African Cassava Mosaic Virus Research Articles

Deeply Sequenced Infectious Clones of Key Cassava Begomovirus Isolates from Cameroon.

We deeply sequenced two pairs of widely used infectious clones (4 plasmids) of the bipartite begomoviruses African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV). The ACMV clones were quite divergent from published sequences. Raw reads, consensus plasmid sequences, and the infectious clones themselves are all publicly available.

South West and North Central Nigeria: Assessment of cassava mosaic disease and field status of African cassava mosaic virus and East African cassava mosaic virus.

Cassava mosaic disease (CMD), caused by cassava mosaic begomoviruses (CMBs), is a major threat to cassava production in Nigeria. The predominant CMBs in Nigeria are African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and East African cassava mosaic Cameroon virus (EACMCV), which are transmitted through infected stem cuttings and whitefly vectors. This study was conducted in 2015 and 2017 to assess the epidemiology of CMD and the current distribution of CMBs in cassava farms in South West (SW) and North Central (NC) Nigeria. A survey of cassava farms was undertaken, and samples representative of disease symptoms were collected and assessed using molecular techniques. A total of 184 and 328 cassava farms were sampled in 2015 and 2017, respectively. CMD incidence for both regions surveyed was 43.80 and 12.25% in 2015 and 2017, respectively. Fields in SW recorded a higher incidence rate in 2015 (SW: 45.11%, NC: 42.47%), while the reverse occurred in 2017 (SW: 10.90%, NC: 14.01%). Overall, the CMD incidence in Benue State (NC) was significantly higher than other locations surveyed in both years. CMD symptom severity and mean whitefly population were higher in SW Nigeria in the two survey years. ACMV was widespread across both zones, occurring in 79.1% (453/613) and 54.8% (386/704) of cassava leaf samples analysed in 2015 and 2017, respectively. EACMV was detected in only 6.0% (37/613) and 4.7% (33/704) of all cassava leaf samples analysed in 2015 and 2017, respectively. Overall, a higher proportion of infected samples were found in NC in both 2015 (NC: 85.2%, SW: 75.4%) and 2017 (NC: 73.6%, SW: 45.2%). Detection using strain‐specific primers revealed that 97% of EACMV positive samples were indeed infected by the EACMCV strain of the virus. As previously reported, samples with mixed infections showed a higher symptom severity than samples with single ACMV or EACMV infections. This study provides an update to the distribution of CMBs in SW and NC Nigeria and will be useful in development of monitoring and management strategies for the disease in both regions.

INCIDENCE AND DISTRIBUTION OF VIRUSES INFECTING CASSAVA IN KADUNA AND SOKOTO STATES, NIGERIA

Field survey was conducted in the 2015 wet season to determine the incidence and distribution of cassava viruses in Kaduna and Sokoto States, Nigeria. Eighteen farms from 3 Local Government Areas namely; Lere, Chikun and Kajuru (Kaduna State) and Tureta, Shagari and Tambuwal (Sokoto State) were surveyed. Symptomatic leaves (180) and asymptomatic leaves (90) were collected from the surveyed farms. Enzyme Linked Immosorbent Assay (ELISA) technique was used to test the presence of viruses infecting cassava. Three viruses: African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and Cassava Congo sequivirus (no acronyms) were detected. Triple Antibody Sandwich (TAS- ELISA) was used to detect ACMV and EACMV while Double Antibody Sandwich (DAS- ELISA) was used to detect Cassava Congo sequivirus. ACMV had the highest incidence and was widely spread, the highest incidence was recorded in Lere Local Government (68.9%) and least was Tambuwal LG (11.0), followed by EACMV with highest incidence in Tambuwal (22.23%) and no occurrence in Kajuru. Cassava Congo sequivirus recorded the highest incidence in Kajuru (43.33%). Mixed infections occur in some of the farms. This the first report of Cassava Congo sequivirus in Nigeria and Africa. Wide spread occurrence of ACMV calls for effective management of the virus.

