Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein.

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 E. coli BL21 (DE3). The optimal expression of the full-length BC1-encoded movement protein (MP) was obtained via induction with 0.25 mM IPTG at an OD600 of ~0.45 at 37 °C for four hours. Denatured protein fractions (dialysed in 4 M urea), passed through an IMAC column, successfully bound to the nickel resin, and eluted using 250 mM imidazole. The protein was refolded using stepwise dialysis. The molecular weight of MP was confirmed to be 35 kDa using SDS-PAGE. The secondary structure of SACMV MP presented as predominantly ß-strands. An ANS (1-anilino-8-naphthalene sulphonate)-binding assay confirmed that MP possesses hydrophobic pockets with the ability to bind ligands such as ANS (8-anilino-1-naphthalenesulphonic acid). A 2' (3')-N-methylanthraniloyl-ATP (mant-ATP) assay showed binding of mant-ATP to MP and indicated that, while hydrophobic pockets are present, MP also exhibits hydrophilic regions. Intrinsic tryptophan fluorescence indicated a significant conformational change in the denatured form of BC1 in the presence of ATP. In addition, a phosphatase assay showed that MP possessed ATPase activity.

Cassava mosaic disease (CMD) in Benin: Incidence, severity and its whitefly abundance from field surveys in 2020.

Cassava mosaic disease (CMD) is the main threat to cassava (Manihot esculenta Crantz) production in Benin. This study was conducted to assess CMD incidence, disease severity, and adult whitefly (Bemisia tabaci) populations in 11 regions of Benin. A total of 180 cassava fields across the country were assessed during June-December 2020 following the harmonized protocol of the Central and West African Virus Epidemiology program. Based on symptoms observation, CMD was present in all surveyed fields in Ouémé and Alibori regions. The highest disease incidence levels were observed in Malanville (100%), Kpomassè (86.67%), Kandi and Zagnanado (both 81.67%), Ségbanan (80%), and Avrankou (76.67%) districts. The highest mean severity scores were in Couffo (3.68), Mono (3.63), and Atlantique (3.33) regions, while the lowest was in Alibori (2.37). Adult whitefly populations (mean number/plant) were highest in Couffo (15.88) and Mono (13.00) regions and lowest in Donga (0.06). Significant relationships were found between CMD severity and whitefly abundance (P = 0.0010) but there was no significant relationship between whitefly numbers and CMD incidence (P = 0.0577). These findings indicate that CMD has expanded its range across Benin. They also provide a basis for designing a response strategy for the control of cassava virus diseases such as CMD.

Phenotypic and molecular screenings for determination of cassava mosaic disease (CMD) status in farmers' fields in Ebonyi State, Nigeria.

African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) are among the major constraints to cassava productivity within tropical and sub-tropical regions, including Ebonyi State, Nigeria. Thus, virus indexing has become imperative to determine the status of cassava mosaic disease (CMD) in Ebonyi State, to implement appropriate preventive and control strategies. Seventy-eight cassava accessions obtained from different locations of Ebonyi State were phenotypically scored, using scales 1-5 depending on CMD symptomatic expressions, followed by multiplex-PCR and sequencing for validation. 11% of cassava accessions cultivated in Afikpo were resistant (RE) to ACMV compared to 8% of moderately-resistant (MR) accessions in Izzi and 55% of tolerant (TO) ACMV accessions in Ebonyi. 100% of cassava accessions in Onicha and 66% in Afikpo South were susceptible (SU) and highly susceptible (HS) to ACMV, respectively. With multiplex-PCR, 97.4% (ACMV) and 2.6% (EACMV) were positive. Dunn's multiple comparison tests of CMD mean incidence demonstrated differences (P < 0.05), except between RE and MR, and TO and MR. More transitions (A/G, C/T) compared to transversions (A/T, G/T), were detected, with nonsynonymous mutations (Leucine/Isoleucine; Valine/Isoleucine; Arginine/Lysine; Methionine/Isoleucine), and good bit-scores (91.13-99.07% identites; e-values of 7.00e-148-0.00e+00). Phylogeny resolved the sequences into five major groups. DNA sequencing validated the detected ACMV and EACMV species. This study revealed variants of ACMV and low adoption of RE and MR cassava accessions in the farmers' fields. The findings will guide in getting disease-free and resistant varieties as planting materials to significantly mitigate the CMD spread in Ebonyi State, Nigeria.

