African Cassava Mosaic Geminivirus Research Articles

Haplotype-resolved genomes of geminivirus-resistant and geminivirus-susceptible African cassava cultivars

BackgroundCassava is an important food crop in tropical and sub-tropical regions worldwide. In Africa, cassava production is widely affected by cassava mosaic disease (CMD), which is caused by the African cassava mosaic geminivirus that is transmitted by whiteflies. Cassava breeders often use a single locus, CMD2, for introducing CMD resistance into susceptible cultivars. The CMD2 locus has been genetically mapped to a 10-Mbp region, but its organization and genes as well as their functions are unknown.ResultsWe report haplotype-resolved de novo assemblies and annotations of the genomes for the African cassava cultivar TME (tropical Manihot esculenta), which is the origin of CMD2, and the CMD-susceptible cultivar 60444. The assemblies provide phased haplotype information for over 80% of the genomes. Haplotype comparison identified novel features previously hidden in collapsed and fragmented cassava genomes, including thousands of allelic variants, inter-haplotype diversity in coding regions, and patterns of diversification through allele-specific expression. Reconstruction of the CMD2 locus revealed a highly complex region with nearly identical gene sets but limited microsynteny between the two cultivars.ConclusionsThe genome maps of the CMD2 locus in both 60444 and TME3, together with the newly annotated genes, will help the identification of the causal genetic basis of CMD2 resistance to geminiviruses. Our de novo cassava genome assemblies will also facilitate genetic mapping approaches to narrow the large CMD2 region to a few candidate genes for better informed strategies to develop robust geminivirus resistance in susceptible cassava cultivars.

Divergent evolutionary and epidemiological dynamics of cassava mosaic geminiviruses in Madagascar.

BackgroundCassava mosaic disease (CMD) in Madagascar is caused by a complex of at least six African cassava mosaic geminivirus (CMG) species. This provides a rare opportunity for a comparative study of the evolutionary and epidemiological dynamics of distinct pathogenic crop-infecting viral species that coexist within the same environment. The genetic and spatial structure of CMG populations in Madagascar was studied and Bayesian phylogeographic modelling was applied to infer the origins of Madagascan CMG populations within the epidemiological context of related populations situated on mainland Africa and other south western Indian Ocean (SWIO) islands.ResultsThe isolation and analysis of 279 DNA-A and 117 DNA-B sequences revealed the presence in Madagascar of four prevalent CMG species (South African cassava mosaic virus, SACMV; African cassava mosaic virus, ACMV; East African cassava mosaic Kenya virus, EACMKV; and East African cassava mosaic Cameroon virus, EACMCV), and of numerous CMG recombinants that have, to date, only ever been detected on this island. SACMV and ACMV, the two most prevalent viruses, displayed low degrees of genetic diversity and have most likely been introduced to the island only once. By contrast, EACMV-like CMG populations (consisting of East African cassava mosaic virus, EAMCKV, EACMCV and complex recombinants of these) were more diverse, more spatially structured, and displayed evidence of at least three independent introductions from mainland Africa. Although there were no statistically supported virus movement events between Madagascar and the other SWIO islands, at least one mainland African ACMV variant likely originated in Madagascar.ConclusionsOur study highlights both the complexity of CMD in Madagascar, and the distinct evolutionary and spatial dynamics of the different viral species that collectively are associated with this disease. Given that more distinct CMG species and recombinants have been found in Madagascar than any other similarly sized region of the world, the risks of recombinant CMG variants emerging on this island are likely to be higher than elsewhere. Evidence of an epidemiological link between Madagascan and mainland African CMGs suggests that the consequences of such emergence events could reach far beyond the shores of this island.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0749-2) contains supplementary material, which is available to authorized users.

Expression of geminiviral AC2 RNA silencing suppressor changes sugar and jasmonate responsive gene expression in transgenic tobacco plants

