Deeply Sequenced Infectious Clones of Key Cassava Begomovirus Isolates from Cameroon

Hoyer, J. Steen; Fondong, Vincent N.; Dallas, Mary M.; Aimone, Catherine D.; Deppong, David O.; Duffy, Siobain; Hanley‐Bowdoin, Linda

doi:10.1128/mra.00802-20

Cited by 8 publications

(7 citation statements)

References 26 publications

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“…For infection, 6- to 7-week-old plants with ca. 30 leaves were inoculated using a microsprayer at 30 lb/in 2 to deliver gold particles coated with SEGS-2 plasmids (600 ng/6 plants) alone or in combination with infectious clones for ACMV (5 μg each of DNA-A and DNA-B/ 6 plants; GenBank accession numbers MT858793.1 and MT858794.1 [ 59 ]) or CaLCuV (2.5 μg each of DNA-A and DNA-B/ 6 plants; accession numbers U65529.2 and U65530.2 [ 60 ]). Nine plants were inoculated for each DNA combination treatment, and a mock inoculation control was included for each experiment.…”

Section: Methodsmentioning

confidence: 99%

A New Type of Satellite Associated with Cassava Mosaic Begomoviruses

Aimone

Leon

Dallas

et al. 2021

J Virol

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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 ( s equence e nhancing g eminivirus s ymptoms). 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 thaliana 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 A. thaliana . 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. IMPORTANCE Cassava is an important root crop in the developing world and a food and income crop for more than 300 million African farmers. Cassava is rising in global importance and trade as the demands for biofuels and commercial starch increase. More than half of the world’s cassava is produced in Africa, where it is primarily grown by smallholder farmers, many of whom are from the poorest villages. Although cassava can grow under high temperature, drought and poor soil conditions, its production is severely limited by viral diseases. Cassava mosaic disease (CMD) is one of the most important viral diseases of cassava and can cause up to 100% yield losses. We provide evidence that SEGS-2, which was originally isolated from cassava crops displaying severe and atypical CMD symptoms in Tanzanian fields, is a novel begomovirus satellite that can compromise the development of durable CMD resistance.

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Section: Methodsmentioning

confidence: 99%

A New Type of Satellite Associated with Cassava Mosaic Begomoviruses

Aimone

Leon

Dallas

et al. 2021

J Virol

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“…Kibandameno and cv. 60444) were co-inoculated with EACMCV (MT856195.1 and MT856192.1) (Fondong et al, 2000 ; Fondong and Chen, 2011 ; Hoyer et al, 2020 ) and ACMV (MT856193.2 and MT856194.1) infectious clone DNA (AddGene plasmids 159134 to 159137) by low-pressure biolistic bombardment (Aimone et al, 2022 ). ACMV and EACMCV produced in plants infected using viral clones can be acquired by whiteflies and subsequently transmitted into sucrose substrates and leaf disks (Kennedy et al, 2023 ).…”

Section: Methodsmentioning

confidence: 99%

Cassava begomovirus species diversity changes during plant vegetative cycles

Dye,

Muga,

Mwangi

et al. 2023

Front. Microbiol.

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Cassava is a root crop important for global food security and the third biggest source of calories on the African continent. Cassava production is threatened by Cassava mosaic disease (CMD), which is caused by a complex of single-stranded DNA viruses (family: Geminiviridae, genus: Begomovirus) that are transmitted by the sweet potato whitefly (Bemisia tabaci). Understanding the dynamics of different cassava mosaic begomovirus (CMB) species through time is important for contextualizing disease trends. Cassava plants with CMD symptoms were sampled in Lake Victoria and coastal regions of Kenya before transfer to a greenhouse setting and regular propagation. The field-collected and greenhouse samples were sequenced using Illumina short-read sequencing and analyzed on the Galaxy platform. In the field-collected samples, African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Kenya virus (EACMKV), and East African cassava mosaic virus-Uganda variant (EACMV-Ug) were detected in samples from the Lake Victoria region, while EACMV and East African mosaic Zanzibar virus (EACMZV) were found in the coastal region. Many of the field-collected samples had mixed infections of EACMV and another begomovirus. After 3 years of regrowth in the greenhouse, only EACMV-like viruses were detected in all samples. The results suggest that in these samples, EACMV becomes the dominant virus through vegetative propagation in a greenhouse. This differed from whitefly transmission results. Cassava plants were inoculated with ACMV and another EACMV-like virus, East African cassava mosaic Cameroon virus (EACMCV). Only ACMV was transmitted by whiteflies from these plants to recipient plants, as indicated by sequencing reads and copy number data. These results suggest that whitefly transmission and vegetative transmission lead to different outcomes for ACMV and EACMV-like viruses.