Evaluation of resistance to cassava mosaic disease in selected African cassava cultivars using combined molecular and greenhouse grafting tools.

Cassava mosaic disease (CMD) threatens cassava (Manihot esculenta) production in Africa. A total of 24 selected cultivars were screened against CMD using combined molecular and greenhouse grafting tools. Disease severity was recorded for 10 weeks after inoculation and the molecular markers associated with CMD2 were detected by PCR. CMD severity significantly differed (P < 0.0001) among cultivars. Twelve cultivars were morphologically resistant and eight of these possessed CMD2 and four did not. These results suggest that there are several CMD-resistant cassava cultivars that could be recommended for on-farm production and for conservation and breeding programs.

A method for generating virus-free cassava plants to combat viral disease epidemics in Africa.

Here, we report a method to clean cassava plants from viral infections that cause cassava mosaic and brown streak diseases in Africa. Infected plants of resistant or tolerant varieties from Malawi, Mozambique, Kenya, Tanzania and Uganda were cleaned in the UK using a combination of tissue culture, chemotherapy and thermotherapy. In the first cycle of our virus-indexing procedure, we successfully cleaned 27 of the 31 varieties (87%), and after an additional three cleaning cycles, all plants were virus-free. Virus-free tissue-cultured plants were shipped back to Africa for distribution to farmers. This first cross-boundary effort provides important lessons for mitigating the two-major cassava viral diseases.

Phylogenetic characterization of East African cassava mosaic begomovirus (Geminiviridae) isolated from Manihot carthaginensis subsp. glaziovii (Müll.Arg.) Allem., from a non-cassava growing region in Tanzania.

Manihot carthaginensis subsp. glaziovii (Müll.Arg.) Allem., a wild relative of cassava, native to Brazil, is one of the popular agroforestry trees used for hedges and/or boundary plants surrounding homesteads and farms and also harbours cassava mosaic begomoviruses (CMBs) and cassava brown streak ipomoviruses. Sequences of the DNA-A component of East African cassava mosaic virus (EACMV) isolates from M. carthaginensis subsp. glaziovii (Müll.Arg.) Allem., collected from non-cassava growing areas of Tanzania were characterized. Thirteen full length DNA-A sequences were analysed together with 15 already reported EACMV sequences and six CMB species reference genomes. The results show 96 to 100% nucleotide sequence identity with EACMV isolates from Kenya. Phylogenetic analysis revealed that EACMV isolates from M. carthaginensis subsp. glaziovii (Müll.Arg.) Allem, belong to a single cassava mosaic begomovirus species. The EACMV monophyletic clade is distinct from all other CMB species. The presence of Cassava infecting begomoviruses in wild cassava relative growing from traditionally non cassava growing region serve as inoculum sources for cassava-infecting begomoviruses and therefore their eradication is key in the sustainable management of CMBs, especially in the non-cassava growing areas.

Resistance gene analogs involved in tolerant cassava--geminivirus interaction that shows a recovery phenotype.

The current literature describes recovery from virus-induced symptoms as a RNA silencing defense, but immunity-related genes, including the structurally specific resistance gene analogs (RGAs) that may play a key role in tolerance and recovery is not yet reported. In this study, the transcriptome data of tolerant cassava TME3 (which exhibits a recovery phenotype) and susceptible cassava T200 infected with South African cassava mosaic virus were explored for RGAs. Putative resistance protein analogs (RPAs) with amide-like indole-3-acetic acid-Ile-Leu-Arg (IAA-ILR) and leucine-rich repeat (LRR)-kinase conserved domains were unique to TME3. Common responsive RPAs in TME3 and T200 were the dirigent-like protein, coil-coil nucleotide-binding site (NBS) and toll-interleukin-resistance, disease resistance zinc finger chromosome condensation-like protein (DZC), and NBS-apoptosis repressor with caspase recruitment (ARC)-LRR domains. Mutations in RPAs in the MHD motif of the NBS-ARC2 subdomain associated with the recovery phase in TME3 were observed. Additionally, a cohort of 25 RGAs mined solely during the recovery process in TME3 was identified. Phylogenetic and expression analyses support that diverse RGAs are differentially expressed during tolerance and recovery. This study reveals that in cassava, a perennial crop, RGAs participate in tolerance and differentially accumulate during recovery as a complementary defense mechanism to natural occurring RNA silencing to impair viral replication.