Characterization of Sirnas Derived from Two Species of Cassava-Infecting Geminiviruses in Nicotiana Benthamiana

The infection dynamics of cassava mosaic geminiviruses was determined in Nicotiana benthamiana. Following inoculation, symptoms were observed at 5 and 10 dpi for African cassava mosaic virus (ACMV-CM) and the Kenyan strain K201 of East African cassava mosaic virus (EACMV KE2 [K201]). Northern and Southern blots indicated higher accumulation of ACMV-CM at 10 dpi compared with EACMV KE2 (K201) correlating with more severe symptoms at this time point. However, unlike for EACMV KE2 (K201), symptom remission characterized by appearance of non-asymptomatic leaves was observed after 20 dpi in plants infected with ACMV-CM. Deep sequencing identified between 969,015 and 1,307,689 total reads of which >80% mapped to the host genome. For both virus species, 22 nt virus derived sRNAs (vsRNAs) reads were the most abundant (34% - 53%) followed by 21 nt (27% - 27%) and 24 nt (9 – 23%) vsRNA reads. EACMV KE2 (K201) vsRNAs were proportionally higher (12.31% and 4.51%) than those derived from ACMV-CM (5.70% and 0.45%) for DNA A and DNA B respectively. vsRNAs were covered the entire virus genome with dense accumulation at regions where the viral ORFs overlapped. Key words; RNA interference, Cassava mosaic disease, Geminiviruses DOI: 10.7176/JNSR/10-8-06 Publication date: April 30 th 2020

Response of cassava cultivars to African cassava mosaic virus infection across a range of inoculum doses and plant ages.

Cassava production in Africa is constrained by cassava mosaic disease (CMD) that is caused by the Cassava mosaic virus (CMV). The aim of this study was to evaluate the responses of a range of commonly cultivated West African cassava cultivars to varying inoculum doses of African cassava mosaic virus (ACMV). We grafted 10 cultivars of cassava plants with different inoculum doses of CMV (namely two, four, or six CMD-infected buds) when the experimental plants were 8, 10, or 12 weeks old, using non-inoculated plants as controls. Three cultivars showed disease symptoms when grafted with two buds, and four cultivars showed disease symptoms when grafted with four or six buds. Most cultivars became symptomatic six weeks after inoculation, but one (‘TMS92/0326’) was symptomatic two weeks after inoculation, and two (‘Ntollo’ and ‘Excel’) were symptomatic after four weeks. Root weight tended to be lower in the six-bud than in the two-bud dose, and disease severity varied with plant age at inoculation. These results indicate that the level of CMD resistance in cassava cultivars varies with inoculum dose and timing of infection. This will allow appropriate cultivars to be deployed in each production zone of Africa in accordance with the prevalence of CMD.

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.

Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses

BackgroundGeminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses.ResultsOur results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find that between 33 and 48% of edited virus genomes evolve a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. We also find that in the model plant Nicotiana benthamiana the replication of the novel, mutant virus is dependent on the presence of the wild-type virus.ConclusionsOur study highlights the risks associated with CRISPR-Cas9 virus immunity in eukaryotes given that the mutagenic nature of the system generates viral escapes in a short time period. Our in-depth analysis of virus populations also represents a template for future studies analyzing virus escape from anti-viral CRISPR transgenics. This is especially important for informing regulation of such actively mutagenic applications of CRISPR-Cas9 technology in agriculture.

Optimal control of a vectored plant disease model for a crop with continuous replanting

ABSTRACTVector-transmitted diseases of plants have had devastating effects on agricultural production worldwide, resulting in drastic reductions in yield for crops such as cotton, soybean, tomato, and cassava. Plant-vector-virus models with continuous replanting are investigated in terms of the effects of selection of cuttings, roguing, and insecticide use on disease prevalence in plants. Previous models are extended to include two replanting strategies: frequencyreplanting and abundance-replanting. In frequency-replanting, replanting of infected cuttings depends on the selection frequency parameter ε, whereas in abundance-replanting, replanting depends on plant abundance via a selection rate parameter also denoted as ε. The two models are analysed and new thresholds for disease elimination are defined for each model. Parameter values for cassava, whiteflies, and African cassava mosaic virus serve as a case study. A numerical sensitivity analysis illustrates how the equilibrium densities of healthy and infected plants vary with parameter values. Optimal control theory is used to investigate the effects of roguing and insecticide use with a goal of maximizing the healthy plants that are harvested. Differences in the control strategies in the two models are seen for large values of ε. Also, the combined strategy of roguing and insecticide use performs better than a single control.

