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  1. Article: Proteome Mapping of South African Cassava Mosaic Virus-Infected Susceptible and Tolerant Landraces of Cassava.

    Ramulifho, Elelwani / Rey, Marie Emma Christine

    Proteomes

    2021  Volume 9, Issue 4

    Abstract: The production of cassava is threatened by the geminivirus South African cassava mosaic virus (SACMV), which causes cassava mosaic disease. Cassava landrace TME3 shows tolerance to SACMV, while T200 is highly susceptible. This study aimed to identify the ...

    Abstract The production of cassava is threatened by the geminivirus South African cassava mosaic virus (SACMV), which causes cassava mosaic disease. Cassava landrace TME3 shows tolerance to SACMV, while T200 is highly susceptible. This study aimed to identify the leaf proteome involved in anti-viral defence. Liquid chromatography mass spectrometry (LC-MS) identified 2682 (54 differentially expressed) and 2817 (206 differentially expressed) proteins in both landraces at systemic infection (32 days post infection) and symptom recovery (67 days post infection), respectively. Differences in the number of differentially expressed proteins (DEPs) between the two landraces were observed. Gene ontology analysis showed that defence-associated pathways such as the chloroplast, proteasome, and ribosome were overrepresented at 67 days post infection (dpi) in SACMV-tolerant TME3. At 67 dpi, a high percentage (56%) of over-expressed proteins were localized in the chloroplast in TME3 compared to T200 (31% under-expressed), proposing that chloroplast proteins play a role in tolerance in TME3. Ribosomal_L7Ae domain-containing protein (Manes.12G139100) was over-expressed uniquely in TME3 at 67 dpi and interacts with the ribosomal protein Sac52 (RPL10). RPL10 is a known key player in the NIK1-mediated effector triggered immunity (ETI) response to geminivirus infection, indicating a possible role for Sac52 in SACMV recovery in TME3. In conclusion, differential protein expression responses in TME3 and T200 may be key to unravel tolerance to CMD.
    Language English
    Publishing date 2021-10-23
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2720995-7
    ISSN 2227-7382
    ISSN 2227-7382
    DOI 10.3390/proteomes9040041
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  2. Article ; Online: Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein.

    Nankoo, Nikita / Achilonu, Ikechukwu Anthony / Rey, Marie Emma Christine

    Current issues in molecular biology

    2022  Volume 44, Issue 6, Page(s) 2717–2729

    Abstract: South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises DNA-A (encodes six genes) and DNA-B (encodes BC1 cell-to-cell movement and BV1 nuclear shuttle proteins) components. A few secondary and tertiary ...

    Abstract South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises DNA-A (encodes six genes) and DNA-B (encodes BC1 cell-to-cell movement and BV1 nuclear shuttle proteins) components. A few secondary and tertiary structural and physicochemical characteristics of partial but not full-length begomovirus proteins have been elucidated to date. The full-length codon-optimised SACMV BC1 gene was cloned into a pET-28a (+) expression vector and transformed into expression host cells
    Language English
    Publishing date 2022-06-15
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2000024-8
    ISSN 1467-3045 ; 1467-3037
    ISSN (online) 1467-3045
    ISSN 1467-3037
    DOI 10.3390/cimb44060186
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  3. Article ; Online: A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus.

    Chatukuta, Patience / Rey, Marie Emma Christine

    Virology journal

    2020  Volume 17, Issue 1, Page(s) 184

    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 ... ...

    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
    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.
    MeSH term(s) Begomovirus/classification ; Begomovirus/genetics ; Begomovirus/pathogenicity ; Cell Culture Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression ; Host Microbial Interactions/genetics ; Manihot/virology ; Phylogeny ; Plant Diseases/virology ; Plant Viruses/genetics ; Protoplasts/virology ; RNA, Plant/genetics ; Ubiquitin-Protein Ligases/genetics
    Chemical Substances RNA, Plant ; Ubiquitin-Protein Ligases (EC 2.3.2.27)
    Language English
    Publishing date 2020-11-23
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1743-422X
    ISSN (online) 1743-422X
    DOI 10.1186/s12985-020-01453-4
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  4. Article ; Online: Nitric oxide associated protein 1 is associated with chloroplast perturbation and disease symptoms in Nicotiana benthamiana infected with South African cassava mosaic virus.

    Mwaba, Imanu / Rey, Marie Emma Christine

    Virus research

    2017  Volume 238, Page(s) 75–83

    Abstract: Nitric oxide associated 1 (NOA1) in plants is a cyclic GTPase involved in protein translation in the chloroplast and has been indirectly linked to nitric oxide (NO) accumulation and response to biotic stress. The association between NOA1 and NO ... ...

