The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection.

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

High-resolution identification and abundance profiling of cassava (Manihot esculenta Crantz) microRNAs.

BACKGROUND: Small RNAs (sRNAs) are endogenous sRNAs that play regulatory roles in plant growth, development, and biotic and abiotic stress responses. In plants, one subset of sRNAs, microRNAs (miRNAs) exhibit tissue-differential expression and regulate gene expression mainly through direct cleavage of mRNA or indirectly via production of secondary phased siRNAs (phasiRNAs) that silence cognate target transcripts in trans. RESULTS: Here, we have identified cassava (Manihot esculenta Crantz) miRNAs using high resolution sequencing of sRNA libraries from leaf, stem, callus, male and female flower tissues. To analyze the data, we built a cassava genome database and, via sequence analysis and secondary structure prediction, 38 miRNAs not previously reported in cassava were identified. These new cassava miRNAs included two miRNAs not previously been reported in any plant species. The miRNAs exhibited tissue-differential accumulation as confirmed by quantitative RT-PCR and Northern blot analysis, largely reflecting levels observed in sequencing data. Some of the miRNAs identified were predicted to trigger production of secondary phased siRNAs (phasiRNAs) from 80 PHAS loci. CONCLUSIONS: Cassava is a woody perennial shrub, grown principally for its starch-rich storage roots, which are rich in calories. In this study, new miRNAs were identified and their expression was validated using qRT-PCR of RNA from five different tissues. The data obtained expand the list of annotated miRNAs and provide additional new resources for cassava improvement research.

Complete genome sequence of a new bipartite begomovirus infecting fluted pumpkin (Telfairia occidentalis) plants in Cameroon.

The complete genome sequence was determined and characterized for a previously unreported bipartite begomovirus from fluted pumpkin (Telfairia occidentalis, family Cucurbitaceae) plants displaying mosaic symptoms in Cameroon. The DNA-A and DNA-B components were ~2.7 kb and ~2.6 kb in size, and the arrangement of viral coding regions on the genomic components was like those characteristic of other known bipartite begomoviruses originating in the Old World. While the DNA-A component was more closely related to that of chayote yellow mosaic virus (ChaYMV), at 78 %, the DNA-B component was more closely related to that of soybean chlorotic blotch virus (SbCBV), at 64 %. This newly discovered bipartite Old World virus is herein named telfairia mosaic virus (TelMV).

Complete genome sequence of a new bipartite begomovirus infecting cotton in the Republic of Benin in West Africa.

Here, we report the complete genome sequence of a novel bipartite begomovirus isolated from cotton plants (Gossypium raimondii, Malvaceae) exhibiting light yellow mosaic symptoms. The genome sequence was determined by Illumina DNA sequencing and confirmed by Sanger sequencing of RCA-enriched, cloned circular genomic components. The DNA-A and DNA-B components were each ~2.7 kb in size, and their genome arrangement was characteristic of other Old World bipartite begomoviruses. While the DNA-A component was most closely related to tobacco leaf curl Comoros virus (TbLCKMV) at 80 %, the DNA-B component had as its closet relative soybean chlorotic blotch virus (SbCBV) at 66 %. This previously undescribed begomovirus is herein named "cotton yellow mosaic virus" (CYMV).

Novel Functional Genomics Approaches: A Promising Future in the Combat Against Plant Viruses.

Advances in functional genomics and genome editing approaches have provided new opportunities and potential to accelerate plant virus control efforts through modification of host and viral genomes in a precise and predictable manner. Here, we discuss application of RNA-based technologies, including artificial micro RNA, transacting small interfering RNA, and Cas9 (clustered regularly interspaced short palindromic repeat-associated protein 9), which are currently being successfully deployed in generating virus-resistant plants. We further discuss the reverse genetics approach, targeting induced local lesions in genomes (TILLING) and its variant, known as EcoTILLING, that are used in the identification of plant virus recessive resistance gene alleles. In addition to describing specific applications of these technologies in plant virus control, this review discusses their advantages and limitations.

An atlas of small RNAs from potato.

