Molecular Variation of Satellite DNAβ Molecules Associated with Malvastrum yellow vein virus and Their Role in Pathogenicity

Previous studies have found that the diversity of begomovirus-associated DNAβ satellites is related to host and geographical origin. In this study, we have cloned and sequenced 20 different isolates of DNAβ molecules associated with Malvastrum yellow vein virus (MYVV) isolated from Malvastrum coromandelianum plants in different geographical locations of Yunnan Province, China. Analyses of their molecular variation indicate that the satellites are clustered together according to their geographical location but that they have only limited sequence diversity. Infectivity tests using infectious clones of MYVV and its associated DNAβ molecule indicate that MYVV DNAβ is indispensable for symptom induction in Nicotiana benthamiana, N. glutinosa, Petunia hybrida, and M. coromandelianum plants. Furthermore, we showed that MYVV interacts functionally with heterologous DNAβ molecules in N. benthamiana plants.     

Short Communication: Ageratum,Croton and Malvastrum harbour geminiviruses: evidence through PCR amplification

Ageratum conyzoides, Croton bonpladianum and Malvastrum coromandelianum are common weeds found around agricultural fields. In several cases these were found to exhibit vein yellowing and yellow mosaic symptoms. Using degenerate primers specific for whitefly-transmitted geminiviruses (WTGs), and total DNA isolated from such infected plants (exhibiting the above symptoms) as a template, 1.2kbp fragments were amplified and were shown to have homology to DNA-A of Indian tomato leaf curl virus (ITLCV) by Southern hybridization. In control experiments the same primers failed to amplify any DNA fragments from the total DNA isolated from healthy plants (no symptoms as above). These results show that Ageratum, Croton and Malvastrum harbour geminivirus(es).     

A melting pot of old world begomoviruses and their satellites infecting a collection of gossypium species in pakistan

CLCuD in southern Asia is caused by a complex of multiple begomoviruses (whitefly transmitted, single-stranded [ss]DNA viruses) in association with a specific ssDNA satellite; Cotton leaf curl Multan betasatellite (CLCuMuB). A further single ssDNA molecule, for which the collective name alphasatellites has been proposed, is also frequently associated with begomovirus-betasatellite complexes. Multan is in the center of the cotton growing area of Pakistan and has seen some of the worst problems caused by CLCuD. An exhaustive analysis of the diversity of begomoviruses and their satellites occurring in 15 Gossypium species (including G. hirsutum, the mainstay of Pakistan's cotton production) that are maintained in an orchard in the vicinity of Multan has been conducted using φ29 DNA polymerase-mediated rolling-circle amplification, cloning and sequence analysis. The non-cultivated Gossypium species, including non-symptomatic plants, were found to harbor a much greater diversity of begomoviruses and satellites than found in the cultivated G. hirsutum. Furthermore an African cassava mosaic virus (a virus previously only identified in Africa) DNA-A component and a Jatropha curcas mosaic virus (a virus occurring only in southern India) DNA-B component were identified. Consistent with earlier studies of cotton in southern Asia, only a single species of betasatellite, CLCuMuB, was identified. The diversity of alphasatellites was much greater, with many previously unknown species, in the non-cultivated cotton species than in G. hirsutum. Inoculation of newly identified components showed them to be competent for symptomatic infection of Nicotiana benthamiana plants. The significance of the findings with respect to our understanding of the role of host selection in virus diversity in crops and the geographical spread of viruses by human activity are discussed.     

