The essential role of clathrin-mediated endocytosis and early endosomes in the trafficking of begomoviruses through the primary salivary glands of their whitefly vectors.

IMPORTANCE: Many plant viruses are transmitted by insect vectors in a circulative manner. For efficient transmission, the entry of the virus from vector hemolymph into the primary salivary gland (PSG) is a step of paramount importance. Yet, vector components mediating virus entry into PSG remain barely characterized. Here, we demonstrate the role of clathrin-mediated endocytosis and early endosomes in begomovirus entry into whitefly PSG. Our findings unravel the key components involved in begomovirus transport within the whitefly body and transmission by their whitefly vectors and provide novel clues for blocking begomovirus transmission.

Phylogenetic and recombination analysis of Begomoviruses associated with Cotton leaf curl disease and in silico analysis of viral-host protein interactions.

Cotton leaf curl disease (CLCuD), caused by numerous begomoviruses (BGVs), is a highly disastrous disease in cotton crops worldwide. To date, several efforts have shown limited success in controlling this disease. CLCuD-associated BGVs (CABs) are known for their high rate of intra and interspecific recombinations, which raises an urgent need to find an efficient and conserved target region to combat disease. In the present study, phylogenetic analysis of selected 11 CABs, along with associated alphasatellites, and betasatellites revealed a close evolutionary relationship among them. Recombination analysis of 1374 isolates of CABs revealed 54 recombination events for the major players of CLCuD in cotton and the Cotton leaf curl Multan virus (CLCuMuV) as the most recombinant CAB. Recombination breakpoints were frequent in all regions except C2 and C3. C3-encoded protein, known as viral replication enhancer (REn), promotes viral replication by enhancing the activity of replicase (Rep) protein. Both proteins were found to contain significantly conserved domains and motifs. The identified motifs were found crucial for their interaction with host protein PCNA (Proliferating cell nuclear antigen), facilitating viral replication. Interruption at the REn-PCNA and Rep-PCNA interactions by targeting the identified conserved motifs is proposed as a prospect to halt viral replication, after suitable experimental validation.

The Role of Extensive Recombination in the Evolution of Geminiviruses.

Mutation, recombination and pseudo-recombination are the major forces driving the evolution of viruses by the generation of variants upon which natural selection, genetic drift and gene flow can act to shape the genetic structure of viral populations. Recombination between related virus genomes co-infecting the same cell usually occurs via template swapping during the replication process and produces a chimeric genome. The family Geminiviridae shows the highest evolutionary success among plant virus families, and the common presence of recombination signatures in their genomes reveals a key role in their evolution. This review describes the general characteristics of members of the family Geminiviridae and associated DNA satellites, as well as the extensive occurrence of recombination at all taxonomic levels, from strain to family. The review also presents an overview of the recombination patterns observed in nature that provide some clues regarding the mechanisms involved in the generation and emergence of recombinant genomes. Moreover, the results of experimental evolution studies that support some of the conclusions obtained in descriptive or in silico works are summarized. Finally, the review uses a number of case studies to illustrate those recombination events with evolutionary and pathological implications as well as recombination events in which DNA satellites are involved.

A Tomato-Free Period Delays and Reduces Begomovirus Disease in Processing Tomato Fields in a Complex Agroecosystem in Central Brazil.

A mandatory tomato-free period (TFP) was implemented in the state of Goiás, Brazil, in 2007 to help manage diseases caused by whitefly-transmitted begomoviruses. The impact of the TFP was examined in five locations across three states in Central Brazil from 2013 to 2016. Surveys revealed significant differences in begomovirus disease incidence among locations, i.e., low in Guaíra-TFP and Patos de Minas-TFP; moderate-high in Itaberaí-TFP and Morrinhos-TFP; and high in the non-TFP (NTFP) control, Cristalina-NTFP. PCR tests and DNA sequencing were used to validate the symptoms and showed that all collected symptomatic plant samples were infected with tomato severe rugose virus (ToSRV), a common indigenous bipartite begomovirus. Early season surveys (20 to 40 days after transplants [DAT]) in Itaberaí-TFP and Morrinhos-TFP revealed significantly less begomovirus disease in fields established sooner after the TFP (0 to 2 months) compared with incidences in (i) equivalent early planted fields in the Cristalina-NTFP control and (ii) fields established longer after the end of the TFP (>2 to 5 months). Whitefly infestation of crops was detected year-round in all locations and years, and all tested adults were classified in the Bemisia tabaci MEAM1 cryptic species. Infestation levels were significantly higher during the summer but did not vary significantly among locations. Results of monthly monitoring of adult whiteflies for general begomovirus and ToSRV were positively correlated and were indicators of disease incidence in the field. Notably, ToSRV was not detected in whiteflies collected from nontomato plants during the TFP, and there was a longer lag period before detection in whiteflies collected from processing tomatoes for Itaberaí-TFP and Morrinhos-TFP compared with Cristalina-NTFP. Taken together with the low levels of ToSRV infection detected in potential nontomato reservoir hosts at all locations, our results revealed low levels of primary inoculum during the TFP. Thus, even in a complex agroecosystem with year-round whitefly infestation of crops, the TFP was beneficial due to delayed and reduced begomovirus disease pressure during a critical stage of plant development (first month) and for favoring low levels of primary inoculum. Thus, we concluded that the TFP should be part of a regional integrated pest management (IPM) program targeting ToSRV in Brazil.