Different forms of African cassava mosaic virus capsid protein within plants and virions

One geminiviral gene encodes the capsid protein (CP), which can appear as several bands after electrophoresis depending on virus and plant. African cassava mosaic virus-Nigeria CP in Nicotiana benthamiana, however, yielded one band (~ 30 kDa) in total protein extracts and purified virions, although its expression in yeast yielded two bands (~ 30, 32 kDa). Mass spectrometry of the complete protein and its tryptic fragments from virions is consistent with a cleaved start M1, acetylated S2, and partial phosphorylation at T12, S25 and S62. Mutants for additional potentially modified sites (N223A; C235A) were fully infectious and formed geminiparticles. Separation in triton acetic acid urea gels confirmed charge changes of the CP between plants and yeast indicating differential phosphorylation. If the CP gene alone was expressed in plants, multiple bands were observed like in yeast. A high turnover rate indicates that post-translational modifications promote CP decay probably via the ubiquitin-triggered proteasomal pathway.

First Report of East African Cassava Mosaic Virus-Uganda Infecting the Nile Tulip Tree in Western Kenya

Whitefly-transmitted cassava mosaic begomoviruses (CMBs; family Geminiviridae; genus Begomovirus) are viruses with bipartite genomes (components referred to as DNA-A and DNA-B) containing a conserved (between the two components) noncoding sequence known as the common region (CR) required for DNA replication and transcription (Fondong 2013). CMBs cause cassava mosaic disease (CMD), a major limitation to cassava production in sub-Saharan Africa. In Kenya, CMD is associated with the begomoviruses East African cassava mosaic virus (EACMV), African cassava mosaic virus (ACMV), East African cassava mosaic Kenya virus, and East African cassava mosaic Zanzibar virus, as well as several recombinant viruses, such as the virulent EACMV-Uganda (EACMV-UG) that so far occurs only in western Kenya (Nyongesa Were et al. 2016). Within the CMB-cassava pathosystem, various noncassava plant species may play an epidemiological role as virus reservoirs for the whitefly vector Bemisia tabaci (Alabi et al. 2008). In May 2017, leaf samples were collected from three plants exhibiting virus-like symptoms including mosaic, mottling, twisted leaves, and reduction in leaf surface area as well as from two nonsymptomatic plants belonging to the family Bignoniaceae growing in three cassava fields in western Kenya. Total genomic DNA was extracted from leaf samples using the ZR Plant/Seed DNA MiniPrep kit as per the manufacturer’s instructions (Zymo Research Corp., U.S.A.). The identity of the plant species was confirmed as Markhamia lutea by phenotypic characteristics and by polymerase chain reaction (PCR) amplification, Sanger sequencing, and BLASTn analysis of the chloroplast rubisco large-chain gene using the primer pair RbcLa-F (5′-ATGTCACCACAAACAGAGACTAAAGC-3′)/RbcLa-R (5′-GTAAAATCAAGTCCACCRCG-3′). Samples were screened by PCR with the diagnostic primer pairs JSP001/JSP002 for ACMV, JSP001/JSP003 for EACMV (Fondong et al. 2000), and UV AL1-F1/ACMV CP-R3 for EACMV-UG (Zhou et al. 1997). Samples from nonsymptomatic plants did not yield amplification products, confirming absence of viral infection. A 1,650-bp fragment of the expected size was amplified from one symptomatic sample with the primer pair UV AL1-F1/ACMV CP-R3, suggesting the presence of EACMV-UG. To confirm the positive PCR result, the genome of the virus was enriched by rolling-circle amplification (RCA) using a TempliPhi 100 RCA Kit (GE Healthcare, U.S.A.). The library was prepared using the NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, U.S.A.) according to the manufacturer’s instructions and sequenced using an Illumina MiSeq platform with the MiSeq Reagent Kit version 3 (600 cycles) (Illumina, U.S.A.). Following de novo sequence assembly, 344 and 89 paired-end reads aligned to generate two contigs identified by BLASTn search as the DNA-A and DNA-B of a bipartite virus. The nucleotide sequences were deposited in GenBank as accession numbers MF974568 and MH379641. The 2,798-nucleotide (nt) DNA-A and 2,774-nt DNA-B had an approximately 160-nt EACMV-like CR with 53%-nt sequence identity. DNA-A shared 99% nucleotide sequence identity with the DNA-A of an EACMV-UG isolate (AJ717522) from western Kenya, whereas the DNA-B was closely related to another EACMV-UG isolate (AJ704962) from western Kenya at 97% nucleotide sequence identity. To the best of our knowledge, this is the first report of occurrence of EACMV-UG in a species of the family Bignoniaceae. Further investigations are required to determine the epidemiological significance of M. lutea in the CMB-cassava pathosystem.

