Bipartite Geminivirus Research Articles

A Bipartite Geminivirus with a Highly Divergent Genomic Organization Identified in Olive Trees May Represent a Novel Evolutionary Direction in the Family Geminiviridae

Olea europaea Geminivirus (OEGV) was recently identified in olive in Italy through HTS. In this work, we used HTS to show the presence of an OEGV isolate in Portuguese olive trees and suggest the evolution direction of OEGV. The bipartite genome (DNA-A and DNA-B) of the OEGV-PT is similar to Old World begomoviruses in length, but it lacks a pre-coat protein (AV2), which is a typical feature of New World begomoviruses (NW). DNA-A genome organization is closer to NW, containing four ORFs; three in complementary-sense AC1/Rep, AC2/TrAP, AC3/REn and one in virion-sense AV1/CP, but no AC4, typical of begomoviruses. DNA-B comprises two ORFs; MP in virion sense with higher similarity to the tyrosine phosphorylation site of NW, but in opposite sense to begomoviruses; BC1, with no known conserved domains in the complementary sense and no NSP typical of bipartite begomoviruses. Our results show that OEGV presents the longest common region among the begomoviruses, and the TATA box and four replication-associated iterons in a completely new arrangement. We propose two new putative conserved regions for the geminiviruses CP. Lastly, we highlight unique features that may represent a new evolutionary direction for geminiviruses and suggest that OEGV-PT evolution may have occurred from an ancient OW monopartite Begomovirus that lost V2 and C4, gaining functions on cell-to-cell movement by acquiring a DNA-B component.

The AC2 Protein of a Bipartite Geminivirus Stimulates the Transcription of the BV1 Gene through Abscisic Acid Responsive Promoter Elements.

Geminiviruses possess single-stranded, circular DNA genomes and control the transcription of their late genes, including BV1 of many bipartite begomoviruses, through transcriptional activation by the early expressing AC2 protein. DNA binding by AC2 is not sequence-specific; hence, the specificity of AC2 activation is thought to be conferred by plant transcription factors (TFs) recruited by AC2 in infected cells. However, the exact TFs AC2 recruits are not known for most viruses. Here, we report a systematic examination of the BV1 promoter (PBV1) of the mungbean yellow mosaic virus (MYMV) for conserved promoter motifs. We found that MYMV PBV1 contains three abscisic acid (ABA)-responsive elements (ABREs) within its first 70 nucleotides. Deleting these ABREs, or mutating them all via site-directed mutagenesis, abolished the capacity of PBV1 to respond to AC2-mediated transcriptional activation. Furthermore, ABRE and other related ABA-responsive elements were prevalent in more than a dozen Old World begomoviruses we inspected. Together, these findings suggest that ABA-responsive TFs may be recruited by AC2 to BV1 promoters of these viruses to confer specificity to AC2 activation. These observations are expected to guide the search for the actual TF(s), furthering our understanding of the mechanisms of AC2 action.

Phosphorylations of the Abutilon Mosaic Virus Movement Protein Affect Its Self-Interaction, Symptom Development, Viral DNA Accumulation, and Host Range.

The genome of bipartite geminiviruses in the genus Begomovirus comprises two circular DNAs: DNA-A and DNA-B. The DNA-B component encodes a nuclear shuttle protein (NSP) and a movement protein (MP), which cooperate for systemic spread of infectious nucleic acids within host plants and affect pathogenicity. MP mediates multiple functions during intra- and intercellular trafficking, such as binding of viral nucleoprotein complexes, targeting to and modification of plasmodesmata, and release of the cargo after cell-to-cell transfer. For Abutilon mosaic virus (AbMV), phosphorylation of MP expressed in bacteria, yeast, and Nicotiana benthamiana plants, respectively, has been demonstrated in previous studies. Three phosphorylation sites (T221, S223, and S250) were identified in its C-terminal oligomerization domain by mass spectrometry, suggesting a regulation of MP by posttranslational modification. To examine the influence of the three sites on the self-interaction in more detail, MP mutants were tested for their interaction in yeast by two-hybrid assays, or by Förster resonance energy transfer (FRET) techniques in planta. Expression constructs with point mutations leading to simultaneous (triple) exchange of T221, S223, and S250 to either uncharged alanine (MPAAA), or phosphorylation charge-mimicking aspartate residues (MPDDD) were compared. MPDDD interfered with MP-MP binding in contrast to MPAAA. The roles of the phosphorylation sites for the viral life cycle were studied further, using plant-infectious AbMV DNA-B variants with the same triple mutants each. When co-inoculated with wild-type DNA-A, both mutants infected N. benthamiana plants systemically, but were unable to do so for some other plant species of the families Solanaceae or Malvaceae. Systemically infected plants developed symptoms and viral DNA levels different from those of wild-type AbMV for most virus-plant combinations. The results indicate a regulation of diverse MP functions by posttranslational modifications and underscore their biological relevance for a complex host plant-geminivirus interaction.