Why mosaic? Gene expression profiling of African cassava mosaic virus-infected cassava reveals the effect of chlorophyll degradation on symptom development.

Cassava mosaic disease, caused by cassava begomoviruses, is the most serious disease for cassava in Africa. However, the pathogenesis of this disease is poorly understood. We employed high throughput digital gene expression profiling based on the Illumina Solexa sequencing technology to investigate the global transcriptional response of cassava to African cassava mosaic virus infection. We found that 3,210 genes were differentially expressed in virus-infected cassava leaves. Gene ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that genes implicated in photosynthesis were most affected, consistent with the chlorotic symptoms observed in infected leaves. The upregulation of chlorophyll degradation genes, including the genes encoding chlorophyllase, pheophytinase, and pheophorbide a oxygenase, and downregulation of genes encoding the major apoproteins in light-harvesting complex II were confirmed by qRT-PCR. These findings, together with the reduction of chlorophyll b content and fewer grana stacks in the infected leaf cells, reveal that the degradation of chlorophyll plays an important role in African cassava mosaic virus symptom development. This study will provide a road map for future investigations into viral pathogenesis.

Differential miRNA profiles in South African cassava mosaic virus-infected cassava landraces reveal clues to susceptibility and tolerance to cassava mosaic disease.

Specific miRNA families are involved in susceptibility or antiviral immunity in plants. Manihot esculenta Crantz (cassava) is a perennial plant that is an important food security crop in sub-Saharan Africa. Cassava is susceptible to several begomoviruses that cause cassava mosaic disease (CMD). In this study, we investigated the leaf miRNAome response in a tolerant (TME3) and susceptible (T200) cassava landrace challenged with South African cassava mosaic virus. RNAseq was performed on leaf samples at 12, 32 and 67 days post infection (dpi), representing early, symptomatic and late persistent stages of CMD infection. Significantly, distinct profiles of conserved miRNA family expression between the T200 and TME3 landraces at the three infection stages were observed. Notably at 12 days post SACMV infection, TME3 exhibited significant downregulation (log2fold<2.0) of 42 %, compared to 9% in T200, of the conserved miRNA families. This demonstrates an overall early response to SACMV in TME3 prior to symptom appearance not observed in T200, and expression of a large cohort of miRNA-regulated genes. Notably, at early infection, downregulation of mes-miR162 and 168 that target antiviral posttransriptional gene silencing (PTGS) regulators DCL1 and AGO1, respectively, was observed in TME3, and AGO1 and DCL1 expression was higher compared to T200 post infection. Early rapid responses prior to symptom development, including RNA silencing, may be key to establishing the tolerance/recovery phenotype exhibited by TME3 landrace later on at 67 dpi. At recovery, TME3 was hallmarked by a highly significant down-regulation of mes-miR167. MiR167 targets an auxin responsive factor which plays a role in auxin signaling and adaptive responses to stress, suggesting the importance of the auxin signaling in recovery of SACMV-induced symptoms. The gene targets of these miRNAs and their associated networks may provide clues to the molecular basis of CMD tolerance in perennial hosts such as cassava.

WRKY Transcription Factors in Cassava Contribute to Regulation of Tolerance and Susceptibility to Cassava Mosaic Disease through Stress Responses.