Small RNAs, including microRNAs (miRNAs), phased secondary small interfering RNAs (phasiRNA), and heterochromatic small interfering RNAs (hc-siRNA) are an essential component of gene regulation. To establish a broad potato small RNA atlas, we constructed an expression atlas of leaves, flowers, roots, and tubers of Desiree and Eva, which are commercially important potato (Solanum tuberosum) cultivars. All small RNAs identified were observed to be conserved between both cultivars, supporting the hypothesis that small RNAs have a low evolutionary rate and are mostly conserved between lineages. However, we also found that a few miRNAs showed differential accumulation between the two potato cultivars, and that hc-siRNAs have a tissue specific expression. We further identified dozens of reproductive and non-reproductive phasiRNAs originating from coding and noncoding regions that appeared to exhibit tissue-specific expression. Together, this study provides an extensive small RNA profiling of different potato tissues that might be used as a resource for future investigations.

Development and Adoption of Genetically Engineered Plants for Virus Resistance: Advances, Opportunities and Challenges.

Plant viruses cause yield losses to crops of agronomic and economic significance and are a challenge to the achievement of global food security. Although conventional plant breeding has played an important role in managing plant viral diseases, it will unlikely meet the challenges posed by the frequent emergence of novel and more virulent viral species or viral strains. Hence there is an urgent need to seek alternative strategies of virus control that can be more readily deployed to contain viral diseases. The discovery in the late 1980s that viral genes can be introduced into plants to engineer resistance to the cognate virus provided a new avenue for virus disease control. Subsequent advances in genomics and biotechnology have led to the refinement and expansion of genetic engineering (GE) strategies in crop improvement. Importantly, many of the drawbacks of conventional breeding, such as long lead times, inability or difficulty to cross fertilize, loss of desirable plant traits, are overcome by GE. Unfortunately, public skepticism towards genetically modified (GM) crops and other factors have dampened the early promise of GE efforts. These concerns are principally about the possible negative effects of transgenes to humans and animals, as well as to the environment. However, with regards to engineering for virus resistance, these risks are overstated given that most virus resistance engineering strategies involve transfer of viral genes or genomic segments to plants. These viral genomes are found in infected plant cells and have not been associated with any adverse effects in humans or animals. Thus, integrating antiviral genes of virus origin into plant genomes is hardly unnatural as suggested by GM crop skeptics. Moreover, advances in deep sequencing have resulted in the sequencing of large numbers of plant genomes and the revelation of widespread endogenization of viral genomes into plant genomes. This has raised the possibility that viral genome endogenization is part of an antiviral defense mechanism deployed by the plant during its evolutionary past. Thus, GM crops engineered for viral resistance would likely be acceptable to the public if regulatory policies were product-based (the North America regulatory model), as opposed to process-based. This review discusses some of the benefits to be gained from adopting GE for virus resistance, as well as the challenges that must be overcome to leverage this technology. Furthermore, regulatory policies impacting virus-resistant GM crops and some success cases of virus-resistant GM crops approved so far for cultivation are discussed.

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.

Role of a geminivirus AV2 protein putative protein kinase C motif on subcellular localization and pathogenicity.

Virus-derived genes or genome fragments are increasingly being used to generate transgenic plants with resistance to plant viruses. There is need to rapidly investigate these genes in plants using transient expression prior to using them as transgenes since they may be pathogenic to plants. In this study, we investigated the AV2 protein encoded by East African cassava mosaic Cameroon virus, a virus associated with a cassava disease epidemic in western Africa. For subcellular localization, AV2 was fused to the yellow fluorescent protein (YFP) and expressed in Nicotiana benthamiana. Confocal analyses showed that AV2-YFP localizes mainly in the cytoplasm. Because it overlaps with the coat protein gene and therefore could be used to generate transgenic plants for resistance to geminiviruses, we investigated its pathogenesis in N. benthamiana by using the Potato virus X (PVX) vector. The chimeric virus PVX-AV2 induced a mild mottling in infected plants and was shown to suppress virus-induced gene silencing (VIGS). Using point mutations, we show here that AV2 pathogenicity is dependent on a conserved putative protein kinase C (PKC) phosphorylation motif. Because of its pathogenicity and ability to suppress RNA silencing, AV2 transgenic plants will less likely provide a control to geminiviruses, indeed it may weaken the resistance of the plant. We therefore suggest the use of the AV2 putative PKC mutants to generate transgenic plants.