Manipulation of the Plant Host by the Geminivirus AC2/C2 Protein, a Central Player in the Infection Cycle.

Geminiviruses are a significant group of emergent plant DNA viruses causing devastating diseases in food crops worldwide, including the Southern United States, Central America and the Caribbean. Crop failure due to geminivirus-related disease can be as high as 100%. Improved global transportation has enhanced the spread of geminiviruses and their vectors, supporting the emergence of new, more virulent recombinant strains. With limited coding capacity, geminiviruses encode multifunctional proteins, including the AC2/C2 gene that plays a central role in the viral replication-cycle through suppression of host defenses and transcriptional regulation of the late viral genes. The AC2/C2 proteins encoded by mono- and bipartite geminiviruses and the curtovirus C2 can be considered virulence factors, and are known to interact with both basal and inducible systems. This review highlights the role of AC2/C2 in affecting the jasmonic acid and salicylic acid (JA and SA) pathways, the ubiquitin/proteasome system (UPS), and RNA silencing pathways. In addition to suppressing host defenses, AC2/C2 play a critical role in regulating expression of the coat protein during the viral life cycle. It is important that the timing of CP expression is regulated to ensure that ssDNA is converted to dsDNA early during an infection and is sequestered late in the infection. How AC2 interacts with host transcription factors to regulate CP expression is discussed along with how computational approaches can help identify critical host networks targeted by geminivirus AC2 proteins. Thus, the role of AC2/C2 in the viral life-cycle is to prevent the host from mounting an efficient defense response to geminivirus infection and to ensure maximal amplification and encapsidation of the viral genome.

Topical application of double-stranded RNA molecules containing sequences of Tomato leaf curl virus and Cucumber mosaic virus confers protection against the cognate viruses

The Tomato leaf curl virus (ToLCV) is a bipartite (DNA) geminivirus whereas Cucumber mosaic virus (CMV) is a tripartite RNA virus, both infecting a wide range of host plants. RNAi that is triggered by double-stranded RNA (dsRNA) molecules, is a powerful means to control plant viruses in transgenic plants, as well as in a non-transgenic manner in a process designated as ‘RNA-based vaccination’. DsRNA molecules were made for AC1/AC4, AV1/AV2 (overlapping regions of ToLCV), AC1/AC4_AV1/AV2 (designated as fusion construct), CMV-2b and CMV-2b_ToLCV-AV1/AV2 (designated as hybrid construct, containing regions from two different viruses). In tomato (Solanum lycopersicum), dsRNAs for AC1/AC4, AV1/AV2, AC1/AC4_AV1/AV2 and CMV-2b_ToLCV-AV1/AV2 conferred 45%, 60%, 50% and 55% protection against ToLCV, respectively. Experiments with tobacco (Nicotiana tabacum) showed that the dsRNA construct CMV-2b_ToLCV-AV1/AV2 conferred 33.3% protection against CMV, while CMV-2b provided 40% protection. The present study reported that the dsRNA exhibits systemic transport in tomato. This is the first case where RNA-based vaccination is functional for a bipartite geminivirus and where a single dsRNA molecule could protect against two tomato-infecting viruses, namely a DNA (ToLCV) and an RNA (CMV) plant virus.