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“…Plants were then inoculated at 30 psi with a microdrop sprayer (Venganza, Inc.) to deliver gold particles coated with plasmid DNA corresponding to viral infectious clones and SEGS-1 constructs as described earlier ( Aimone et al., 2021a ). Plants were inoculated with 1.5 µg of cloned SEGS-1 DNA (S1-1.0, S1-1.5a, S1-1.5b and S1-2.0) and with 0.75 µg of each viral replicon plasmid corresponding to ACMV DNA-A or DNA-B (Accession Numbers: MT858793.1 and MT858794.1) ( Hoyer et al., 2020 ).…”

Section: Methodsmentioning

confidence: 99%

“…Total DNA was purified 48 h post transfection, and 30 μg was digested with Dpn I and linearized with Bsu 36I. Viral DNA was resolved using native gel conditions followed by DNA blotting using a 32 P-labeled ACMV DNA-A probe (948-bp Nco I/ Bam H1 fragment from MT858793.1) ( Hoyer et al., 2020 ). Blots were scanned by using a PhosphorImager and quantified by using IQMacV1.2 software (Storm; Amersham, Inc.).…”

Section: Methodsmentioning

confidence: 99%

SEGS-1 a cassava genomic sequence increases the severity of African cassava mosaic virus infection in Arabidopsis thaliana

Rajabu,

Dallas,

Chiunga

et al. 2023

Front. Plant Sci.

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Cassava is a major crop in Sub-Saharan Africa, where it is grown primarily by smallholder farmers. Cassava production is constrained by Cassava mosaic disease (CMD), which is caused by a complex of cassava mosaic begomoviruses (CMBs). A previous study showed that SEGS-1 (sequences enhancing geminivirus symptoms), which occurs in the cassava genome and as episomes during viral infection, enhances CMD symptoms and breaks resistance in cassava. We report here that SEGS-1 also increases viral disease severity in Arabidopsis thaliana plants that are co-inoculated with African cassava mosaic virus (ACMV) and SEGS-1 sequences. Viral disease was also enhanced in Arabidopsis plants carrying a SEGS-1 transgene when inoculated with ACMV alone. Unlike cassava, no SEGS-1 episomal DNA was detected in the transgenic Arabidopsis plants during ACMV infection. Studies using Nicotiana tabacum suspension cells showed that co-transfection of SEGS-1 sequences with an ACMV replicon increases viral DNA accumulation in the absence of viral movement. Together, these results demonstrated that SEGS-1 can function in a heterologous host to increase disease severity. Moreover, SEGS-1 is active in a host genomic context, indicating that SEGS-1 episomes are not required for disease enhancement.

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Deeply Sequenced Infectious Clones of Key Cassava Begomovirus Isolates from Cameroon

Cited by 8 publications

References 26 publications

A New Type of Satellite Associated with Cassava Mosaic Begomoviruses

A New Type of Satellite Associated with Cassava Mosaic Begomoviruses

Cassava begomovirus species diversity changes during plant vegetative cycles

SEGS-1 a cassava genomic sequence increases the severity of African cassava mosaic virus infection in Arabidopsis thaliana

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