Unraveling different begomoviruses, DNA satellites and cryptic species of Bemisia tabaci and their endosymbionts in vegetable ecosystem.

Bemisia tabaci species complex contains more than 46 cryptic species. It has emerged as an important pest causing significant yield loss in many cultivated crops. This pest is also a vector for more than 100 species of begomoviruses, that are a major threat for the cultivation of many crops in different regions of the world. The relation between cryptic species of the B. tabaci species complex and associated begomoviruses that infect different crops remains unclear. In the present study, four cryptic species (Asia I, China 3, Asia II 5 and Asia II-1) of B. tabaci and four associated endosymbionts (Arsenophonus, Cardinium, Rickettsia and Wolbachia) were identified in different vegetable crops. The vector-based PCR detection revealed five different begomoviruses such as okra enation leaf curl virus (OELCuV), tomato leaf curl Palampur virus (ToLCPalV), squash leaf curl China virus (SLCCNV), chilli leaf curl virus (ChiLCuV), and tomato leaf curl New Delhi virus (ToLCNDV). Of these begomoviruses, the maximum infection rate was observed (9.1%) for OELCuV, followed by 7.3% for ToLCNDV. The infection rate of the other three viruses (SLCCNV, ChiLCuV, ToLCPalV) ranged from 0.9 to 2.7% in cryptic species of B. tabaci. Further, each cryptic species was infected with multiple virus species and the virus infection rate of Asia I, Asia II-5, China 3 and Asia II-1 was 21.2%, 15.1%, 15.1% and 0.6% respectively. Similarly, in case of betasatellites the highest infection rate was 12% for ToLCBDB, followed by 6% for OLCuB and PaLCB. With regard to alphasatellites, the highest infection rate was 18.2% for AEV and 3% for CLCuMuA. This study demonstrates the distribution of cryptic species of whitefly and their endosymbionts, and associated begomoviruses and DNA satellites in vegetable ecosystem. We believe that the information generated here is useful for evolving an effective pest management strategies for vegetable production.

A C2H2 zinc finger transcription factor of the whitefly Bemisia tabaci interacts with the capsid proteins of begomoviruses and inhibits virus retention.

Begomoviruses are a group of ssDNA viruses exclusively transmitted by the whitefly Bemisia tabaci and constrain vegetable production in the old and new worlds. Although multiple molecular determinants governing the transmission of begomoviruses by whiteflies have been unravelled, factors critical for transmission majorly remain unknown. In this study, a whitefly C2H2 zinc finger (ZF) protein, 100% identical to the vascular endothelial ZF-like gene (vezf) protein was confirmed to interact with the CP of both old- and new-world begomoviruses. This was achieved by a yeast two-hybrid (Y2H) system screening of a whitefly cDNA library using capsid protein (CP) of TYLCV as a bait. In silico annotation of vezf protein revealed that it contains a N-terminal ZF-associated domain (ZAD) alongside multiple C2H2 ZF domains on the C-terminal end. ZAD-ZF proteins form the most abundant class of transcription factors within insects. Herein, we validated the interaction of vezf with four diverse begomoviruses and its functional role in begomovirus transmission. Silencing of the vezf gene of B. tabaci led to increased retention of three diverse begomoviruses tested. Vezf is the first insect transcription factor identified to interact with plant viruses and can be crucial to understand the possible mechanisms by which plant viruses modulate transcription of their insect vectors during transmission.