Among the numerous biological constraints that hinder cassava (Manihot esculenta Crantz) production, foremost is cassava mosaic disease (CMD) caused by virus members of the family Geminiviridae, genus Begomovirus. The mechanisms of CMD tolerance and susceptibility are not fully understood; however, CMD susceptible T200 and tolerant TME3 cassava landraces have been shown to exhibit different large-scale transcriptional reprogramming in response to South African cassava mosaic virus (SACMV). Recent identification of 85 MeWRKY transcription factors in cassava demonstrated high orthology with those in Arabidopsis, however, little is known about their roles in virus responses in this non-model crop. Significant differences in MeWRKY expression and regulatory networks between the T200 and TME3 landraces were demonstrated. Overall, WRKY expression and associated hormone and enriched biological processes in both landraces reflect oxidative and other biotic stress responses to SACMV. Notably, MeWRKY11 and MeWRKY81 were uniquely up and downregulated at 12 and 67 days post infection (dpi) respectively in TME3, implicating a role in tolerance and symptom recovery. AtWRKY28 and AtWRKY40 homologs of MeWRKY81 and MeWRKY11, respectively, have been shown to be involved in regulation of jasmonic and salicylic acid signaling in Arabidopsis. AtWRKY28 is an interactor in the RPW8-NBS resistance (R) protein network and downregulation of its homolog MeWRKY81 at 67 dpi in TME3 suggests a negative role for this WRKY in SACMV tolerance. In contrast, in T200, nine MeWRKYs were differentially expressed from early (12 dpi), middle (32 dpi) to late (67 dpi) infection. MeWRKY27 (homolog AtWRKY33) and MeWRKY55 (homolog AtWRKY53) were uniquely up-regulated at 12, 32 and 67 dpi in T200. AtWRKY33 and AtWRKY53 are positive regulators of leaf senescence and oxidative stress in Arabidopsis, suggesting MeWRKY55 and 27 contribute to susceptibility in T200.

Epidemiological assessment of cassava mosaic disease in Burkina Faso.

Surveys were conducted in 2016 and 2017 across the main cassava-growing regions of Burkina Faso to assess the status of cassava mosaic disease (CMD) and to determine the virus strains causing the disease, using field observation and phylogenetic analysis. CMD incidence varied between regions and across years but was lowest in Hauts-Bassins (6.0%, 2016 and 5.4%, 2017) and highest in Centre-Sud (18.5%, 2016) and in Boucle du Mouhoun (51.7%, 2017). The lowest CMD severity was found in Est region (2.0) for both years and the highest in Sud-Ouest region (3.3, 2016) and Centre-Sud region (2.8, 2017). The CMD infection was primarily associated with contaminated cuttings in all regions except in Hauts-Bassins, where whitefly-borne infection was higher than cuttings-borne infection in 2016. PCR screening of 687 samples coupled with sequence analysis revealed the presence of African cassava mosaic-like (ACMV-like) viruses and East African cassava mosaic-like (EACMV-like) viruses as single infections at 79.5% and 1.1%, respectively. Co-infections of ACMV-like and EACMV-like viruses were detected in 19.4% of the tested samples. In addition, 86.7% of the samples positive for EACMV-like virus were found to be positive for East African cassava mosaic Cameroon virus (EACMCMV). Phylogenetic analysis revealed the segregation of cassava mosaic geminiviruses (CMGs) from Burkina Faso into three clades specific to ACMV, African cassava mosaic Burkina Faso virus (ACMBFV), and EACMCMV, confirming the presence of these viruses. The results of this study show that EACMCMV occurrence may be more prevalent in Burkina Faso than previously thought.

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

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.

Virus-Induced Gene Silencing (VIGS) in Cassava Using Geminivirus Agroclones.

Virus-induced gene silencing (VIGS) is an efficient, low-cost, and rapid functional validation tool for candidate genes in planta. The VIGS approach is particularly suitable to perform reverse genetics studies in crop species. Here we present a detailed method to perform VIGS in cassava, from target gene fragment to agroinoculation and VIGS quantitation.

RNA silencing of South African cassava mosaic virus in transgenic cassava expressing AC1/AC4 hp- RNA induces tolerance.

Cassava mosaic disease (CMD), caused by geminiviruses, is a major hurdle to cassava production. Due to the heterozygous nature of cassava, breeding for virus resistance is difficult, but cassava has been shown to be a good candidate for genetic engineering using RNA interference (RNAi). T This study reports on the ability of a transgene-derived RNA hairpin, homologous to an overlapping region of the SACMV replication associated protein and putative virus suppressor of silencing protein (AC1/AC4), to confer tolerance in the CMD-susceptible model cassava cultivar 60444. Three of the fourteen transgenic lines expressing SACMV AC1/AC4 hairpin-derived siRNAs showed decreased symptoms and viral loads compared to untransformed control plants. Expression of SACMV AC1/AC4 homologous siRNAs showed that this tolerance is most likely associated with post-transcriptional gene silencing of the virus. This is the first report of targeting the overlapping AC1 and AC4 genes of SACMV conferring CMD tolerance in cassava.

Differential response of cassava genotypes to infection by cassava mosaic geminiviruses.

