Deeply sequenced infectious clones of key cassava begomovirus isolates from Cameroon

Leon

Dallas

et al. 2021

Preprint

Self Cite

Cassava mosaic disease (CMD), which is caused by single-stranded DNA begomoviruses, severely limits cassava production across Africa. A previous study showed that CMD symptom severity and viral DNA accumulation increase in cassava in the presence of a DNA sequence designated as SEGS-2 (sequence enhancing geminivirus symptoms). We report here that when SEGS-2 is co-inoculated with African cassava mosaic virus (ACMV) onto Arabidopsis thaliana, viral symptoms increase. Transgenic Arabidopsis with an integrated copy of SEGS-2 inoculated with ACMV also display increased symptom severity and viral DNA levels. Moreover, SEGS-2 enables Cabbage leaf curl virus (CaLCuV) to infect a geminivirus resistant Arabidopsis accession. Although SEGS-2 is related to cassava genomic sequences, an earlier study showed that it occurs as episomes and is packaged into virions in CMD-infected cassava and viruliferous whiteflies. We identified SEGS-2 episomes in SEGS-2 transgenic Arabidopsis. The episomes occur as both double-stranded and single-stranded DNA, with the single-stranded form packaged into virions. In addition, SEGS-2 episomes replicate in tobacco protoplasts in the presence, but not the absence, of ACMV DNA-A. SEGS-2 episomes contain a SEGS-2 derived promoter and an open reading frame with the potential to encode a 75-amino acid protein. An ATG mutation at the beginning of the SEGS-2 coding region does not enhance ACMV infection in Arabidopsis. Together, the results established that SEGS-2 is a new type of begomovirus satellite that enhances viral disease through the action of a SEGS-2 encoded protein that may also be encoded in the cassava genome.

Section: Methodsmentioning

confidence: 99%

A New Type of Satellite associated with Cassava Mosaic Begomoviruses

Leon

Dallas

et al. 2021

Preprint

Self Cite

“…Cassava plants ( Manihot esculenta cv. Kibandameno) were propagated at 28°C and 30°C under a 12-h light/dark cycle representing the predicted 2°C temperature shift in Africa by 2030 (47) and inoculated using a microsprayer to deliver plasmids (100 ng) containing partial tandem dimers of DNA-A or DNA-B of ACMV (Accession Numbers: MT858793.1 and MT858794.1) and EACMCV (Accession Numbers: MT856195 and MT856192) (48, 49). Three plants were coinoculated with ACMV and EACMCV clones for each temperature treatment with each plant representing a biological replicate.…”

Section: Methodsmentioning

confidence: 99%

“…Raw sequencing data were processed according to Aimone et al (2020); the workflow is also available on Galaxy (ViralSeq, https://cassavavirusevolution.vcl.ncsu.edu/). Raw Illumina data is available from the NCBI Sequence Read Archive for Veg2 (PRJNA667210) and Veg6 (PRJNA658475).…”

Section: Analysis Of Raw Readsmentioning

confidence: 99%

Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation

Lavington

Hoyer

et al. 2021

Preprint

Self Cite

Cassava mosaic disease (CMD) represents a serious threat to cassava, a major root crop for more than 300 million Africans. CMD is caused by single-stranded DNA begomoviruses that evolve rapidly, making it challenging to develop durable disease resistance. In addition to the evolutionary forces of mutation, recombination, and reassortment, factors such as climate, agriculture practices, and the presence of DNA satellites may impact viral diversity. To gain insight into the factors that alter and shape viral diversity in planta, we used high-throughput sequencing to characterize the accumulation of nucleotide diversity after inoculation of infectious clones corresponding to African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in the susceptible cassava landrace Kibandameno. We found that vegetative propagation had a significant effect on viral nucleotide diversity, while temperature and a satellite DNA did not have measurable impacts in our study. EACMCV diversity increased linearly with the number of vegetative propagation passages, while ACMV diversity increased for a time and then decreased in later passages. We observed a substitution bias toward C->T and G->A for mutations in the viral genomes consistent with field isolates. Non-coding regions excluding the promoter regions of genes showed the highest levels of nucleotide diversity for each genome component. Changes in the 5' intergenic region of DNA-A resembled the sequence of the cognate DNA-B sequence. The majority of nucleotide changes in coding regions were non-synonymous, most with predicted deleterious effects on protein structure, indicative of relaxed selection pressure over 6 vegetative passages. Overall, these results underscore the importance of knowing how cropping practices affect viral evolution and disease progression.

“…Cassava plants (Manihot esculenta cv. Kibandameno) were propagated at 28 and 30 °C under a 12 h light/dark cycle representing the predicted 2 °C temperature shift in Africa by 2030 [47] and inoculated using a microsprayer to deliver plasmids (100 ng) containing partial tandem dimers of DNA-A or DNA-B of ACMV (accession numbers: MT858793.1 and MT858794.1) and EACMCV (accession numbers: MT856195 and MT856192) [48,49]. Three plants were coinoculated with ACMV and EACMCV clones for each temperature treatment with each plant representing a biological replicate.…”

Section: Vegetation Propagation Study With Two Passages (Veg2) At Two Temperaturesmentioning

confidence: 99%

Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation

Journal of General Virology

Lavington

Hoyer

et al. 2021

Self Cite

Cassava mosaic disease (CMD) represents a serious threat to cassava, a major root crop for more than 300 million Africans. CMD is caused by single-stranded DNA begomoviruses that evolve rapidly, making it challenging to develop durable disease resistance. In addition to the evolutionary forces of mutation, recombination and reassortment, factors such as climate, agriculture practices and the presence of DNA satellites may impact viral diversity. To gain insight into the factors that alter and shape viral diversity in planta, we used high-throughput sequencing to characterize the accumulation of nucleotide diversity after inoculation of infectious clones corresponding to African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in the susceptible cassava landrace Kibandameno. We found that vegetative propagation had a significant effect on viral nucleotide diversity, while temperature and a satellite DNA did not have measurable impacts in our study. EACMCV diversity increased linearly with the number of vegetative propagation passages, while ACMV diversity increased for a time and then decreased in later passages. We observed a substitution bias toward C→T and G→A for mutations in the viral genomes consistent with field isolates. Non-coding regions excluding the promoter regions of genes showed the highest levels of nucleotide diversity for each genome component. Changes in the 5′ intergenic region of DNA-A resembled the sequence of the cognate DNA-B sequence. The majority of nucleotide changes in coding regions were non-synonymous, most with predicted deleterious effects on protein structure, indicative of relaxed selection pressure over six vegetative passages. Overall, these results underscore the importance of knowing how cropping practices affect viral evolution and disease progression.