A Begomovirus DNAbeta-encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus

Our previous results demonstrated that the DNAbeta satellite (Y10beta) associated with Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) is essential for induction of leaf curl symptoms in plants and that transgenic expression of its betaC1 gene in Nicotiana plants induces virus-like symptoms. In the present study, in vitro DNA binding activity of the betaC1 proteins of Y10beta and DNAbeta (Y35beta) found in the Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) were studied following their expression as six-His fusion proteins in Escherichia coli. Electrophoretic mobility shift assays and UV cross-linking experiments revealed that betaC1 proteins could bind both single-stranded and double-stranded DNA without size or sequence specificity. Suppression of green fluorescent protein (GFP) transgene silencing was observed with the new leaves of GFP-expressing Nicotiana benthamiana plants coinoculated by TYLCCNV-Y10 plus Y10beta or by TbCSV-Y35 plus Y35beta. In a patch agroinfiltration assay, the transiently expressed betaC1 gene of Y10beta or Y35beta was able to suppress host RNA silencing activities and permitted the accumulation of high levels of GFP mRNA in the infiltrated leaf patches of GFP transgenic N. benthamiana plants. The betaC1 protein of Y10beta accumulated primarily in the nuclei of plant and insect cells when fused with beta-glucuronidase or GFP and immunogold labeling showed that the betaC1 protein is present in the nuclei of infected N. benthamiana plants. A mutant version of Y10beta carrying the mutations within the putative nuclear localization sequence of the Y10 betaC1 protein failed to induce disease symptoms, suppress RNA silencing, or accumulate in the nucleus, suggesting that nuclear localization of the betaC1 protein is a key requirement for symptom induction and silencing suppression.     

Genetic structure and population variability of tomato yellow leaf curl China virus

Geminiviruses have circular single-stranded DNA genomes and are important pathogens in tropical and subtropical regions, but their population diversity and variability are poorly understood. Here, we have investigated variations accumulating in Tomato yellow leaf curl China virus (TYLCCNV), a geminivirus in the genus Begomovirus of the family Geminiviridae. The population variation was analyzed in a naturally infected tomato (Solanum lycopersicom) plant and in Nicotiana benthamiana and tomato plants experimentally infected with a swarm of TYLCCNV DNA clones to provide an identical sequence for initiation of infection. Our results demonstrate that the population of TYLCCNV in a naturally infected tomato plant was genetically heterogeneous and that rapid mutation occurred in the populations amplified from N. benthamiana and tomato plants that had been infected with cloned DNA. This feature of the population of TYLCCNV in these plants consisted of the consensus sequence and a pool of mutants that are not identical but are closely related to the consensus sequence, and it coincides with the quasispecies concept described for many RNA viruses. The mutation frequency was circa 10(-4) in N. benthamiana and tomato at 60 days postinoculation, a value comparable to that reported for plant RNA viruses. The quasispecies-like nature of the TYLCCNV populations suggested that TYLCCNV is capable of rapid evolution and adaptation in response to changing agricultural practices.     

Mixed Infection of Two Begomoviruses in Malvastrum coromandelianum in Fujian, China

Virus isolates were obtained from three Malvastrum coromandelianum plants showing vein thickening symptoms in Fujian Province, China. A fragment of approximately 500 bp was amplified from all the samples by PCR using the special degenerate primer pair PA/PB for begomoviruses. Sequence differences among the partial DNA-A fragments revealed that all three samples contained two virus isolates. Isolate I and isolate II share the highest nucleotide sequence identity (98–99%), respectively, with Malvastrum leaf curl Guangdong virus (MLCuGdV) and Ageratum yellow vein virus (AYVV). The complete nucleotide sequences of Fs1 and Fs2 isolates representing each virus were determined to be 2741 and 2756 nucleotides, respectively. Alignment and phylogenetic analysis showed that the complete DNA-A sequences of Fs1 and Fs2 were most closely to those of MLCuGdV (AM503104) and AYVV (AB100305), with 90.4% and 93.3% nucleotide sequence identity, respectively. Fs1 and Fs2 are considered therefore to be isolates of MLCuGdV and AYVV, respectively. This is the first report of AYVV in M. coromandelianum.     

Geminivirus disease complexes: an emerging threat

Small circular single-stranded DNA satellites have recently been isolated from plants infected with whitefly-transmitted monopartite begomoviruses. The satellites, named DNA beta, depend on the helper viruses for their proliferation and, in turn, are required for helper virus accumulation and symptom expression. They are highly diverse yet retain an overall conserved structure with respect to potential coding regions and regulatory elements. The begomovirus-satellite disease complexes are associated with economically important diseases, and have been isolated from vegetable and fibre crops, ornamental plants and weeds throughout Africa and Asia. Their widespread distribution and diversity, coupled to the global movement of plant material and the dissemination of the whitefly vector, suggests that these disease complexes pose a serious threat to tropical and sub-tropical agro-ecosystems worldwide.     