Identification of a Major-Effect Quantitative Trait Loci Associated with Begomovirus Resistance in Cucurbita moschata.

Begomoviruses, viz. squash leaf curl China virus and tomato leaf curl New Delhi virus causative diseases are major concerns of quantitative and qualitative losses in pumpkin (Cucurbita moschata) worldwide. Punjab Agricultural University (PAU) in India has identified a resistant source (PVR-1343) against mixed infection (MI-Sq/To) of these begomoviruses. Introgression of resistance in diverse genetic backgrounds requires the identification of quantitative trait loci (QTLs) associated with MI-Sq/To resistance. Phenotyping of 229 F2:3 progenies derived from the PVR-1343 × P-135 cross revealed digenic recessive inheritance against MI-Sq/To resistance in PVR-1343. To identify the genomic region, resistant and susceptible bulks were subjected to whole-genome resequencing along with their parents. The whole-genome resequence analysis of parents and bulks using QTLseq/QTLseqr approaches identified an overlapping 1.52 Mb region on chromosome 7 (qMI-Sq/To7.1), while chromosomal region spanning 0.87 Mb on chromosome17 (qMI-Sq/To17.1) was additionally identified by QTLseqr. However, the highest peak value on chromosome 7 with three algorithms {G', ∆(SNP-index) and -log10 (P value)} highlighted the major contribution of qMI-Sq/To7.1 in MI-Sq/To resistance. Nine polymorphic SNPs identified within the highly significant qMI-Sq/To7.1 region were converted into KASP markers. KASP genotyping of F2 individuals narrowed down the qMI-Sq/To7.1 interval to 103 kb region flanked by two markers, Cmo3914729 and Cmo4018182, which contained 16 annotated genes and accounted for 59.84% of phenotypic variation. The Cmo4018182 KASP marker accurately predicted disease reaction in 91% of diverse Cucurbita genotypes and showed nonsynonym substitutions in the coding region of putative candidate SYNTAXIN-121 gene. These findings pave the way for marker-assisted breeding and elucidating the underlying mechanism of begomovirus resistance in C. moschata.

Characteristics of the Complete Genome of Ageratum Yellow Vein China Virus Infecting Sonchus oleraceus.

Sonchus (Sonchus oleraceus) originated from Europe and is now cultivated worldwide. The wild resources of sonchus are very abundant, and it has rich nutritional and medicinal value. In this study, 15 sonchus samples with typical symptoms showing leaf curling, vein thickening, and enations were collected from Guigang and Baise City of Guangxi, China. Diseased sonchus were identified by PCR detection, whole genome sequence amplification, and phylogenetic and recombination analysis. The results showed that all the samples were confirmed infected by begomoviruses, and three full-length viral genomes were obtained from 15 sonchus, named GG7-13, GG8-6, and BS63-5. The full genome lengths were 2,584, 2,735, and 2,746 nt, respectively. The nucleotide identities among the three isolates ranged from 92.67 to 99.93%. All of them shared the highest identities (greater than 91.69%) with other isolates of ageratum yellow vein China virus (AYVCNV) (available on GenBank). According to the guidelines of classification of begomoviruses, the virus isolates obtained in this study are different isolates of AYVCNV; a phylogenetic tree analysis showed that these isolates formed a large branch with three other Guangxi isolates of AYVCNV, indicating their close evolution. The genome structures of GG8-6 and BS63-5 are consistent with the monopartite genome virus of the begomoviruses, and both have six open reading frames (ORFs), while GG7-13 has a 151-nt deletion between C2 and C3, resulting in a mutant strain of only five ORFs. This study is the first report on S. oleraceus infected by ageratum yellow vein China virus.

Cassava mosaic disease and its management in Southeast Asia.