BackgroundRNA-silencing is a conserved gene regulation and surveillance machinery, which in plants, is also used as major defence mechanism against viruses. Various virus-specific dsRNA structures are recognized by the silencing machinery leading to degradation of the viral RNAs or, as in case of begomoviruses, to methylation of their DNA genomes. Viruses produce specific RNA silencing suppressor (RSS) proteins to prevent these host defence mechanisms, and as these interfere with the silencing machinery they also disturb the endogenous silencing reactions. In this paper, we describe how expression of AC2 RSS, derived from African cassava mosaic geminivirus changes transcription profile in tobacco (Nicotiana tabacum) leaves and in flowers.ResultsExpression of AC2 RSS in transgenic tobacco plants induced clear phenotypic changes both in leaves and in flowers. Transcriptomes of these plants were strongly altered, with total of 1118 and 251 differentially expressed genes in leaves and flowers, respectively. The three most up-regulated transcript groups were related to stress, cell wall modifications and signalling, whereas the three most down-regulated groups were related to translation, photosynthesis and transcription. It appears that many of the gene expression alterations appeared to be related to enhanced biosynthesis of jasmonate and ethylene, and consequent enhancement of the genes and pathways that are regulated by these hormones, or to the retrograde signalling caused by the reduced photosynthetic activity and sugar metabolism. Comparison of these results to a previous transcriptional profiling of HC-Pro RSS-expressing plants revealed that some of same genes were induced by both RSSs, but their expression levels were typically higher in AC2 than in HC-Pro RSS expressing plants. All in all, a large number of transcript alterations were found to be specific to each of the RSS expressing transgenic plants.ConclusionsAC2 RSS in transgenic tobacco plants interferes with the silencing machinery. It causes stress and defence reactions for instance via induction of the jasmonate and ethylene biosynthesis, and by consequent gene expression alteration regulated by these hormones. The changed sugar metabolism may cause significant down-regulation of genes encoding ribosomal proteins, thus reducing the general translation level.

English

Field-infected cassava stems whose leaves were identified with differential symptoms ofAfrican cassava mosaic geminivirus (ACMV) disease were assessed for index of severity of symptoms (ISS) to determine their infection status by scoring young resultant plants in the laboratory. Extracts of young stem tissues and leaves of emergent shoots from topped stems were also assayed for occurrence of ACMV by the enzyme-linked immunosorbent assay (ELISA). Geminivirus disease incidence and severity of symptoms were most abundant on leaves of plants from cuttings of field-symptomatic (I) stems of the moderately-resistant TMS 4(2)1425 (63.2%) and the susceptible TMS 60506 (51.1%) genotypes while those from apparently symptom-free (H) stems of the resistant genotype TMS 30001 recorded significantly the least disease (0%). Similarly, the index of severity of symptoms on all plants (ISSAP) and diseased plants only (ISSDP) were significantly highest on leaves of TMS 4(2)1425 (2.42 and 2.83), and were significantly the least (1) on leaves of TMS 30001, respectively. ACMV was not detected in extracts of all sections of lignified cassava stems by ELISA as all absorbance values were below threshold (0.0890). However, the virus was detected at greater concentrations in leaves of emergent axillary shoots regenerating on topped plants of all node types of TMS 60506 and TMS 4(2)1425 as well as the base (node 1) of TMS 30001 stems but not on those from middle and uppermost nodes (10 and 20, respectively) of the latter genotype. The highest absorbance values were recorded on shoots on node 1 of TMS 60506 (0.1720 ± 0.096), TMS 4(2)1425 (0.1640 ± 0.115) and TMS 30001 (0.1580 ± 0.080) in that order, while the least values were on nodes 10 (0.0298 ± 0.020) and 20 (0.0289 ± 0.019) of TMS 30001.   Key words: African cassava mosaic geminivirus, infection status, cassava genotypes, detection limitations, enzyme-linked-immunosorbent assay.

RNAi dependent epigenetic marks on a geminivirus promoter

Nicotiana benthamiana plants were stably transformed with an intron-spliced dsRNA producing construct cognate to bidirectional promoter of African cassava mosaic geminivirus (ACMV) DNA A. Transgenic lines expressed multiple siRNAs species upon ACMV infection. The de novo DNA methylation and an increased proportion of histone H3 Lysine-9 methylation (H3K9) at intergenic region (IGR) of ACMV DNA A were observed.

Effects of cassava genotype, climate and the Bemisia tabaci vector population on the development of African cassava mosaic geminivirus (ACMV)

A survey was carried out in the 1996/97 and 1997/98 growing seasons on a field planted in three replicates with five clones of cassava at the International Institute of Tropical Agriculture, Ibadan, located in a transition forest, to determine the effects of cassava genotype and climate on the development of African cassava mosaic geminivirus (ACMV) and changes in the Bemisiatabaci population. Cassava genotype, climate and their interactions have significant (P<0.01) effects on the population of B. tabaci and the development of ACMV. The incidence of ACMV was significantly (P<0.01) higher in clones 81/01635 and 92/0520 than in TMS 30572 and 94/0239, while 91/02327 showed the greatest resistance. A positive correlation between the incidence and severity of ACMV was observed, but this did not correlate with the whitefly population density.