‘Abnormal vertical growth’: a disorder threatening the viability of the Australian macadamia industry

'Abnormal vertical growth' (AVG) was recognised in Australia as a dysfunction of macadamia (Macadamia spp.) in the mid-1990s. Affected trees displayed unusually erect branching, and poor flowering and yield. Since 2002, the commercial significance of AVG, its cause, and strategies to alleviate its affects, has been studied. The cause is still unknown, and AVG remains a serious threat to orchard viability. AVG affects both commercial and urban macadamia. It occurs predominantly in the warmer-drier production regions of Queensland and New South Wales. An estimated 100,000 orchard trees are affected, equating to an annual loss of $ 10.5 M. In orchards, AVG occurs as aggregations of affected trees, affected tree number can increase by 4.5% per year, and yield reduction can exceed 30%. The more upright cultivars 'HAES 344' and '741' are highly susceptible, while the more spreading cultivars 'A4', 'A16' and 'A268' show tolerance. Incidence is higher (p<0.05) in soils of high permeability and good drainage. No soil chemical anomaly has been found. Fine root dry weight of AVG trees (0-15 cm depth) was found lower (p<0.05) than non-AVG. Next generation sequencing has led to the discovery of a new Bacillus sp. and a bipartite Geminivirus, which may have a role in the disease. Trunk cinctures will increase (p<0.05) yield of moderately affected trees. Further research is needed to clarify whether a pathogen is the cause, the role of soil moisture in AVG, and develop a varietal solution.

Mapping and identification of cassava mosaic geminivirus DNA-A and DNA-B genome sequences for efficient siRNA expression and RNAi based virus resistance by transient agro-infiltration studies

Geminiviruses are among the most serious pathogens of many economically important crop plants and RNA interference (RNAi) is an important strategy for their control. Although any fragment of a viral genome can be used to generate a double stranded (ds) RNA trigger, the precursor for generation of siRNAs, the exact sequence and size requirements for efficient gene silencing and virus resistance have so far not been investigated. Previous efforts to control geminiviruses by gene silencing mostly targeted AC1, the gene encoding replication-associated protein. In this study we made RNAi constructs for all the genes of both the genomic components (DNA-A and DNA-B) of African cassava mosaic virus (ACMV-CM), one of the most devastating geminiviruses causing cassava mosaic disease (CMD) in Africa. Using transient agro-infiltration studies, RNAi constructs were evaluated for their ability to trigger gene silencing against the invading virus and protection against it. The results show that the selection of the DNA target sequence is an important determinant for the amount of siRNA produced and the extent of resistance. The ACMV genes AC1, AC2, AC4 from DNA-A and BC1 from DNA-B were effective targets for RNAi-mediated resistance and their siRNA expression was higher compared to other RNAi constructs. The RNAi construct targeting AC2, the suppressor of gene silencing of ACMV-CM gave highest level of resistance in the transient studies. This is the first report of targeting DNA-B to confer resistance to a bipartite geminivirus infection.

A Population Genetics Perspective on Geminivirus Infection.

The selective accumulation of both DNA components of a bipartite geminivirus, Abutilon mosaic virus, was recorded during early systemic infection of Nicotiana benthamiana plants. Purified nuclei were diagnosed for viral DNA using hybridization specific for DNA A or DNA B to detect these individual genome components either alone or both simultaneously by dual-color staining. Although this virus needs both components for symptomatic infection, DNA A alone was transported to upper leaves, where it was imported into phloem nuclei and replicated autonomously. The coinfection with DNA A and DNA B revealed an independent spread of both molecules, which resulted in a stochastic distribution of DNA A- and DNA A/B-infected nuclei. A population genetics evaluation of the respective frequencies was compared to a model computation. This elucidated a surprisingly simple relationship between the initial frequencies of the viral DNA components and the number of susceptible cells during the course of early systemic infection. For bipartite begomoviruses, DNA B-independent long-distance spread of DNA A has been described before, but it has never been shown whether viral DNA A alone invades nuclei of systemic tissues and replicates therein. This is demonstrated now for the first time. During infection with DNA A and DNA B, a similar solitary spread of DNA A can be recognized at early stages. We describe a population genetics model of how the hit probabilities of DNA A and DNA B for susceptible cells determine the relative frequencies of either genome component during the course of infection.

Genome-wide SNP identification and characterization in two soybean cultivars with contrasting Mungbean Yellow Mosaic India Virus disease resistance traits.