    Abstract Nitric oxide associated 1 (NOA1) in plants is a cyclic GTPase involved in protein translation in the chloroplast and has been indirectly linked to nitric oxide (NO) accumulation and response to biotic stress. The association between NOA1 and NO accumulation in Arabidopsis noa1 mutants has been linked to the inability of noa1 mutants to accumulate carbon reserves such as fumarate, leading to chloroplast dysfunction and a pale green leaf phenotype. To understand the role played by NOA1 in response to South African cassava mosaic virus infection in Nicotiana benthamiana, the expression of NbNOA1 and the accumulation of NO in leaf samples was compared between south african cassava mosaic (SACMV)-infected and mock-infected plants at 14 and 28 dpi. Real-time qPCR was used to measure SACMV viral load which increased significantly by 20% from 14 to 28 dpi as chlorosis and symptom severity progressed. At 14 and 28 dpi, NbNOA1 expression was significantly lower than mock inoculated plants (2-fold lower at 14 dpi, p-value=0.01 and 5-fold lower at 28, p-value=0.00). At 14 dpi, NO accumulation remained unchanged in infected leaf tissue compared to mock inoculated, while at 28 dpi, NO accumulation was 40% lower (p-value=0.01). At 28 dpi, the decrease in NbNOA1 expression and NO accumulation was accompanied by chloroplast dysfunction, evident from the significant reduction in chlorophylls a and b and carotenoids in SACMV-infected leaves. Furthermore, the expression of chloroplast translation factors (chloroplast RNA binding, chloroplast elongation factor G, translation elongation factor Tu, translation initiation factor 3-2, plastid-specific ribosomal protein 6 and plastid ribosome recycling factor) were found to be repressed in infected N. benthamiana. GC-MS analysis showed a decrease in fumarate and an increase in glucose in SACMV-infected N. benthamiana in comparison to mock samples suggesting a decrease in carbon stores. Collectively, these results provide evidence that in response to SACMV infection, a decrease in photopigments and carbon stores, accompanied by an increase in glucose and decrease in fumarate, leads to a decline in NbNOA1expression and NO levels. This is manifested by suppressed translation factors and disruption of chloroplast function, thereby contributing to chlorotic disease symptoms.
    MeSH term(s) Begomovirus/growth & development ; Chloroplasts/metabolism ; Gene Expression Profiling ; Host-Pathogen Interactions ; Nitric Oxide/analysis ; Nitric Oxide Synthase/genetics ; Nitric Oxide Synthase/metabolism ; Plant Diseases/virology ; Plant Proteins/metabolism ; Quantitative Structure-Activity Relationship ; Real-Time Polymerase Chain Reaction ; Nicotiana
    Chemical Substances Plant Proteins ; Nitric Oxide (31C4KY9ESH) ; Nitric Oxide Synthase (EC 1.14.13.39)
    Language English
    Publishing date 2017-05-31
    Publishing country Netherlands
    Document type Journal Article
    ZDB-ID 605780-9
    ISSN 1872-7492 ; 0168-1702
    ISSN (online) 1872-7492
    ISSN 0168-1702
    DOI 10.1016/j.virusres.2017.05.022
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  5. Article: Molecular analysis of six segments of tobacco leaf enation virus, a novel phytoreovirus from tobacco.

    Picton, Anabela / Potgieter, Christiaan / Rey, Marie Emma Christine

    Virus genes

    2007  Volume 35, Issue 2, Page(s) 387–393

    Abstract: Tobacco leaf enation virus (TLEV) is a putative member of the genus Phytoreovirus within the family Reoviridae. Previous western blot analysis of structural viral proteins (apparent molecular weights of 93 kDa; 58 kDa; 48 kDa; 39 kDa and 36 kDa) ... ...