Cassava Mosaic Disease and Associated Gemini Viruses in Bauchi State, Nigeria: Occurrence and Distribution

Cassava mosaic disease is one of the most important biotic constraint affecting Cassava limiting the production potential of the crop in Northern Nigeria. This study was carried out to evaluate the current status of Cassava mosaic disease incidence, severity, infection type, whitefly abundance in the farmers’ field as well as to determine the virus strains causing the disease in Bauchi State, North eastern Nigeria. A total of thirty three (33) farmers’ fields were surveyed and in each field, thirty plants were assessed along the two diagonals with respect to disease incidence, severity, infection type and whitefly abundance and leaf samples were collected from each field for the virus diagnosis. The results revealed that Cassava mosaic disease incidence was highest in Ganjuwa (88.66%) and lowest in Darazo (43.33%). The disease symptom severity was generally mild. It was also highest in Ganjuwa (2.85) and lowest in Darazo (1.34). Whitefly infection is most prevalent (59.73%) than cutting borne infection (2.67%) in the Districts. Adult whitefly (<i>Bemisia tabaci</i>) population was highest in Ganjuwa (56.33) followed by Kirfi (40.93) and lowest in Toro (27.63). Polymerase chain reaction (PCR) results using specific primers for African Cassava mosaic virus (ACMV) and East African Cassava mosaic virus (EACMV) detected single infections of ACMV and EACMV in 62.4% and 12.9% of the positive samples respectively. Co-infections of ACMV and EACMV were detected in 10.6% of the tested samples. Continuous monitoring of Cassava Mosaic Viruses and whiteflies population is required to provide appropriate management strategies of the disease in Nigeria.

An overview of cassava mosaic disease and its implications for the productivity of cassava in Sierra Leone.

Abstract This paper provides current information on the importance of cassava, characteristics of cassava farms, distribution of cassava mosaic disease (CMD), management practices and potential of resistant varieties to mitigate against the effects of CMD in Sierra Leone. There is a high prevalence of African cassava mosaic virus in Sierra Leone and East African cassava mosaic virus has also been detected recently. Improvement in the deployment of CMD-resistant varieties has had a positive effect of increasing the area under cultivation of improved varieties. Much has to be done in the area of early detection and diagnosis for the management of CMD. More effort must be placed on the breeding of cassava genotypes that meet the food, traditional and cultural needs of farmers as well as the industrial needs for commercialization.