Mungbean yellow mosaic India virus (MYMIV) is a bipartite Geminivirus, which causes severe yield loss in soybean (Glycine max). Considering this, the present study was conducted to develop large-scale genome-wide single nucleotide polymorphism (SNP) markers and identify potential markers linked with known disease resistance loci for their effective use in genomics-assisted breeding to impart durable MYMIV tolerance. The whole-genome re-sequencing of MYMIV resistant cultivar ‘UPSM-534’ and susceptible Indian cultivar ‘JS-335’ was performed to identify high-quality SNPs and InDels (insertion and deletions). Approximately 234 and 255 million of 100-bp paired-end reads were generated from UPSM-534 and JS-335, respectively, which provided ~98% coverage of reference soybean genome. A total of 3083987 SNPs (1559556 in UPSM-534 and 1524431 in JS-335) and 562858 InDels (281958 in UPSM-534 and 280900 in JS-335) were identified. Of these, 1514 SNPs were found to be present in 564 candidate disease resistance genes. Among these, 829 non-synonymous and 671 synonymous SNPs were detected in 266 and 286 defence-related genes, respectively. Noteworthy, a non-synonymous SNP (in chromosome 18, named 18-1861613) at the 149th base-pair of LEUCINE-RICH REPEAT RECEPTOR-LIKE PROTEIN KINASE gene responsible for a G/C transversion [proline (CCC) to alanine(GCC)] was identified and validated in a set of 12 soybean cultivars. Taken together, the present study generated a large-scale genomic resource such as, SNPs and InDels at a genome-wide scale that will facilitate the dissection of various complex traits through construction of high-density linkage maps and fine mapping. In the present scenario, these markers can be effectively used to design high-density SNP arrays for their large-scale validation and high-throughput genotyping in diverse natural and mapping populations, which could accelerate genomics-assisted MYMIV disease resistance breeding in soybean.

Dowstream events in the NIK-mediated defense associated with resistance to begomovirus

The NSP-interacting kinase (NIK) receptor-mediated antiviral signaling has been identified as a virulence target of the begomovirus nuclear shuttle protein (NSP) [1,2]. NSP suppresses the activity of the NIK receptor through specific binding to the kinase domain and hence enhances begomovirus pathogenicity [1,3]. NIK receptors belong to the plant defense group of the leucine-rich repeat (LRR) receptor-like kinase (RLK) subfamily, designated LRR-RLK II. This subfamily of RLKs from tomato and Arabidopsis is constituted by 14 proteins harboring four complete LRRs (with 24 residues) and a fifth incomplete LRR (with 16 residues) arranged in a single continuous block within the extracellular domain [4]. Based on sequence conservation and structural features, the members of the LRR-RLK II subfamily are clustered into three distinct branches: (i) antiviral defense proteins (ii) developmental and defense proteins, such as the somatic embryogenesis receptor-like kinases (SERK-like) including SERK1 and SERK3/BAK1 and (iii) functionally unassigned proteins. The Arabidopsis NSP-interacting kinase 1, NIK1 (At5g16000), NIK2 (At3g25560) and NIK3 (At1g60800) are in the defense group I of the LRR-RLKII subfamily and are virulence targets of the bipartite geminivirus nuclear shuttle protein, NSP [1,4]. NSP from CaLCuV (Cabbage leaf curl virus interacts with all three NIKs from Arabidopsis to suppress their kinase activity [4]. The NSP-NIK interaction is also conserved among geminivirus NSPs and NIK homologs from different hosts [2]. Tomato and soybean NIK homologs also interact stably with NSP from CaLCuV [2] and with NSPs from the tomato-infecting geminiviruses TGMV (Tomato golden mosaic virus), TCrLYV (Tomato crinkle leaf yellows virus) and ToYSV (Tomato yellow spot virus) [1,2]. Several lines of evidence indicate that NIK functions in defense. NSP from CaLCuV acts as a virulence factor to suppress the kinase activity of transmembrane receptor NIKs [1]. Second, loss of NIK1, NIK2 or NIK3 function in Arabidopsis is also linked to an enhanced susceptibility phenotype to CaLCuV infection [1,3]. In addition, overexpression of NIK1 from Arabidopsis in tomato plants attenuates symptom development and delays ToYSV infection [5]. NIK exhibits trans-autophosphorylation activity in vitro and substrate phosphorylation activity in vitro and in vivo, and interacts with the ribosomal proteins L10 (rpL10) and L18 (rpL18) [5]. NIK-mediated phosphorylation of rpL10 promotes translocation of the ribosomal protein to the nucleus where it may function to mount a defense response that negatively impacts virus infection. This is consistent with the notion that the regulated nucleocytoplasmic shuttling of rpL10 links the antiviral response to receptor activation. We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A-loop) with distinct roles for different threonine residues [6]. The conserved Thr-474 and Thr-469 were found to be phosphorylated in vitro and mutations at Thr-474 impaired autophosphorylation and were defective for kinase activation in vitro and in vivo. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. Our results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response [6,7]. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation. To identify novel regulators of NIK-mediated defense response, we screened a two-hybrid library for partners of rpL10. We discovered a novel transcriptional factor, which interacts with rpL10 in the nucleus of plant cells to down-regulate the expression of ribosomal genes. These data are consistent with the observation that constitutive activation of the NIK receptor by replacing Thr-474 with aspartate impairs global translation in Arabidopsis and tomato transgenic lines and confers broad-spectrum tolerance to begomovirus infection. Our data also indicate that the NIK immune receptor-mediated antiviral signaling operates through a bipartite module. One defense signal-transducing branch is mediated by the regulated nucleocytoplasmic transport of ribosomal protein to impair translation and the second branch of the antiviral signaling transduces a typical defense signal through induction of the plant immune system. We will discuss novel insights into the regulation of the NIK-mediated antiviral signaling and an efficient-acquired defense against begomovirus by modulating the activity of the immune defense receptor NIK in tomato plants.

