Nitric oxide associated protein 1 is associated with chloroplast perturbation and disease symptoms in Nicotiana benthamiana infected with South African cassava mosaic virus

2017 ◽  
Vol 238 ◽  
pp. 75-83 ◽  
Author(s):  
Imanu Mwaba ◽  
Marie Emma Christine Rey
Keyword(s):  
Nitric Oxide ◽  
Mosaic Virus ◽  
South African ◽  
Nicotiana Benthamiana ◽  
African Cassava Mosaic Virus ◽  
Disease Symptoms ◽  
Cassava Mosaic Virus

scholarly journals Viruses Infecting Cassava in Kenya

Plant Disease ◽  
2004 ◽  
Vol 88 (1) ◽  
pp. 17-22 ◽  
Author(s):  
H. K. Were ◽  
S. Winter ◽  
E. Maiss
Keyword(s):  
Mosaic Virus ◽  
Viral Disease ◽  
African Cassava Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Stem Cuttings ◽  
East African ◽  
Western Kenya ◽  
Disease Symptoms ◽  
Future Work ◽  
Cassava Mosaic Virus

A survey of cassava viruses was conducted in major cassava-growing regions of Kenya. A total of 185 leaf samples and 62 stem cuttings from plants with viral disease symptoms were collected and analyzed by biological, electron microscopy, enzyme-linked immunosorbent assay, and polymerase chain reaction. All samples from western Kenya had cassava begomoviruses (African cassava mosaic virus [ACMV], East African cassava mosaic virus [EACMV], and Uganda variant [EACMV-UG]) in either single or in mixed infection. However, all samples from the Coast region were infected with only EACMV, a begomovirus. In addition, 15 samples had mixed infections of EACMV and three other hitherto unidentified filamentous viruses. The viruses observed were 200, 500, 650, and 750 nm long, respectively. In addition to rod-shaped and some flexuous viruses, as seen in a crude sap preparation, pinwheels also were observed, indicating a possible association of some of the viruses with the Potyviridae family. The symptoms induced by these viruses in Nicotiana benthamiana were very severe and often caused about 50% death of the test plants. Back inoculation onto cassava resulted in 100% infections. This finding provides evidence that, other than begomoviruses that cause serious diseases of cassava in Africa, filamentous viruses also are present and, despite their limited distribution, they could reach local significance and, most probably, be as serious as begomoviruses. The implications of these findings are discussed and recommendations for future work suggested.

scholarly journals A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Patience Chatukuta ◽  
Marie Emma Christine Rey
Keyword(s):  
Gene Expression ◽  
Tissue Culture ◽  
Mosaic Virus ◽  
South African ◽  
High Throughput ◽  
E3 Ligase ◽  
Turnaround Time ◽  
African Cassava Mosaic Virus ◽  
Likelihood Method ◽  
Cassava Mosaic Virus

Abstract Background The study of transient gene expression in cassava plants during virus infection using existing protocols is laborious and may take approximately fifteen weeks due to cassava’s recalcitrance to transformation. The combination of a protoplast system with CRISPR-mediated gene editing promises to shorten the turnaround time from plant tissue culture to high-throughput gene expression screening for candidate genes. Here, we detail a protocol for screening genes associated with the response to South African cassava mosaic virus (SACMV) in cassava protoplasts, with reference to the ubiquitin E3 ligase gene, MeE3L. Methods Cassava protoplasts of model, and SACMV-susceptible and -tolerant genotypes, were transformed with SACMV infectious clones and/or a CRISPR-editing construct targeting the MeE3L using PEG4000-mediated transfection. DNA and RNA were extracted from transformed protoplasts at 24 h post-transfection. Relative SACMV DNA accumulation was determined via qPCR using DpnI-digested total DNA, MeE3L relative expression was determined via reverse transcriptase qPCR, and results were analysed using one-way ANOVA, Tukey’s HSD test and the 2−ΔΔCTstatistical method. The MeE3L exonic region was sequenced on the ABI 3500XL Genetic Analyzer platform; and sequences were analysed for mutations using MAFTT and MEGA-X software. Construction of a phylogenetic tree was done using the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model. Results The differential expression of unedited and mutant MeE3L during SACMV infection of model, susceptible and tolerant cassava protoplasts was determined within 7 weeks after commencement of tissue culture. The study also revealed that SACMV DNA accumulation in cassava protoplasts is genotype-dependent and induces multiple mutations in the tolerant landrace MeE3L homolog. Notably, the susceptible cassava landrace encodes a RINGless MeE3Lwhich is silenced by SACMV-induced mutations. SACMV also induces mutations which silence the MeE3L RING domain in protoplasts from and tolerant cassava landraces. Conclusions This protocol presented here halves the turnaround time for high-throughput screening of genes associated with the host response to SACMV. It provides evidence that a cassava E3 ligase is associated with the response to SACMV and forms a basis for validation of these findings by in planta functional and interaction studies.

scholarly journals The size of encapsidated single-stranded DNA determines the multiplicity of African cassava mosaic virus particles

2001 ◽  
Vol 82 (3) ◽  
pp. 673-676 ◽  
Author(s):  
Thomas Frischmuth ◽  
Margit Ringel ◽  
Cornelia Kocher
Keyword(s):  
Electron Microscopy ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Southern Blotting ◽  
African Cassava Mosaic Virus ◽  
Dna Virus ◽  
Virus Particles ◽  
20 Nm ◽  
And Control ◽  
Cassava Mosaic Virus

Transgenic Nicotiana benthamiana plants harbouring a defective interfering (DI) DNA of African cassava mosaic virus (ACMV) and control plants were inoculated with ACMV. Virus particles were purified from infected plants, separated in sucrose gradients and fractions were analysed by Southern blotting. Transgenic plant-derived virus particles taken from the top fractions of sucrose gradients contained DI DNA, middle fractions contained a mixture of genomic and DI DNA and bottom fractions contained a mixture of multimeric, genomic and DI DNA. Virus particles from selected top, middle and bottom fractions were analysed by electron microscopy. In fractions containing only DI DNA, isometric particles of 18–20 nm were detected. In fractions containing DI DNA as well as genomic size DNA, isometric and geminate particles were found. Fractions containing multimeric size DNA were found to comprise particles consisting of three subunits adjacent to geminate particles. From these data, it is concluded that the size of encapsidated DNA determines the multiplicity of ACMV particles.

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