KEY MESSAGE: Status of the current outbreak of cassava mosaic disease (CMD) in Southeast Asia was reviewed. Healthy cassava seed production and dissemination systems have been established in Vietnam and Cambodia, along with integrated disease and pest management systems, to combat the outbreak. Cassava (Manihot esculenta Crantz) is one of the most important edible crops in tropical and subtropical regions. Recently, invasive insect pests and diseases have resulted in serious losses to cassava in Southeast Asia. In this review we discuss the current outbreak of cassava mosaic disease (CMD) caused by the Sri Lankan cassava mosaic virus (SLCMV) in Southeast Asia, and summarize similarities between SLCMV and other cassava mosaic begomoviruses. A SATREPS (Science and Technology Research Partnership for Sustainable Development) project "Development and dissemination of sustainable production systems based on invasive pest management of cassava in Vietnam, Cambodia and Thailand", was launched in 2016, which has been funded by The Japan International Cooperation Agency (JICA) and The Japan Science and Technology Agency (JST), Japan. The objectives of SATREPS were to establish healthy seed production and dissemination systems for cassava in south Vietnam and Cambodia, and to develop management systems for plant diseases and insect pests of cassava. To achieve these goals, model systems of healthy seed production in Vietnam and Cambodia have been developed incorporating CMD-resistant planting materials through international networks with The International Center for Tropical Agriculture (CIAT) and The International Institute of Tropical Agriculture (IITA).

Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation.

Cassava mosaic disease (CMD) represents a serious threat to cassava, a major root crop for more than 300 million Africans. CMD is caused by single-stranded DNA begomoviruses that evolve rapidly, making it challenging to develop durable disease resistance. In addition to the evolutionary forces of mutation, recombination and reassortment, factors such as climate, agriculture practices and the presence of DNA satellites may impact viral diversity. To gain insight into the factors that alter and shape viral diversity in planta, we used high-throughput sequencing to characterize the accumulation of nucleotide diversity after inoculation of infectious clones corresponding to African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in the susceptible cassava landrace Kibandameno. We found that vegetative propagation had a significant effect on viral nucleotide diversity, while temperature and a satellite DNA did not have measurable impacts in our study. EACMCV diversity increased linearly with the number of vegetative propagation passages, while ACMV diversity increased for a time and then decreased in later passages. We observed a substitution bias toward C→T and G→A for mutations in the viral genomes consistent with field isolates. Non-coding regions excluding the promoter regions of genes showed the highest levels of nucleotide diversity for each genome component. Changes in the 5' intergenic region of DNA-A resembled the sequence of the cognate DNA-B sequence. The majority of nucleotide changes in coding regions were non-synonymous, most with predicted deleterious effects on protein structure, indicative of relaxed selection pressure over six vegetative passages. Overall, these results underscore the importance of knowing how cropping practices affect viral evolution and disease progression.

Two different begomovirus species are associated with yellow vein mosaic disease of okra in Sri Lanka.

Yellow vein mosaic disease is the major biotic constraint of okra cultivation in Sri Lanka. Identification and detailed molecular characterization of associated pathogen is needed for effective disease management. The genome of the begomovirus and betasatellite were amplified in symptomatic plant samples using specific degenerate primers. DNA-A genome of twelve isolates representing different locations in Sri Lanka were cloned, sequenced and deposited in GenBank database (Accession No- KX698087- KX698092 and MH455207- MH455212). Size of the complete nucleotide sequences ranged from 2735 to 2786 bp. The genome organization showed characteristics of begomoviruses. The pairwise sequence identity revealed the association of two different begomovirus species. Five of the isolates showed > 91% of sequences identity with Bhendi yellow vein mosaic virus, and the rest of the seven isolates were around 92% of identity with Okra enation leaf curl virus. This is further supported by phylogenetic analysis where both of these group of isolates were in different cluster. Recombination analysis showed the presence of recombinant fragments in the virus isolates associated with okra yellow vein mosaic disease (OYVMD) in Sri Lanka. Attempts to amplify DNA- B were failed in any of the samples tested. However, both type of the begomovirus species associated with betasatellite species, Bhendi yellow vein mosaic betasatellite. The present study has revealed the association of two distinct monopartite begomovirus species, Bhendi yellow vein mosaic virus or Okra enation leaf curl virus, with OYVMD in Sri Lanka.

Generation and Characterization of Nanobodies Against Tomato Leaf Curl Sudan Virus.

Begomoviruses infect food, fiber, and vegetable crop plants, including tomato, potato, bean, cotton, cucumber, and pumpkin, and damage many economically important crop plants worldwide. Tomato leaf curl Sudan virus (ToLCSDV) is the most widespread tomato-infecting begomovirus in Saudi Arabia. Using phage display technology, this study isolated two camel-derived nanobodies against purified ToLCSDV virions from a library of antigen-binding fragments (VHH or nanobody) of heavy-chain antibodies built from an immunized camel. The isolated nanobodies also cross-reacted with purified tomato yellow leaf curl virus virions and showed significant enzyme-linked immunosorbent assay reactivity with extracts from plants with typical begomovirus infection symptoms. The results can pave the way to developing diagnostics for begomovirus detection, design, and characterization of novel nanomaterials based on virus-like particles, in addition to nanobody-mediated begomovirus resistance in economically important crops, such as tomato, potato, and cucumber.