East African cassava mosaic-like viruses from Africa to Indian ocean islands: molecular diversity, evolutionary history and geographical dissemination of a bipartite begomovirus

BackgroundCassava (Manihot esculenta) is a major food source for over 200 million sub-Saharan Africans. Unfortunately, its cultivation is severely hampered by cassava mosaic disease (CMD). Caused by a complex of bipartite cassava mosaic geminiviruses (CMG) species (Family: Geminivirideae; Genus: Begomovirus) CMD has been widely described throughout Africa and it is apparent that CMG's are expanding their geographical distribution. Determining where and when CMG movements have occurred could help curtail its spread and reveal the ecological and anthropic factors associated with similar viral invasions. We applied Bayesian phylogeographic inference and recombination analyses to available and newly described CMG sequences to reconstruct a plausible history of CMG diversification and migration between Africa and South West Indian Ocean (SWIO) islands.ResultsThe isolation and analysis of 114 DNA-A and 41 DNA-B sequences demonstrated the presence of three CMG species circulating in the Comoros and Seychelles archipelagos (East African cassava mosaic virus, EACMV; East African cassava mosaic Kenya virus, EACMKV; and East African cassava mosaic Cameroon virus, EACMCV). Phylogeographic analyses suggest that CMG’s presence on these SWIO islands is probably the result of at least four independent introduction events from mainland Africa occurring between 1988 and 2009. Amongst the islands of the Comoros archipelago, two major migration pathways were inferred: One from Grande Comore to Mohéli and the second from Mayotte to Anjouan. While only two recombination events characteristic of SWIO islands isolates were identified, numerous re-assortments events were detected between EACMV and EACMKV, which seem to almost freely interchange their genome components.ConclusionsRapid and extensive virus spread within the SWIO islands was demonstrated for three CMG complex species. Strong evolutionary or ecological interaction between CMG species may explain both their propensity to exchange components and the absence of recombination with non-CMG begomoviruses. Our results suggest an important role of anthropic factors in CMGs spread as the principal axes of viral migration correspond with major routes of human movement and commercial trade. Finer-scale temporal analyses of CMGs to precisely scale the relative contributions of human and insect transmission to their movement dynamics will require further extensive sampling in the SWIO region.

Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing

In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3′-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5′-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs.

Enhanced Viral Intergenic Region–Specific Short Interfering RNA Accumulation and DNA Methylation Correlates with Resistance Against a Geminivirus

RNA silencing mediated by short-interfering RNA (siRNA) is used by plants as a defense against viruses. In the case of geminiviruses, viral DNA is targeted at the transcriptional level, while virus-derived transcripts are targeted by posttranscriptional silencing. Mungbean yellow mosaic India virus (MYMIV), a bipartite geminivirus, causes yellow mosaic disease in soybean (Glycine max). A soybean variety resistant to this disease has been identified (line PK416). To understand the molecular mechanism underlying this resistance, distribution of MYMIV-derived siRNAs along the viral genome was compared in resistant and susceptible plants, using samples obtained in the first few days following inoculation. We observed that, in the resistant soybean variety, most of the virus-derived siRNAs were complementary to the intergenic region (IR), while in the susceptible variety (line JS335), a majority of the siRNAs corresponded to coding regions of the viral genome. Most of the IR-specific siRNA molecules produced in the resistant plants were 24 nt in size. Bisulfite sequencing showed that, in the resistant plants, a higher level of methylation occurred in the IR of viral DNA.