Two sub-Saharan Africa 1 populations of Bemisia tabaci exhibit distinct biological differences in fecundity and survivorship on cassava

A study was conducted to determine the fecundity (number of eggs laid) and survivorship of two sub-Saharan Africa 1 (SSA1) populations of Bemisia tabaci: subgroup 1 (SSA1-SG1) obtained from the Lake Victoria Basin, and subgroup 3 (SSA1-SG3) from Coast Region, on healthy and cassava mosaic begomovirus (CMB)-infected cassava plants in Tanzania in April (first trial) and June (repeat trial) 2012. Colonies of the two SSA1 B. tabaci populations, SSA1-SG1 and SSA1-SG3 were established on healthy cassava plants in separate insect-proof cages within a screen house at Mikocheni Agricultural Research Institute, Dar es Salaam, Tanzania. Field-collected cassava plants infected with African cassava mosaic virus-[Tanzania:2001] (ACMV-[TZ:01]) and East African cassava mosaic virus-Kenya (EACMV-KE) were raised in containment as sources of viruses. Single female, 1–4-day old adults of SSA1-SG1 and SSA1-SG3 B. tabaci populations were allowed to feed and oviposit on healthy and CMB-infected cassava for 3 days. Results show that there were significant differences (P < 0.05) in the mean number of eggs laid and emerged adults between SSA1-SG1 and SSA1-SG3 in both the first and repeat trials. Overall, SSA1-SG1 produced a higher number of eggs and emerged adults (30.5 ± 3.3 & 17.7 ± 2.5) than SSA1-SG3 (23.4 ± 3.1 & 13.6 ± 2.3). Interestingly, CMB-infection had no significant effect on the number of eggs laid and emerged adults in both the first and repeat trials. Overall, there were no significant differences (P > 0.05) between the survivorship of the 1st, 2nd & 3rd, 4th instars and emerged adults of both SSA1-SG1 and SSA1-SG3 populations on healthy and CMB-infected cassava plants. Irrespective of the virus treatment, survivorship of SSA1-SG1 and SSA1-SG3 populations was similar across all developmental stages. In conclusion, our study did not reveal any synergistic interaction between CMB infection and whitefly fecundity and survivorship. However, the results point to genetic differences as the likely explanation of the differences in the biology of the two SSA1 (SSA1-SG1 and SSA1-SG3) B. tabaci populations.

Resistance of advanced cassava breeding clones to infection by major viruses in Uganda

Cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) are two viral diseases that cause severe yield losses in cassava of up to 100%, thereby persistently threatening food and income security in sub-Saharan Africa. For effective management of these diseases, there is a critical need to develop and deploy varieties with dual resistance to CBSD and CMD. In this study, we determined the response of advanced breeding lines to field infection by cassava brown streak viruses (CBSVs) and cassava mosaic begomoviruses (CMBs). This aim helped in identifying superior clones for downstream breeding. In total, 220 cassava clones, three in uniform yield trials (UYTs) and 217 in a crossing block trial (CBT), were evaluated for virus and disease resistance. Field data were collected on disease incidence and severity. To detect and quantify CBSVs, 448 and 128 leaf samples from CBSD symptomatic and symptomless plants were analyzed by reverse transcription PCR and real-time quantitative PCR, respectively. In addition, 93 leaf samples from CMD symptomatic plants in the CBT were analyzed by conventional PCR using CMB species-specific primers. In the CBT, 124 (57%) cassava clones did not express CMD symptoms. Of the affected plants, 44 (55%) had single African cassava mosaic virus infection. Single Cassava brown streak virus (CBSV) infections were more prevalent (81.6%) in CBT clones than single Ugandan cassava brown streak virus (UCBSV) infection (3.2%). Of the three advanced clones in the UYT, NAROCASS 1 and NAROCASS 2 had significantly lower (P < 0.05) CBSD severity, incidence, and CBSV load than MH04/0300. In the UYT, only 22% of samples tested had CBSVs, and all showed a negative result for CMBs. The low disease incidence, severity, and viral load associated with NAROCASS 1 and NAROCASS 2 is evidence of their tolerance to both CBSD and CMD. Therefore, these two cassava clones should be utilized in CBSD and CMD management in Uganda, including their utilization as progenitors in further virus resistance breeding.

