited, most noticeably in eastern Uganda.
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