A transient assay for recombination demonstrates that Arabidopsis SNM1 and XRCC3 enhance non-homologous recombination

Replacement of endogenous genes by homologous recombination is rare in plants; the majority of genetic modifications are the result of transforming DNA molecules undergoing random genomic insertion by way of non-homologous recombination. Factors that affect chromatin remodeling and DNA repair are thought to have the potential to enhance the frequency of homologous recombination in plants. Conventional tools to study the frequencies of genetic recombination often rely on stable transformation-based approaches, with these systems being rarely capable of high-throughput or combinatorial analysis. We developed a series of vectors that use chemiluminescent (LUC and REN) reporter genes to assay the relative frequency of homologous and non-homologous recombination in plants. These transient assay vectors were used to screen 14 candidate genes for their effects on recombination frequencies in Nicotiana benthamiana plants. Over-expression of Arabidopsis genes with sequence similarity to SNM1 from yeast and XRCC3 from humans enhanced the frequency of non-homologous recombination when assayed using two different donor vectors. Transient N. benthamiana leaf systems were also used in an alternative assay for preliminary measurements of homologous recombination frequencies, which were found to be enhanced by over-expression of RAD52, MIM and RAD51 from yeast, as well as CHR24 from Arabidopsis. The findings for the assays described here are in line with previous studies that analyzed recombination frequencies using stable transformation. The assays we report have revealed functions in non-homologous recombination for the Arabidopsis SNM1 and XRCC3 genes, so the suppression of these genes' expression offers a potential means to enhance the gene targeting frequency in plants. Furthermore, our findings also indicate that plant gene targeting frequencies could be enhanced by over-expression of RAD52, MIM, CHR24, and RAD51 genes.     

Phylogenetic beta diversity of native and alien species in European urban floras

Aim Human activities have weakened biogeographical barriers to dispersal, increasing the rate of introduction of alien plants. However, their impact on beta diversity and floristic homogenization is poorly understood. Our goal is to compare the phylogenetic beta diversity of native species with that of two groups of alien species, archaeophytes and neophytes (introduced before and after ad 1500, respectively), across European urban floras to explore how biological invasions affect phylogenetic turnover at a continental scale. Location Twenty European cities located in six countries between 49 and 53° N latitude in continental Europe and the British Isles. Methods To compare the phylogenetic beta diversity of native and alien species we use the average phylogenetic dissimilarity of individual floras from their group centroid in multivariate space. Differences in phylogenetic beta diversity among different species groups are then assessed using a randomization test for homogeneity of multivariate dispersions. Results Across European urban floras, and when contrasted with natives, archaeophytes are usually associated with lower levels of phylogenetic beta diversity while neophytes tend to increase phylogenetic differentiation. Main conclusions While archaeophytes tend to promote limited homogenization in phylogenetic beta diversity, because of their diverse geographical origin together with short residence times in the invaded regions, neophytes are not promoting biotic homogenization of urban floras across Europe. Therefore, in spite of the increasing rate of alien invasion, an intense phylogenetic homogenization of urban cities is not to be expected soon.     

烟草曲茎病毒卫星DNA在不同寄主中的分子变异比较

近年来对一些单组分菜豆金色花叶病毒属病毒(begomoviruses)的研究发现,这类单组分begomoviruses伴随着一种新型卫星分子-DNAβ。对我国云南省发生的双生病毒的研究表明,云南begomoviruses普遍伴随DNAβ分子,DNAβ分子与致病性紧密相关,且begomoviruses与其伴随的卫星DNA的是共进化的。为了弄清卫星DNAβ是否存在类似于卫星RNA的高度变异,将烟草曲茎病毒(Tobacco curly shoot virus,TbCSV)及其伴随卫星DNAβ的侵染性克隆共同接种本氏烟(Nicotiana benthamiana)和心叶烟(N．glutinosa),对TbCSV卫星DNAβ在不同寄主中的分子变异进行了比较。     

Bacterial cyclic beta-(1,2)-glucan acts in systemic suppression of plant immune responses