Transmission of Begomoviruses and Other Whitefly-Borne Viruses: Dependence on the Vector Species.

Most plant viruses require a biological vector to spread from plant to plant in nature. Among biological vectors for plant viruses, hemipteroid insects are the most common, including phloem-feeding aphids, whiteflies, mealybugs, planthoppers, and leafhoppers. A majority of the emerging diseases challenging agriculture worldwide are insect borne, with those transmitted by whiteflies (Hemiptera: Aleyrodidae) topping the list. Most damaging whitefly-transmitted viruses include begomoviruses (Geminiviridae), criniviruses (Closteroviridae), and torradoviruses (Secoviridae). Among the whitefly vectors, Bemisia tabaci, now recognized as a complex of cryptic species, is the most harmful in terms of virus transmission. Here, we review the available information on the differential transmission efficiency of begomoviruses and other whitefly-borne viruses by different species of whiteflies, including the cryptic species of the B. tabaci complex. In addition, we summarize the factors affecting transmission of viruses by whiteflies and point out some future research prospects.

African Basil (Ocimum gratissimum) Is a Reservoir of Divergent Begomoviruses in Uganda.

Begomoviruses are plant viruses that cause major losses to many economically important crops. Although they are poorly understood, begomoviruses infecting wild plants may have an important role as reservoirs in the epidemiology of viral diseases. This study reports the discovery and genomic characterization of three novel bipartite begomoviruses from wild and cultivated African basil (Ocimum gratissimum) plants collected in Uganda, East Africa. Based on the symptoms shown by the infected plants, the names proposed for these viruses are Ocimum yellow vein virus (OcYVV), Ocimum mosaic virus (OcMV), and Ocimum golden mosaic virus (OcGMV). Genome and phylogenetic analyses suggest that DNA-A of OcGMV is mostly related to begomoviruses infecting tomato in Africa, whereas those of OcYVV and OcMV are closely related to one another and highly divergent within the Old World begomoviruses. The DNA-A of all characterized begomovirus isolates are of a recombinant nature, revealing the role of recombination in the evolution of these begomoviruses. The viruses characterized here are the first identified in O. gratissimum and the first in Ocimum spp. in the African continent and could have important epidemiological consequences for cultivated basils and other important crops.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

A thioredoxin-like protein of Bemisia tabaci interacts with coat protein of begomoviruses.

Bemisia tabaci (whitefly) is the sole vector of begomoviruses, which transmits them in a persistent and circulative manner from infected to healthy plants. During this process, begomoviruses interact with various proteins in the insect vector B. tabaci that would play a specific role in the virus transmission. Identification and characterization of such proteins are important to understand the complete process of virus transmission. Coat protein (CP) of begomoviruses is the only protein which is reported to interact with proteins of the insect vector B. tabaci. In this study, we performed yeast two-hybrid assay using CP of cotton leaf curl Rajasthan virus (CLCuV) and Tomato leaf curl New Delhi virus (ToLCNDV) as bait in separate experiments and cDNA prepared from total RNA of B. tabaci was used as prey. Yeast two-hybrid assay resulted in identification of a thioredoxin-like protein (TLP) from CLCuV yeast two-hybrid library. Later TLP was also found to interact with CP of ToLCNDV. In vitro pull-down assay showed TLP interaction with CP of both CLCuV and ToLCNDV. TLP was found to interact with ToLCNDV virus particles isolated from tomato leaves.

Cassava mosaic disease: a review of a threat to cassava production in Zambia.

Cassava (Manihot esculenta Crantz) is one of the most important root staple crops in Zambia. An estimated 30% of Zambians, over 4 million people, consume cassava as part of their daily diet. Cassava is mostly grown by subsistence farmers on fields of less than 1 ha. Cultivation of cassava is hampered by several biotic constraints, of which cassava mosaic disease (CMD) is currently the most important factor limiting cassava production in Zambia. CMD occurs in all the cassava-growing provinces and accounts for 50% to 70% of yield losses countrywide. Strategies to counter CMD were initiated in the early 1990s and included the release of CMD-resistant cassava cultivars. However, efforts to control CMD are limited because few growers plant these cultivars. More recently, to address the CMD problem, regular disease monitoring and diagnostic capabilities have been strengthened, and there is increased support for screening breeders materials. CMD is a rising threat to cassava production in Zambia. This review of CMD research on disease surveillance, CMD spread, yield losses, awareness campaigns and control options in Zambia over the past 25 years informs future control efforts and management strategies.