Cassava geminivirus agroclones for virus-induced gene silencing in cassava leaves and roots

AimWe report the construction of a Virus-Induced Gene Silencing (VIGS) vector and an agroinoculation protocol for gene silencing in cassava (Manihot esculenta Crantz) leaves and roots. The African cassava mosaic virus isolate from Nigeria (ACMV-[NOg]), which was initially cloned in a binary vector for agroinoculation assays, was modified for application as VIGS vector. The functionality of the VIGS vector was validated in Nicotiana benthamiana and subsequently applied in wild-type and transgenic cassava plants expressing the uidA gene under the control of the CaMV 35S promoter in order to facilitate the visualization of gene silencing in root tissues. VIGS vectors were targeted to the Mg2+-chelatase gene in wild type plants and both the coding and promoter sequences of the 35S::uidA transgene in transgenic plants to induce silencing. We established an efficient agro-inoculation method with the hyper-virulent Agrobacterium tumefaciens strain AGL1, which allows high virus infection rates. The method can be used as a low-cost and rapid high-throughput evaluation of gene function in cassava leaves, fibrous roots and storage roots.BackgroundVIGS is a powerful tool to trigger transient sequence-specific gene silencing in planta. Gene silencing in different organs of cassava plants, including leaves, fibrous and storage roots, is useful for the analysis of gene function.ResultsWe developed an African cassava mosaic virus—based VIGS vector as well as a rapid and efficient agro-inoculation protocol to inoculate cassava plants. The VIGS vector was validated by targeting endogenous genes from Nicotiana benthamiana and cassava as well as the uidA marker gene in transgenic cassava for visualization of gene silencing in cassava leaves and roots.ConclusionsThe African cassava mosaic virus—based VIGS vector allows efficient and cost-effective inoculation of cassava for high-throughput analysis of gene function in cassava leaves and roots.

Efficacy of chemotherapy and thermotherapy in elimination of East African cassava mosaic virus from Tanzanian cassava landrace.

Cassava mosaic disease is caused by cassava mosaic begomoviruses (CMBs) and can result in crop losses up to 100% in cassava (Manihot esculenta) in Tanzania. We investigated the efficacy of chemotherapy and thermotherapy for elimination of East African cassava mosaic virus (EACMV) of Tanzanian cassava. In vitro plantlets from EACMV‐infected plants obtained from coastal Tanzania were established in the greenhouse. Leaves were sampled from the plants and tested to confirm the presence of EACMV. Plantlets of plants positive for EACMV were initiated in Murashige and Skoog (MS) medium. On the second subculture, they were subjected into chemical treatment in the medium containing salicylic acid (0, 10, 20, 30 and 40 mg/L) and ribavirin (0, 5, 10, 15 and 20 mg/L). In the second experiment, EACMV‐infected plantlets were subjected to temperatures between 35 and 40°C with 28°C as the control. After 42 days of growth, DNA was extracted from plant leaves and PCR amplification was performed using EACMV specific primers. It was found that plant survival decreased with increasing levels of both salicylic acid and ribavirin concentrations. In general, plants treated with salicylic acid exhibited a lower plant survival % than those treated with ribavirin. However, the percentage of virus‐free plants increased with an increase in the concentration of both ribavirin and salicylic acid. The most effective concentrations were 20 mg/L of ribavirin and 30 mg/L of salicylic acid; these resulted in 85.0% and 88.9% virus‐free plantlets, respectively. With regard to thermotherapy, 35°C resulted in 79.5% virus‐free plantlets compared to 69.5% at 40°C. Based on virus elimination, ribavirin at 20 mg/L, salicylic acid 30 mg/L and thermotherapy at 35°C are recommended for production of EACMV free cassava plantlets from infected cassava landraces.