Although cyclic glucans have been shown to be important for a number of symbiotic and pathogenic bacterium-plant interactions, their precise roles are unclear. Here, we examined the role of cyclic beta-(1,2)-glucan in the virulence of the black rot pathogen Xanthomonas campestris pv campestris (Xcc). Disruption of the Xcc nodule development B (ndvB) gene, which encodes a glycosyltransferase required for cyclic glucan synthesis, generated a mutant that failed to synthesize extracellular cyclic beta-(1,2)-glucan and was compromised in virulence in the model plants Arabidopsis thaliana and Nicotiana benthamiana. Infection of the mutant bacterium in N. benthamiana was associated with enhanced callose deposition and earlier expression of the PATHOGENESIS-RELATED1 (PR-1) gene. Application of purified cyclic beta-(1,2)-glucan prior to inoculation of the ndvB mutant suppressed the accumulation of callose deposition and the expression of PR-1 in N. benthamiana and restored virulence in both N. benthamiana and Arabidopsis plants. These effects were seen when cyclic glucan and bacteria were applied either to the same or to different leaves. Cyclic beta-(1,2)-glucan-induced systemic suppression was associated with the transport of the molecule throughout the plant. Systemic suppression is a novel counterdefensive strategy that may facilitate pathogen spread in plants and may have important implications for the understanding of plant-pathogen coevolution and for the development of phytoprotection measures.     

Beet curly top virus DI DNA-mediated resistance is linked to its size

Beet curly top virus (BCTV) infection is associated with the de novo synthesis of a heterogeneous population of subgenomic viral DNAs. Nicotiana benthamiana plants transformed with a partial repeat of one such subgenomic DNA remained susceptible to infection but produced ameliorated symptoms when agroinoculated with BCTV. Symptom amelioration is associated with the mobilization of subgenomic DNA from the integrated copy. In an attempt to improve the resistance, N. benthamiana has been transformed with a partial repeat of a much smaller subgenomic DNA. However, transgenic plants showed almost no resistance although subgenomic DNA was mobilised from the host genome. To further understand the molecular basis of the interference phenomenon, we compared the ability of BCTV to replicate and accumulate in leaf discs derived from resistant and non-resistant transgenic plants. Both subgenomic DNAs were able to interfere with virus replication but only in case of resistant plants the DI DNA efficiently suppressed viral accumulation.     

Induction of plant cell division by beet curly top virus gene C4

Beet curly top virus (BCTV) is a small DNA virus that causes tumorigenic growths (enations) in infected plants by inducing division of phloem parenchyma cells (hyperplasia). It has previously been shown that BCTV C4 plays an important role in symptom development in sugarbeet and Nicotiana benthamiana , and it has been suggested that this gene is responsible for the induction of hyperplasia. Using in situ hybridization, we show that BCTV infection is closely associated with the vascular system in these hosts, although hyperplastic cells associated with wild-type virus infection frequently do not contain detectable levels of viral DNA. Extensive hyperplasia was not observed in plants infected with a C4 mutant, demonstrating a role for C4 in virus-induced cell proliferation. Ectopic expression of C4 in transgenic N. benthamiana resulted in abnormal plant development and the production of tumorigenic growths, confirming that this gene alone is sufficient to initiate cell division in permissive cells when removed from the context of the viral genome.     

Interaction between a Nanovirus-like Component and the Tobacco Curly Shoot Virus／Satellite Complex

The biological role of DNA1, a nanovirus-like component shown to be associated with the begomovirus/satellite complex, has not yet been identified. Here, we demonstrated that DNA1 of Tobacco curly shoot virus isolate Y35 (TbCSV-Y35) attenuated leaf-curling symptoms induced by TbCSV-Y35 or TbCSV-Y35 plus Y35 DNAβ in the early stage of symptom development and induced leaf cluster at a later stage of symptom development in Nicotiana benthamiana plants. The leaf disc assay demonstrated that TbCSV-Y35 DNA1 replicated autonomously. Southern blot analysis revealed that TbCSV-Y35 DNA1 reduced viral DNA accumulation. Viral DNA accumulation was not reduced when plants were co-inoculated with TbCSV-Y35 DNAβ, but the TbCSV-Y35 DNAβ level was dramatically reduced in the presence of TbCSV-Y35 DNA1. To determine whether the interaction between TbCSV/satellite complex and DNA1 had isolate specificity, DNA1 of TbCSV isolate Y132 was cloned and sequenced. It was found to have 75% nucleotide sequence identity with TbCSV-Y35 DNA1. Infectivity tests showed that TbCSV-Y132 DNA1 had no effect on the symptoms induced by TbCSV-Y35 or TbCSV-Y35 and Y35 DNAβ in N. benthamiana plants, although Y 132 DNA1 could replicate in these plants.     

