Vincent N. Fondong

Molecular evidence for five distinct Bemisia tabaci (Homoptera: Aleyrodidae) geographic haplotypes associated with cassava plants in Sub-Saharan Africa

Abstract The Bemisia tabaci (Gennadius) complex contains the only known whitefly vector of plant-infecting begomoviruses, which are the causal agents of mosaic diseases of cassava in Africa and India. Widespread phenotypic variability, together with the absence of definitive morphological taxonomic characters for this whitefly complex, has confounded both the systematics and the study of its virus vector biology. Substantial genetic variability and phylogeographical relationships have been shown for phenotypic, but morphologically identical, variants of B. tabaci based on the mitochondrial (mt) cytochrome oxidase I (COI) sequence, leading to the suggestion that they represent a species complex. Here, phylogenetic relationships were explored, using the mtCOI sequence (780 bp) as a molecular marker, for B. tabaci collected from cassava plants in southern and western Africa, including Cameroon, Zambia, Mozambique, Zimbabwe, Swaziland, and South Africa. Maximum likelihood analyses of mtCOI sequences revealed that most B. tabaci examined were placed into one of three subgroups within the major sub-Saharan African clade, which also contains previously reported populations indigenous to Malawi and Uganda, and collectively shared on overall nucleotide (nt) identity at 88.9–99.7%. Two other reference populations, the monophagous Benin haplotype from Asystasia spp. and a B. tabaci from cassava in the Ivory Coast (IC), were the most divergent outliers of the sub-Saharan clade, each representing the only member of their respective clade (I and V), at the present time. Members of the sub-Saharan clade associated with cassava had as their closest relatives haplotypes I and II of the Mediterranean-Northern Africa clade, with which they shared a collective 84.2–92.9% nt identity (not including the IC cassava reference haplotype). In contrast, the sub-Saharan African clade diverged from the Americas and Southeast Asia/Far East clades at 79.7–85.1 and 77.5–84.9%, respectively. Within the sub-Saharan clade, subclade II contained B. tabaci from Zambia, Mozambique, South Africa, and Swaziland at 95–99% identity. The sub-Saharan subcluster III contained haplotypes from southern and western Africa. Counter to the otherwise phylogeographical relationships observed for cassava-associated B. tabaci from southern Africa, one and two populations from Cameroon (okra) and Zimbabwe (cassava), respectively, grouped with the major Mediterranean-North Africa clade, together with their closest relative associated with okra from IC, are included here as a reference sequence for the first time, with which they collectively formed a new, third subclade. Thus, phylogenetic analysis of B. tabaci mtCOI haplotypes examined thus far from the African continent has revealed five major cassava-associated haplotypes, which grouped primarily based on extant geography, with the exception of one and two collections from Cameroon and Zimbabwe, respectively. Hypotheses explaining the potential distributions of haplotypes are discussed.

Geminivirus protein structure and function

Summary 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.

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.

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.

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).

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.

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.

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

BackgroundSmall 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.ResultsHere, 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.ConclusionsCassava 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.

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.

Molecular characterization and experimental host range of an isolate of Macroptilium golden mosaic virus that infects Wissadula amplissima in Jamaica

Partial genome sequences for the tentative begomovirus Macroptilium golden mosaic virus (MGMV) have been previously reported and were originally obtained for an isolate that infected Macroptilium lathyroides in Jamaica. In this study, we PCR-amplified, cloned and determined the sequence for the complete genome of isolates of MGMV that we found infecting Wissadula amplissima collected from August Town and Spanish Town, Jamaica. Sequence analysis confirmed that MGMV is a distinct begomovirus species, based on the ICTV 89% rule for species demarcation. MGMV shared its highest nucleotide identity at 79% for DNA-A component and 66% for DNA-B component to Corchorus yellow spot virus [Mexico:Yucatan:2005]. The names Macroptilium golden mosaic virus [Jamaica1:Wissadula:AugustTown] (MGMV [JM1:Wd:AT]) and Macroptilium golden mosaic virus [Jamaica1:Wissadula:SpanishTown] (MGMV [JM1:Wd:ST]) are proposed herein for the MGMV isolates from August Town and Spanish Town, respectively. The genome organization of MGMV [JM1:Wd:AT] and MGMV [JM1:Wd:ST] is characteristic of Western Hemisphere bipartite begomoviruses. Excluding the replication enhancer protein (REn), all proteins encoded by the MGMV [JM1:Wd:AT] and MGMV [JM1:Wd:ST] genomes are most similar to their counterparts in Western Hemisphere begomoviruses. The REn proteins of MGMV [JM1:Wd:AT] and MGMV [JM1:Wd:ST], share greatest similarity to the REn protein of Corchorus yellow vein virus [Vietnam:Hoa Binh:2000], a New World-like begomovirus identified in Asia. Phylogenetic reconstruction places MGMV in a clade containing Potato yellow mosaic virus. Results of an experimental host range study indicated that MGMV [JM1:Wd:AT] can infect kidney bean, hot pepper and tomato.