The consensus N-myristoylation motif of a geminivirus AC4 protein is required for membrane binding and pathogenicity.

Some geminiviruses encode a small protein, AC4, whose role in pathogenesis has only recently attracted attention. A few studies have shown that this protein is involved in pathogenesis and suppresses RNA silencing. Here, using Nicotiana benthamiana, we show that East African cassava mosaic Cameroon virus (EACMCV) AC4 is a pathogenicity determinant and that it suppresses the systemic phase of RNA silencing. Furthermore, confocal imaging analyses show that it binds preferentially to the plasma membrane as well as to cytosolic membranes including the perinucleus but is excluded from the nucleus. A computational examination of the AC4 protein encoded by the EACMCV, a bipartite geminivirus, shows that it encodes a consensus N-myristoylation motif and is likely posttranslationally myristoylated and palmitoylated. Replacement of Gly-2 and Cys-3 (sites of posttranslational attachment of myristic and palmatic acids, respectively) with alanine affected AC4 membrane binding and pathogenesis. Furthermore, replacement of Ile-5, a nonessential myristoylation residue, with alanine did not affect AC4 function. Together, these data indicate that EACMCV AC4 is likely dually acylated at Gly-2 and Cys-3 and that these modifications are intrinsic signals for membrane targeting and pathogenesis. This is the first report of a membrane protein to be involved in pathogenesis and RNA silencing suppression.

The Search for Resistance to Cassava Mosaic Geminiviruses: How Much We Have Accomplished, and What Lies Ahead.

The cassava mosaic disease (CMD), which occurs in all cassava growing regions of Africa and the Indian subcontinent, is caused by cassava mosaic geminiviruses (CMGs). CMGs are considered to be the most damaging vector-borne plant pathogens. So far, the most successful approach used to control these viruses has been the transfer of a polygenic recessive resistance locus, designated CMD1, from wild cassava to cassava cultivars. Further progress in harnessing natural resistance to contain CMGs has come from the discovery of the dominant monogenic resistance locus, CMD2, in some West African cassava cultivars. CMD2 has been combined with CMD1 through genetic crosses. Because of the limitations of the cassava breeding approach, especially with regard to time required to produce a variety and the loss of preferred agronomic attributes, efforts have been directed toward the deployment of genetic engineering approaches. Most of these approaches have been centered on RNA silencing strategies, developed mainly in the model plant Nicotiana benthamiana. Early RNA silencing platforms assessed for CMG resistance have been use of viral genes for co-suppression, antisense suppression or for hairpin RNAs-mediated gene silencing. Here, progress and challenges in the deployment of these approaches in the control of CMGs are discussed. Novel functional genomics approaches with potential to overcome some of the drawbacks of the current strategies are also discussed.

Somatic embryogenesis and plant regeneration of cassava (Manihot esculenta Crantz) landraces from Cameroon.

A procedure to regenerate cassava (Manihot esculenta Crantz) cultivars from Cameroon via somatic embryogenesis (SE) was developed. Shoot apical meristems and immature leaf lobes were used as explants on Murashige and Skoog (MS) basal medium containing 33 or 50 µM of the auxins Picloram (Pic), 2,4-Dichlorophenoxyacetic acid (2,4-D), Dicamba (Dic), and α-Naphthalene acetic acid. Cultivar performance was assessed using SE and number of somatic embryos produced. Overall, the frequency of primary somatic embryogenesis (PSE) and the mean number of somatic embryos produced varied considerably with genotype, type of auxin and concentration tested. For example, cultivar (cv.) Ngan Mbada showed the best performance on MS medium supplemented with 50 µM Pic with a SE frequency of 40 % and an average number of somatic embryos of 90. The second best performance was recorded in cv. Local Red on MS medium supplemented with 33 µM 2,4-D, where the SE frequency was 40 % and an average number of somatic embryos of 60.5. Cultivar Ekona Red recorded the best performance on medium supplemented with 50 µM Pic showing a SE frequency of 47 % and an average number of somatic embryos of 45. We further examined secondary and cyclic somatic embryogenesis (SSE, CSE) and both were also observed to vary with genotype, however, both exhibited significantly higher frequencies of SE compared with PSE. SE started to decline at the fourth cycle of embryogenesis. Examination of organogenesis showed that shoot bud induction from green cotyledons varied across cultivars and benzylaminopurine was shown to outperform Thidiazuron in the ability to induce organogenesis. Furthermore, the frequencies of bud induction were identical under light and dark conditions. Finally, regenerated plants grew easily in the greenhouse with 90-100 % survival rate and did not display detectable variation in morphology.