Subcellular targeting of the coat protein of African cassava mosaic geminivirus.

The coat protein (CP) of geminiviruses is involved in a number of processes during the life cycle of the virus. The predominant function is encapsidation of single-stranded DNA and formation of the virus particle to protect viral DNA during transmission. The CP of monopartite geminiviruses is absolutely essential for virus movement, whereas CP mutants of bipartite geminiviruses are able to infect some host plants systemically, indicating an involvement of the CP in host specificity. During the life cycle of geminiviruses, the viral DNA enters the nucleus of the infected cell where virus replication, transcription, and encapsidation occur. For systemic infection, the virus moves cell-to-cell from the site of inoculation to vascular tissue and via phloem to other plant tissues. To move, viral DNA has to shuttle in and out of the nucleus and through plasmodesmata. Parts of the bipartite African cassava mosaic virus (ACMV) CP were fused with green fluorescent protein (GFP) or beta-glucuronidase (GUS). CP domains were identified that mediate both nuclear import and export, as well as targeting of CP-fusion proteins to the cell periphery. These results indicate that domains of the CP facilitate several aspects of geminivirus movement, including nuclear import and export and transport of the viral genome to the cell periphery.

Identification of genes directly and indirectly involved in the insect transmission of African cassava mosaic geminivirus by Bemisia tabaci.

The inability to transmit progeny virus resulting from the cloned components of an isolate of African cassava mosaic virus originating from Kenya (ACMV-K) has been shown to be due to defects in both genomic components. This was achieved by the production of infectious pseudorecombinants between ACMV-K and the cloned components of a whitefly-transmissible ACMV isolate originating from Nigeria (ACMV-NOg). The exchange of gene fragments between ACMV-K and ACMV-NOg has been used to demonstrate that the defects responsible for lack of transmissibility reside on the coat protein and DNA B C1 gene of ACMV-K. The significance of these finding with respect to the present understanding of the function of these gene products are discussed.

Recombination between viral DNA and the transgenic coat protein gene of African cassava mosaic geminivirus.

Nicotiana benthamiana was transformed with three different constructs (pCRA1, pCRA2 and pJC1) containing the coat protein coding sequence of African cassava mosaic virus (ACMV). Transformed plants were inoculated with a coat protein deletion mutant of ACMV that induces mild systemic symptoms in control plants. Several inoculated plants of transgenic lines CRA1/3, CRA1/4, CRA2/1 and CRA2/2 developed severe systemic symptoms typical of ACMV. DNA analysis revealed that, in these plants, recombination had occurred between the mutant viral DNA and the integrated construct DNA, resulting in the production of recombinant virus progeny with 'wild-type' characteristics. No reversion of mutant to 'wild-type' virus was observed in pJC1-transformed plants. Recombinant virus from several transgenic plants was analysed by PCR and parts of DNA A of virus progeny were cloned. Sequence analysis revealed that only a few nucleotides were changed from the published sequence.

Survey of cassava virus diseases in Tanzania

A survey was carried out of African cassava mosaic geminivirus and cassava brown streak virus diseases (ACMD and CBSD) in Tanzania. ACMD occurred throughout the country at low to moderate incidences in the fifteen different regions surveyed (1- 64%). The incidence was generally higher along the coastalplain than in higher altitude areas in the interior. There was a significant correlation between numbers ofadults ofthe whitefly vector, Bemisia tabaci, and incidence of recently infected plants ( R = 0.77, p < 0.01), although most infection was attributable to the use of infected cuttings. CBSD was more restricted in distribution and, where present, incidence was low to moderate (1- 36%). Cassava plantings along the southern border with Mozambique link the highest incidence area on the south-east coast near Mtwara, with the other important area ofCBSD incidence along the shore ofLake Malawi. The effects of climatic parameters on ACMD and CBSD incidence are discussed. The survey data suggest that both diseases could be controlled in Tanzania by the use of phytosanitation which involves the use of disease-free planting material and the removal (roguing) of diseased plants.