Virus-induced gene silencing of argonaute genes in Nicotiana benthamiana demonstrates that extensive systemic silencing requires Argonaute1-like and Argonaute4-like genes

Several distinct pathways of RNA silencing operate in plants with roles including the suppression of virus accumulation, control of endogenous gene expression, and direction of DNA and chromatin modifications. Proteins of the Dicer-Like and Argonaute (AGO) families have key roles within these silencing pathways and have distinct biochemical properties. We are interested in the relationships between different silencing pathways and have used Nicotiana benthamiana as a model system. While not being an amenable plant for traditional genetics, N. benthamiana is extensively used for RNA-silencing studies. Using virus-induced gene silencing technology we demonstrate that both NbAGO1- and NbAGO4-like genes are required for full systemic silencing but not for silencing directed by an inverted repeat transgene. Phenotypic differences between the virus-induced gene silencing plants indicate that NbAGO1 and NbAGO4 like act at different stages of the silencing pathways. Suppression of NbAGO1 expression recapitulated the hypomorphic mutant phenotype of certain Arabidopsis (Arabidopsis thaliana) ago1 alleles, however, suppression of NbAgo4 like resulted in phenotypes differing in some respects from those reported for Arabidopsis ago4. We suggest that the small interfering RNA amplification step required for full systemic silencing is dependent upon a nuclear event requiring the activity of NbAGO4 like.     

Viroid-induced symptoms in Nicotiana benthamiana plants are dependent on RDR6 activity

Viroids are small self-replicating RNAs that infect plants. How these noncoding pathogenic RNAs interact with hosts to induce disease symptoms is a long-standing unanswered question. Recent experimental data have led to the suggestive proposal of a pathogenic model based on the RNA silencing mechanism. However, evidence of a direct relation between key components of the RNA silencing pathway and symptom expression in infected plants remains elusive. To address this issue, we used a symptomatic transgenic line of Nicotiana benthamiana that expresses and processes dimeric forms of Hop stunt viroid (HSVd). These plants were analyzed under different growing temperature conditions and were used as stocks in grafting assays with the rdr6i-Nb line, in which the RNA-dependent RNA polymerase 6 (RDR6) is constitutively silenced. Here, we show that the symptom expression in N. benthamiana plants is independent of HSVd accumulation levels but dependent on an active state of the viroid-specific RNA silencing pathway. The scion of rdr6i-Nb plants remained asymptomatic when grafted onto symptomatic plants, despite an accumulation of a high level of mature forms of HSVd, indicating the requirement of RDR6 for viroid-induced symptom production. In addition, the RDR6 requirement for symptom expression was also observed in wild-type N. benthamiana plants mechanically infected with HSVd. These results provide biological evidence of the involvement of the viroid-specific RNA silencing pathway in the symptom expression associated with viroid pathogenesis.     

DspA/E, a type III effector of Erwinia amylovora, is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell death

DspA/E is a pathogenicity factor of Erwinia amylovora that is translocated into the plant cell cytoplasm through an Hrp type III secretion system. Transient expression of dspA/E in Nicotiana benthamiana or yeast induced cell death, as it does in N. tabacum and apple as described previously. DspA/E-induced cell death in N. benthamiana was not inhibited by coexpression of AvrPtoB of Pseudomonas syringae pv. tomato , which inhibits programmed cell death (PCD) induced by several other elicitors in plants. Silencing of NbSGT1 , the expression of which is required for PCD mediated by several resistance proteins of plants, prevented DspA/E-induced cell death in N. benthamiana. However, silencing of NbRAR1 , or two MAP kinase kinase genes, which are required for PCD associated with many resistance genes in plants, did not prevent cell death induced by DspA/E. Silencing of NbSGT1 also compromised non-host resistance against E. amylovora . E. amylovora grew rapidly within the first 24 h after infiltration in N. benthamiana , and DspA/E was required for this early rapid growth. However, bacterial cell numbers decreased after 24 h in TRV-vector-transformed plants, whereas a dspA/E mutant strain grew to high populations in NbSGT1 -silenced plants. Our results indicate that DspA/E enhances virulence of E. amylovora in N. benthamiana, but the bacteria are then recognized by the plant, resulting in PCD and death of bacterial cells or restriction of bacterial cell growth.