Geminivirus protein structure and function.

Geminiviruses are a family of plant viruses that cause economically important plant diseases worldwide. These viruses have circular single-stranded DNA genomes and four to eight genes that are expressed from both strands of the double-stranded DNA replicative intermediate. The transcription of these genes occurs under the control of two bidirectional promoters and one monodirectional promoter. The viral proteins function to facilitate virus replication, virus movement, the assembly of virus-specific nucleoprotein particles, vector transmission and to counteract plant host defence responses. Recent research findings have provided new insights into the structure and function of these proteins and have identified numerous host interacting partners. Most of the viral proteins have been shown to be multifunctional, participating in multiple events during the infection cycle and have, indeed, evolved coordinated interactions with host proteins to ensure a successful infection. Here, an up-to-date review of viral protein structure and function is presented, and some areas requiring further research are identified.

Molecular interaction between two cassava geminiviruses exhibiting cross-protection.

There are increasing reports of geminivirus mixed infections of field plant hosts. These mixed infections have been suggested to result in recombinations, emergence of new viruses and new disease epidemics. We previously reported the occurrence of mixed infection between African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) resulting in severe symptoms in cassava fields in Cameroon. Here, we show that reassortment of DNA-A and DNA-B components of ACMV and EACMCV does not form viable recombinants. However, in the presence of both components of either virus, the DNA-A component of the other virus replicated and spread in the absence of its DNA-B component. This result suggests that failure of ACMV and EACMCV to form viable recombinants is due to the inability of each DNA-A component to trans-replicate the heterologous DNA-B component. This study also shows that ACMV DNA-A induces a resistance to ACMV and EACMCV as indicated by absence or late symptom development. Moreover, this resistance enabled plants to recover from severe symptoms caused by EACMCV in Nicotiana benthamiana, suggesting that the resistance induced is not specific to ACMV and is consistent with the phenomenon of cross-protection between related viruses.

Genetic variability of East African cassava mosaic Cameroon virus under field and controlled environment conditions.

Cassava geminiviruses occur in all cassava growing areas of Africa and are considered to be the most damaging vector-borne plant pathogens. At least seven species of these viruses have been identified. We investigated genetic variation in East African cassava mosaic cassava Cameroon virus (EACMCV) from naturally infected cassava and from experimentally infected Nicotiana benthamiana. Results showed that the populations of EACMCV in cassava and in N. benthamiana were genetically heterogeneous. Mutation frequencies in the order of 10(-4), comparable to that reported for plant RNA viruses, were observed in both hosts. We also produced an EACMCV mutant that induces reversion and second site mutations, thus suggesting that a high mutation frequency facilitates the maintenance of genome structure and function. This is direct experimental evidence showing that cassava geminiviruses exhibit a high mutation frequency and that a single clone quickly transforms into a collection of mutant sequences upon introduction into the host.

Characterization of the cassava geminivirus transcription activation protein putative nuclear localization signal.

The geminivirus transcription activation protein (TrAP) localizes to the nucleus and contains a putative nuclear localization signal (NLS) ((28)PRRRR(32)) on the N-terminus. The role of individual residues of this putative NLS on nuclear localization and symptom induction was investigated using TrAP of East African cassava mosaic Cameroon virus (EACMCV). Subcellular localization was conducted using the green fluorescent protein (GFP). Results showed that the proline residue at position 28 (Pro-28) is essential for nuclear localization whereas individually, none of the four contiguous arginines is necessary for nuclear targeting. The role of each of the five NLS amino acid residues on TrAP-mediated disease phenotype and gene silencing suppression was investigated by expressing these mutants in Nicotiana benthamiana from the PVX vector and under the control of the Cauliflower mosaic virus 35S promoter. Results showed that all five residues of the NLS play a role on disease phenotype production in N. benthamiana plants. Furthermore, each of the NLS residues appeared to be required for suppression of VIGS but appeared not to be required for the ability of TrAP to transactivate transcription and interact with adenosine kinase (ADK).