A plastid-targeted heat shock cognate 70kDa protein interacts with the Abutilon mosaic virus movement protein

The movement protein (MP) of bipartite geminiviruses facilitates cell-to-cell as well as long-distance transport within plants and influences viral pathogenicity. Yeast two-hybrid assays identified a chaperone, the nuclear-encoded and plastid-targeted heat shock cognate 70kDa protein (cpHSC70-1) of Arabidopsis thaliana, as a potential binding partner for the Abutilon mosaic virus (AbMV) MP. In planta, bimolecular fluorescence complementation (BiFC) analysis showed cpHSC70-1/MP complexes and MP homooligomers at the cell periphery and co-localized with chloroplasts. BiFC revealed cpHSC70-1 oligomers associated with chloroplasts, but also distributed at the cellular margin and in filaments arising from plastids reminiscent of stromules. Silencing the cpHSC70 gene of Nicotiana benthamiana using an AbMV DNA A-derived gene silencing vector induced minute white leaf areas, which indicate an effect on chloroplast stability. Although AbMV DNA accumulated within chlorotic spots, a spatial restriction of these occurred, suggesting a functional relevance of the MP-chaperone interaction for viral transport and symptom induction.

Characterization of signals that dictate nuclear/nucleolar and cytoplasmic shuttling of the capsid protein of Tomato leaf curl Java virus associated with DNAβ satellite

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of geminiviruses. Capsid proteins (CPs) of geminiviruses are multifunctional proteins thought to be involved in this process. The CP of monopartite geminiviruses is absolutely essential for virus movement. To more precisely examine the role of CP, we have constructed a series of single and double deletions into the coding sequence of Tomato leaf curl Java virus (ToLCJAV) CP and examined sub-cellular localization using transient expression of GFP fusion proteins. In this report, the domains of the CP encoded by ToLCJAV localized in the nucleus/nucleolus and cytoplasm in transfected cells were mapped. Deletion analysis revealed that the Arg-rich cluster from amino acids (aa) 16KVRRR 20 in the N-terminal region of CP functioned as nuclear/nucleolar localization signals (NLSs). The region from aa 52RKPR 55 contained basic amino acid cluster was capable to redirect the CP to the nucleus. Further, both transient expression and yeast hybrid assays demonstrated that CP was capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property was attributed to a nuclear export signal (NES) sequence consisted of aa ( 245LKIRIY 250) reside at C-terminal part of CP. This hydrophobic region caused transport of GFP to the cytoplasm. However, ToLCJAV CP NLSs and NES show peculiarities in the number and position of basic residues. Taken together, these results demonstrated that ToLCJAV CP shuttles between the nucleus and cytoplasm, such an activity homolog to bipartite geminivirus BV1 ORF.

Soybean cultivar resistant to Mungbean Yellow Mosaic India Virus infection induces viral RNA degradation earlier than the susceptible cultivar

Yellow mosaic disease caused by whitefly-transmitted bipartite Geminiviruses is one of the major constraints on productivity of a number of pulse crops. We have cloned the bipartite genome of Mungbean Yellow Mosaic India Virus isolated from infected Soybean. We report here that agroinfection of Soybean seedlings with a single uncut recombinant binary plasmid containing tandem dimers of both DNA A and DNA B resulted in 100% infectivity in susceptible varieties. To understand the mechanism of natural resistance in a Soybean variety, we compared the abundance of the viral RNAs in a resistant and a susceptible variety at the early time points after agroinfection. Whilst the resistant variety displayed synthesis but rapid degradation of the early viral RNAs; the degradation in the susceptible variety was delayed resulting in accumulation of those transcripts later in infection. Accumulation of the late viral transcripts and DNA replication were detectable only in the susceptible variety. This indicates that rapid degradation of the early viral transcripts, possibly through siRNA mechanism, is one of the probable mechanisms of natural resistance against geminivirus.