Formation of Circular Satellite Tobacco Ringspot Virus RNA in Protoplasts Transiently Expressing the Linear RNA

The most abundant form of the satellite RNA of tobacco ringspot virus (sTRSV RNA) is a linear, unit length molecule of 359 nucleotide residues, designated L−(+)M. A postulated replication scheme for the satellite RNA has as its first, and apparently virus-independent, step the ligation of L−(+)M into the corresponding circular form C−(+)M. We transiently expressed L−(+)M wild type and L−(+)M mutants in tobacco protoplasts using an African cassava mosaic geminivirus vector. Measured extents of C−(+)M accumulation were correlated with computer-predicted folding to suggest wild-type secondary structure elements that might be deleted without reducing ligation. A 127-nucleotide residue mutant L−(+)M was created by replacing, with 7 and 3 residues, respectively, nucleotide residues 53–211 and 268–350, each of which was predicted to form a set of three adjacent imperfect stem-loops in wild-type L−(+)M. The mutant L−(+)M was found to be extensively ligated to C−(+)M in protoplasts and to retain a calculated helix of the wild-type molecule that incorporates the 3′ terminal sequence. A trinucleotide in the 3′ region was mutated so as to disrupt and restore, respectively, the calculated helix, reducing and restoring, respectively, C−(+)M formation. These results suggest that the 3′ stem contributes to the suitability of the small L−(+)M molecules as a substrate for a protoplast RNA ligase and that computed folding of sTRSV RNA may be predictive of sTRSV RNA structurein vivo.

Efficient whitefly transmission of African cassava mosaic geminivirus requires sequences from both genomic components.

Clones of two subgroup III geminiviruses, the common strain of tomato golden mosaic virus (csTGMV) and African cassava mosaic virus originating from Kenya (ACMV-K), were shown to be non-transmissible by whitefiles. Lack of transmissibility of cloned ACMV-K was investigated by exchanging genomic components with a whitefly-transmissible ACMV isolate from Nigeria (ACMV-NOg). Neither pseudorecombinant was transmissible, indicating that defects in both genomic components contributed to the lack of transmissibility. Analysis of the acquisition of the pseudorecombinats by Bemisia tabaci indicated that accumulation of virus within the insect was DNA B dependent. Return of virus to plants was determined by DNA A, although the coat protein was essential for acquisition. Repeated passaging of both the wild strain of ACMV-NOg and the cloned virus led to loss of insect transmissibility of the wild isolate but not the cloned virus. Products encoded on both genomic components are required for transmission of bipartite geminiviruses by B. tabaci.

Host‐associated strains within Ugandan populations of the whitefly Bemisia tabaci (Genn.),(Hom., Aleyrodidae)

Abstract: Studies were undertaken to investigate host relationships of populations of Bemisia tabaci taken from the major crop hosts in Uganda. B. tabaci adults taken from cotton and sweet potato did not colonize cassava. None survived for longer than two days and nymphs emerging from eggs laid on cassava died. Cassava B. tabaci in contrast showed limited colonization on cotton and sweet potato and adults emerging from cotton retained the ability to colonize cassava, confirming the genetic basis of the distinction between ‘strains’ suggested by previous isozyme studies. The implications of these findings with regard to the role of B. tabaci in the epidemiology of African cassava mosaic geminivirus are discussed.

Effective ribozyme delivery in plant cells.

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.

Particle purification, properties and epitope variability of Indian tomato leaf curl geminivirus

SUMMARYA whitefly‐transmissible stock isolate of Indian tomato leaf curl geminivirus (ITmLCV) was cultured in graft‐inoculated tomato plants and its particles purified from chloroform‐clarified extracts in citrate buffer by precipitation with 70 g/litre polyethylene glycol, ultracentrifugation and sucrose density gradient centrifugation. Contaminating helical filaments were eliminated by banding in caesium sulphate gradients. ITmLCV particles had the shape typical for geminiviruses, measured c. 30 × 20 nm and contained a single major protein of estimated mol. wt c. 32 000. They reacted in immunosorbent electron microscopy with antisera to four other whitefly‐transmitted geminiviruses. ITmLCV reacted with one out of 17 monoclonal antibodies specific for different epitopes in the particle protein of African cassava mosaic geminivirus and five or six out of 10 monoclonal antibodies to the particle protein of Indian cassava mosaic geminivirus. Virus isolates from tomato at nine locations in Karnataka State showed only slight differences in epitope profile, and isolates from four weed species in tomato fields were similar or identical to those from tomato.