Mitigation of cassava mosaic disease (CMD) focuses on the introgression of resistance imparted by the polygenic recessive (CMD1), dominant monogenic (CMD2) and CMD3 loci. The mechanism(s) of resistance they impart, however, remain unknown. Two CMD susceptible and nine CMD resistant cassava genotypes were inoculated by microparticle bombardment with infectious clones of African cassava mosaic virus Cameroon strain (ACMV-CM) and the Kenyan strain K201 of East African cassava mosaic virus (EACMV KE2 [K201]). Genotypes carrying the CMD1 (TMS 30572), CMD2 (TME 3, TME 204 and Oko-iyawo) and CMD3 (TMS 97/0505) resistance mechanisms showed high levels of resistance to ACMV-CM, with viral DNA undetectable by PCR beyond 7days post inoculation (dpi). In contrast, all genotypes initially developed severe CMD symptoms and accumulated high virus titers after inoculation with EACMV KE2 (K201). Resistant genotypes recovered to become asymptomatic by 65dpi with no detectable virus in newly formed leaves. Genotype TMS 97/2205 showed highest resistance to EACMV KE2 (K201) with <30% of inoculated plants developing symptoms followed by complete recovery by 35dpi. Deep sequencing of small RNAs confirmed production of 21-24 nt virus derived small RNAs (vsRNA) that mapped to cover the entire ACMV-CM and EACMV KE2 (K201) viral genomes in both polarities, with hotspots seen within gene coding regions. In resistant genotypes, total vsRNAs were most abundant at 20 and 35dpi but reduced significantly upon recovery from CMD. In contrast, CMD susceptible genotypes displayed abundant vsRNAs throughout the experimental period. The percentage of vsRNAs reads ranked by class size were 21nt (45%), 22 nt (28%) and 24 nt (18%) in all genotypes studied. The number of vsRNA reads directly correlated with virus titer and CMD symptoms.

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

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.

Quantification of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV-UG) in single and mixed infected Cassava (Manihot esculenta Crantz) using quantitative PCR.

The quantity of genomic DNA-A and DNA-B of African cassava mosaic virus (ACMV) and East African cassava mosaic virus Uganda (Uganda variant, EACMV-UG) was analysed using quantitative PCR to assess virus concentrations in plants from susceptible and tolerant cultivars. The concentrations of genome components in absolute and relative quantification experiments in single and mixed viral infections were determined. Virus concentration was much higher in symptomatic leaf tissues compared to non-symptomatic leaves and corresponded with the severity of disease symptoms. In general, higher titres were recorded for EACMV-UG Ca055 compared to ACMV DRC6. The quantitative assessment also showed that the distribution of both viruses in the moderately resistant cassava cv. TMS 30572 was not different from the highly susceptible cv. TME 117. Natural mixed infections with both viruses gave severe disease symptoms. Relative quantification of virus genomes in mixed infections showed higher concentrations of EACMV-UG DNA-A compared to ACMV DNA-A, but a marked reduction of EACMV-UG DNA-B. The higher concentrations of EACMV-UG DNA-B compared to EACMV DNA-A accumulation in single infections were consistent. Since DNA-B is implicated in virus cell-to-cell spread and systemic movement, the abundance of the EACMV-UG DNA-B may be an important factor driving cassava mosaic disease epidemic.

Form follows function in geminiviral minichromosome architecture.

A comprehensive survey on the viral minichromosomes of the begomoviruses Abutilon mosaic virus, tomato yellow leaf curl Sardinia virus, African cassava mosaic virus, Indian cassava mosaic virus (family Geminiviridae) during the course of infections in Nicotiana benthamiana is summarized. Using optimized one-dimensional and two-dimensional gel systems combined with blot hybridization and a standardized evaluation, discrete and heterogeneous virus-specific signals with different DNA forms were compared to trace functions of viral multiplication with inactive/active replication and/or transcription. A quantitative approach to compare the distantly related viruses during the course of infection with the aim to generalize the conclusions for geminiviruses has been developed. Focussing on the distribution of topoisomers of viral supercoiled DNA, which reflect minichromosomal stages, predominant minichromosomes with 12 nucleosomes, less with 13 nucleosomes and no with 11 nucleosomes were found. These results indicate that chromatin with only one open gap to bind transcription factors is the favourite form. The dynamics during infections in dependence on the experimental conditions is discussed with reference to the design of experiments for resistance breeding and molecular analyses.