Identification and comparative analysis of microRNAs from tomato varieties showing contrasting response to ToLCV infections.

Increasing incidence of viral infections in crop plants adversely affects their growth and yield. Tomato (Solanum lycopersicum) is considered to be a favorite host for viruses with over 50 species of begomoviruses naturally infecting this crop. Tomato leaf curl virus (ToLCV) is among the most widespread and devastating begomoviruses affecting tomato production. microRNAs (miRs) have been established as key regulators of gene expression and plant development. The miR pathways are disturbed during infection by viruses. Thus, comprehension of regulatory miR networks is crucial in understanding the effect of viral pathogenicity. To identify key miRs involved in ToLCV infection, a high throughput approach involving next generation sequencing was employed. Healthy and infected leaf tissues of two tomato varieties, differing in their susceptibility to ToLCV infection were analyzed. NGS data analysis followed by computational predictions, led to identification of 91 known miRs, 15 novel homologs and 53 novel miRs covering two different varieties of tomato, susceptible (Pusa Ruby) and tolerant (LA1777) to ToLCV infection. The cleaved targets of these miRs were identified using online available degradome libraries from leaf, flower and fruit of tomato and showed their involvement in various biological pathways through KEGG Orthology. With detailed comparative profiling of expression pattern of these miRs, we could associate the specific miRs with the resistant and infected genotypes. This study depicted that in depth analysis of miR expression patterns and their functions will help in identification of molecules that can be used for manipulation of gene expression to increase crop production and developing resistance against diseases.

NIK1, a host factor specialized in antiviral defense or a novel general regulator of plant immunity?

NIK1 is a receptor-like kinase involved in plant antiviral immunity. Although NIK1 is structurally similar to the plant immune factor BAK1, which is a key regulator in plant immunity to bacterial pathogens, the NIK1-mediated defenses do not resemble BAK1 signaling cascades. The underlying mechanism for NIK1 antiviral immunity has recently been uncovered. NIK1 activation mediates the translocation of RPL10 to the nucleus, where it interacts with LIMYB to fully down-regulate translational machinery genes, resulting in translation inhibition of host and viral mRNAs and enhanced tolerance to begomovirus. Therefore, the NIK1 antiviral immunity response culminates in global translation suppression, which represents a new paradigm for plant antiviral defenses. Interestingly, transcriptomic analyses in nik1 mutant suggest that NIK1 may suppress antibacterial immune responses, indicating a possible opposite effect of NIK1 in bacterial and viral infections.

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

BACKGROUND: Cassava 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. RESULTS: The 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. CONCLUSIONS: Our 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.

Sustained NIK-mediated antiviral signalling confers broad-spectrum tolerance to begomoviruses in cultivated plants.

Begomovirus-associated epidemics currently threaten tomato production worldwide due to the emergence of highly pathogenic virus species and the proliferation of a whitefly B biotype vector that is adapted to tomato. To generate an efficient defence against begomovirus, we modulated the activity of the immune defence receptor nuclear shuttle protein (NSP)-interacting kinase (NIK) in tomato plants; NIK is a virulence target of the begomovirus NSP during infection. Mutation of T474 within the kinase activation loop promoted the constitutive activation of NIK-mediated defences, resulting in the down-regulation of translation-related genes and the suppression of global translation. Consistent with these findings, transgenic lines harbouring an activating mutation (T474D) were tolerant to the tomato-infecting begomoviruses ToYSV and ToSRV. This phenotype was associated with reduced loading of coat protein viral mRNA in actively translating polysomes, lower infection efficiency and reduced accumulation of viral DNA in systemic leaves. Our results also add some relevant insights into the mechanism underlying the NIK-mediated defence. We observed that the mock-inoculated T474D-overexpressing lines showed a constitutively infected wild-type transcriptome, indicating that the activation of the NIK-mediated signalling pathway triggers a typical response to begomovirus infection. In addition, the gain-of-function mutant T474D could sustain an activated NIK-mediated antiviral response in the absence of the virus, further confirming that phosphorylation of Thr-474 is the crucial event that leads to the activation of the kinase.