    Abstract Tobacco leaf enation virus (TLEV) is a putative member of the genus Phytoreovirus within the family Reoviridae. Previous western blot analysis of structural viral proteins (apparent molecular weights of 93 kDa; 58 kDa; 48 kDa; 39 kDa and 36 kDa) associated with TLEV, isolated from infected tobacco in South Africa, suggested that these proteins may correspond to structural Wound tumor virus (WTV) proteins. To further establish the nature of this novel virus disease phenotype in tobacco, molecular characterization of six dsRNA components was undertaken. Full-length cDNA clones were obtained by an optimized modified single-primer amplification sequence-independent dsRNA cloning method. Results of this study revealed the conserved terminal sequence: 5'GG(U/C)...UGAU 3' of segments S6-S12, while adjacent to these conserved terminal sequences are imperfect inverted repeats (7-15 bp in length), both features being common to reoviruses. The complete nucleotide sequences of segments S5 (2,610 bp), S7 (1,740 bp), S8 (1,439 bp), S10 (1,252 bp), S11 (1,187 bp) and S12 (836 bp) were determined. Comparison of full-length nucleotide sequences with corresponding segments of other phytoreoviruses, Rice gall dwarf virus (RGDV), Rice dwarf virus (RDV) and WTV has shown nucleotide and predicted amino acid identities within the range of 30-60%. TLEV consistently shows a higher identity to WTV than to other phytoreovirus species where sequence data is available. Each segment had a single predicted open reading frame encoding proteins with calculated molecular weights of S5 (90.6 kDa); S7 (58.1 kDa); S8 (47.7 kDa); S10 (39.8 kDa); S11 (35 kDa) and S12 (19.5 kDa). The relatively low nucleotide and amino acid identity to other members of the genus demonstrates that TLEV is a novel phytoreovirus, distinct from the only other reported dicotyledenous-infecting WTV and is the first phytoreovirus reported to emerge in Africa.
    MeSH term(s) Amino Acid Sequence ; Molecular Sequence Data ; Phylogeny ; Plant Diseases/virology ; Plant Leaves/virology ; Plant Viruses/classification ; Plant Viruses/genetics ; Reoviridae/classification ; Reoviridae/genetics ; Nicotiana/virology
    Language English
    Publishing date 2007-03-14
    Publishing country United States
    Document type Journal Article
    ZDB-ID 639496-6
    ISSN 1572-994X ; 0920-8569
    ISSN (online) 1572-994X
    ISSN 0920-8569
    DOI 10.1007/s11262-007-0088-x
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  6. Article: Molecular analysis of six segments of Tobacco leaf enation virus, a novel phytoreovirus from tobacco

    Picton, Anabela / Potgieter, Christiaan / Rey, Marie Emma Christine

    Virus genes. 2007 Oct., v. 35, no. 2

    2007  

    Abstract: Tobacco leaf enation virus (TLEV) is a putative member of the genus Phytoreovirus within the family Reoviridae. Previous western blot analysis of structural viral proteins (apparent molecular weights of 93 kDa; 58 kDa; 48 kDa; 39 kDa and 36 kDa) ... ...

    Abstract Tobacco leaf enation virus (TLEV) is a putative member of the genus Phytoreovirus within the family Reoviridae. Previous western blot analysis of structural viral proteins (apparent molecular weights of 93 kDa; 58 kDa; 48 kDa; 39 kDa and 36 kDa) associated with TLEV, isolated from infected tobacco in South Africa, suggested that these proteins may correspond to structural Wound tumor virus (WTV) proteins. To further establish the nature of this novel virus disease phenotype in tobacco, molecular characterization of six dsRNA components was undertaken. Full-length cDNA clones were obtained by an optimized modified single-primer amplification sequence-independent dsRNA cloning method. Results of this study revealed the conserved terminal sequence: 5'GG(U/C)...UGAU 3' of segments S6-S12, while adjacent to these conserved terminal sequences are imperfect inverted repeats (7-15 bp in length), both features being common to reoviruses. The complete nucleotide sequences of segments S5 (2,610 bp), S7 (1,740 bp), S8 (1,439 bp), S10 (1,252 bp), S11 (1,187 bp) and S12 (836 bp) were determined. Comparison of full-length nucleotide sequences with corresponding segments of other phytoreoviruses, Rice gall dwarf virus (RGDV), Rice dwarf virus (RDV) and WTV has shown nucleotide and predicted amino acid identities within the range of 30-60%. TLEV consistently shows a higher identity to WTV than to other phytoreovirus species where sequence data is available. Each segment had a single predicted open reading frame encoding proteins with calculated molecular weights of S5 (90.6 kDa); S7 (58.1 kDa); S8 (47.7 kDa); S10 (39.8 kDa); S11 (35 kDa) and S12 (19.5 kDa). The relatively low nucleotide and amino acid identity to other members of the genus demonstrates that TLEV is a novel phytoreovirus, distinct from the only other reported dicotyledenous-infecting WTV and is the first phytoreovirus reported to emerge in Africa.
    Keywords Phytoreovirus ; tobacco ; sequence analysis
    Language English
    Dates of publication 2007-10
    Size p. 387-393.
    Publisher Kluwer Academic Publishers-Plenum Publishers
    Publishing place New York
    Document type Article
    ZDB-ID 639496-6
    ISSN 1572-994X ; 0920-8569
    ISSN (online) 1572-994X
    ISSN 0920-8569
    DOI 10.1007/s11262-007-0088-x
    Database NAL-Catalogue (AGRICOLA)

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