NSP-Interacting GTPase

Despite the significant progress in the identification of essential components of the nuclear transport machinery, some events of this process are still unclear. Particularly, functional information about the release of nuclear-exported macromolecules at the cytoplasmic side of the nuclear pore complex and their subsequent trans-cytoplasmic movement is lacking. Recently, we identified a cytoplasmic GTPase, designated NIG (NSP-interacting GTPase), which may play a relevant role in these processes. NIG interacts in vivo with the geminivirus NSP and promotes the translocation of the viral protein from the nucleus to the cytoplasm where it is redirected to the cell surface to interact with the viral movement protein, MP. Here we position the NIG function into the mechanistic model for the intracellular trafficking of viral DNA and discuss the putative role of NIG in general cellular nucleocytoplasmic transport of nucleic acid-protein complexes.Addendum to: Carvalho CM, Fontenelle MR, Florentino LH, Santos AA, Zerbini FM, Fontes EPB. A novel nucleocytoplasmic traffic GTPase identified as a functional target of the bipartite geminivirus nuclear shuttle protein. Plant J 2008; DOI: 10.1111/j.1365-313X.2008.03556.x.

A novel nucleocytoplasmic traffic GTPase identified as a functional target of the bipartite geminivirus nuclear shuttle protein

In contrast to the accumulated data on nuclear transport mechanisms of macromolecules, little is known concerning the regulated release of nuclear-exported complexes and their subsequent trans-cytoplasmic movement. The bipartite begomovirus nuclear shuttle protein (NSP) facilitates the nuclear export of viral DNA and cooperates with the movement protein (MP) to transport viral DNA across the plant cell wall. Here, we identified a cellular NSP-interacting GTPase (NIG) with biochemical properties consistent with a nucleocytoplasmic transport role. We show that NIG is a cytosolic GTP-binding protein that accumulates around the nuclear envelope and possesses intrinsic GTPase activity. NIG interacts with NSP in vitro and in vivo (under transient expression), and redirects the viral protein from the nucleus to the cytoplasm. We propose that NIG acts as a positive contributor to geminivirus infection by modulating NSP nucleocytoplasmic shuttling and hence facilitating MP-NSP interaction in the cortical cytoplasm. In support of this, overexpression of NIG in Arabidopsis enhances susceptibility to geminivirus infection. In addition to highlighting the relevance of NIG as a cellular co-factor for NSP function, our findings also have implications for general nucleocytoplasmic trafficking of cellular macromolecules.

RNA-silencing suppressors of geminiviruses

Geminiviruses are single-stranded (ss) DNA viruses that not only cause devastating diseases of important food and fiber crops worldwide, but also are important models to study fundamental aspects of virus-induced gene silencing and RNA interference. As a counterdefense mechanism, viruses have evolved various antisilencing strategies that are being progressively unraveled. The geminiviruses, ssDNA molecules that replicate inside the nucleus and therefore have no dsRNA phase during replication, can both induce and become targets of gene silencing. Proteins AC2 (encoding the transcriptional activator protein) and AC4 of bipartite geminiviruses and protein C2, a positional homolog of AC2 of monopartite viruses, have been identified as suppressors of posttranscriptional gene silencing. The majorities of geminiviral suppressors characterized to date do not share any obvious structural or sequence similarity across families and groups except that they have been identified as pathogenicity determinants. This review mainly focuses on the geminivirus-encoded suppressors of RNA-silencing—the βC1 and V2 proteins—and their possible role in the interference of silencing at different steps in the pathways.

DNA recognition properties of the cell-to-cell movement protein (MP) of soybean isolate of Mungbean yellow mosaic India virus (MYMIV-Sb)

Bipartite geminiviruses possess two movement proteins (NSP and MP), which mediate the intra- and intercellular movement. In order to accomplish the transport process the movement proteins interact with viral nucleic acids in a sequence non-specific manner. To investigate the nucleic acid recognition properties of MP of MYMIV-Sb, the protein was expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP) and purified in native condition. Gel mobility shift assay was employed for analyzing the DNA recognition properties of purified MBP–MP fusion protein. The analyses demonstrated the sequence non-specific binding of MYMIV-Sb MP to both ds and ssDNA and its high affinity for ssDNA. MP of MYMIV-Sb did not show any specificity towards various forms of plasmid DNA but displayed size selection towards linear dsDNA.