Using Open Source Tools for Analysis of the Mutation Rate of African Cassava Mosaic Virus

Popular tools used in studies in life sciences are often costly. This often pauses challenges to researchers in spite of the fact that research continues to be a key to the successful systematic development of new knowledge and a fundamental aspect to the usefulness of all higher education. Particularly, higher education also aims to advance, create and disseminate knowledge through research. Such critical studies like mutation studies therefore require affordable and fast results yielding software. In such research, open source software tools become handy in place of expensive proprietary tools. In order to provide alternative software tools for research, we decided to use a case study of the mutation of the African Cassava Mosaic Virus (ACMV) done by researchers in Zambia. The study of ACMV mutation is hampered by fragmented and non-user-friendly tools, which are currently available. A number of the tools used also depend on network connection, especially the Internet, to access and analyze data. To help alleviate this problem this research proposes the use of open source libraries in biopython to generate cost efficient and user-friendly solutions. Additionally, we propose the use of an open standard using XML as a standard protocol to share data between applications or stages in genomic data analysis of the ACMV. In our strife to provide open source solutions we analysed various tools and noted that biopython is quite popular. During our study of biopython our initial results show that it’s possible to use free tools to analyze data in the life sciences and consequently reduce the time and cost required to analyze ACMV. Based on this case study we propose the adoption of such open source libraries in order to make research much more affordable for scientists in the life sciences for researches that operate within a constrained budget.

Near-Atomic Resolution Structure of a Plant Geminivirus Determined by Electron Cryomicroscopy

African cassava mosaic virus is a whitefly-transmitted geminivirus which forms unique twin particles of incomplete icosahedra that are joined at five-fold vertices, building an unusual waist. How its 22 capsomers interact within a half-capsid or across the waist is unknown thus far. Using electron cryo-microscopy and image processing, we determined the virion structure with a resolution of 4.2Å and built an atomic model for its capsid protein. The inter-capsomer contacts mediated by the flexible N termini and loop regions differed within the half-capsids and at the waist, explaining partly the unusual twin structure. The tip of the pentameric capsomer is sealed by a plug formed by a turn region harboring the evolutionary conserved residue Y193. Basic amino acid residues inside the capsid form a positively charged pocket next to the five-fold axis of the capsomer suitable for binding DNA. Within this pocket, density most likely corresponding to DNA was resolved.

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.

A rapid virus-induced gene silencing (VIGS) method for assessing resistance and susceptibility to cassava mosaic disease

BackgroundCassava mosaic disease (CMD) is a major constraint to cassava production in sub-Saharan Africa. Under field conditions, evaluation for resistance to CMD takes 12–18 months, often conducted across multiple years and locations under pressure from whitefly-mediated transmission. Under greenhouse or laboratory settings, evaluation for resistance or susceptibility to CMD involves transmission of the causal viruses from an infected source to healthy plants through grafting, or by using Agrobacterium-mediated or biolistic delivery of infectious clones. Following inoculation, visual assessment for CMD symptom development and recovery requires 12–22 weeks. Here we report a rapid screening system for determining resistance and susceptibility to CMD based on virus-induced gene silencing (VIGS) of an endogenous cassava gene.ResultsA VIGS vector was developed based on an infectious clone of the virulent strain of East African cassava mosaic virus (EACMV-K201). A sequence from the cassava (Manihot esculenta) ortholog of Arabidopsis SPINDLY (SPY) was cloned into the CP position of the DNA-A genomic component and used to inoculate cassava plants by Helios® Gene Gun microparticle bombardment. Silencing of Manihot esculenta SPY (MeSPY) using MeSPY1-VIGS resulted in shoot-tip necrosis followed by death of the whole plant in CMD susceptible cassava plants within 2–4 weeks. CMD resistant cultivars were not affected and remained healthy after challenge with MeSPY1-VIGS. Significantly higher virus titers were detected in CMD-susceptible cassava lines compared to resistant controls and were correlated with a concomitant reduction in MeSPY expression in susceptible plants.ConclusionsA rapid VIGS-based screening system was developed for assessing resistance and susceptibility to CMD. The method is space and resource efficient, reducing the time required to perform CMD screening to as little as 2–4 weeks. It can be employed as a high throughput rapid screening system to assess new cassava cultivars and for screening transgenic, gene edited and breeding lines under controlled growth conditions.