Analysis of Pathogenicity and Virulence Factors of Ageratum leaf curl Sichuan virus.
ABSTRACT: Ageratum leaf curl Sichuan virus (ALCScV) is a novel monopartite begomovirus, which was identified from Ageratum conyzoides plants in Sichuan Province, China. In this study, we showed that ALCScV can induce typical dwarf and downward leaf-curling symptoms in Ageratum conyzoides, Helianthus annuus, and Nicotiana benthamiana plants and that the noncognate betasatellite can enhance disease symptoms and increase viral accumulation. Expression of the ALCScV-encoded V2, C1, and C4 proteins through a Potato virus X (PVX) vector caused severe symptoms in N. benthamiana. Further study revealed no symptoms in N. benthamiana plants inoculated with infectious ALCScV clones lacking the C4 protein and that the relative viral DNA accumulation levels significantly decreased when compared with ALCScV-inoculated plants. Thus, our mutational analyses demonstrated that C4 is a pathogenicity determinant that plays key roles in symptom formation and virus accumulation. Furthermore, we also demonstrated that the second glycine of C4 was critical for ALCScV pathogenicity.
Project description:Samples of two Ageratum conyzoides, one Sonchus oleraceus and one turnip (Brassica rapa var. rapa) exhibiting virus-like symptoms were collected from Pakistan and Nepal. Full-length begomovirus clones were obtained from the four plant samples and betasatellite clones from three of these. The begomovirus sequences were shown to be isolates of Ageratum enation virus (AEV) with greater than 89.1% nucleotide sequence identity to the 26 AEV sequences available in the databases. The three betasatellite sequences were shown to be isolates of Ageratum yellow leaf curl betasatellite (AYLCB) with greater than 90% identity to the 18 AYLCB sequences available in the databases. The AEV sequences were shown to fall into two distinct strains, for which the names Nepal (consisting of isolates from Nepal, India, and Pakistan-including the isolates identified here) and India (isolates occurring only in India) strains are proposed. For the clones obtained from two AEV isolates, with their AYLCB, infectivity was shown by Agrobacterium-mediated inoculation to Nicotiana benthamiana, N. tabacum, Solanum lycopersicon and A. conyzoides. N. benthamiana plants infected with AEV alone or betasatellite alone showed no symptoms. N. benthamiana plants infected with AEV with its associated betasatellite showed leaf curl symptoms. The findings show that AEV is predominantly a virus of weeds that has the capacity to infect crops. AYLCB appears to be the common partner betasatellite of AEV and is associated with diseases with a range of very different symptoms in the same plant species. The inability to satisfy Koch's postulates with the cloned components of isolate SOL in A. conyzoides suggests that the etiology may be more complex than a single virus with a single betasatellite.
Project description:Ageratum conyzoides L., a weed species widely distributed throughout southeast Asia, frequently exhibits striking yellow vein symptoms associated with infection by Ageratum yellow vein virus (AYVV), a member of the Geminiviridae (genus Begomovirus). Most begomoviruses have bipartite genomes (DNAs A and B), but only a DNA A has been identified for AYVV. We demonstrate that yellow vein disease of A. conyzoides results from co-infection by AYVV DNA A (2,741 nt) and a circular DNA that is approximately half its size (1,347 nt) that we designate DNA beta. Apart from the sequence TAATATTAC, common to all geminiviruses and containing the initiation site of rolling circle replication, DNA beta shows negligible sequence homology either to AYVV DNA A or to DNA B associated with bipartite begomoviruses. DNA beta depends on DNA A for replication and is encapsidated by DNA A-encoded coat protein and so has characteristics of a DNA satellite. However, systemic infection of A. conyzoides by DNA A alone is sporadic and asymptomatic, and DNA A accumulation is reduced to 5% or less of its accumulation in the presence of DNA beta. Therefore, DNA A and DNA beta together form a previously unrecognized disease-inducing complex. Our data also demonstrate that the nanovirus-like DNA 1 component associated with infected A. conyzoides plays no essential role in the disease and represents a satellite-like DNA. Furthermore, the satellite DNA previously found associated with tomato leaf curl virus is probably a defective DNA beta homologue.
Project description:<i>Ageratum conyzoides</i> L. is an important Chinese medicinal plant. In this study, we reported the complete chloroplast genome of <i>A</i>. <i>conyzoides</i>. The chloroplast genome sequence is 151,309?bp in length and consisted of a large single copy (LSC) region (83,884?bp), a small single copy (SSC) region (17,771?bp), and two inverted repeats (IRs) (24,827?bp). It was composed of 126 genes and they were 81 protein-coding genes, 30 tRNA genes, 8 rRNA genes, and 7 pseudogene. Phylogenetic analysis with reported chloroplast genomes can not only show that <i>A. conyzoides</i> has a close genetic relationship with <i>Centaurea diffusa</i> and <i>Carthamus tinctorius</i>, but also provide new evidence for the identification of <i>Praxelis clematidea</i> and <i>A. conyzoides</i>.
Project description:BACKGROUND:Giardia duodenalis causes giardiasis in humans, particularly in developing countries. Despite the availability of treatments, resistance to some of the commercial anti-Giardia drugs has been reported in addition to their harmful side effects. Therefore, novel treatments for giardiasis are required. In this study, we aimed to assess the in vitro activity of crude extracts of Ageratum conyzoides against G. duodenalis trophozoites. METHODS:Plants were classified into three groups based on their flower colors: white (W), purple (P), and white-purple (W-P). Plants were separately cut into leaf (L) and flower (F) parts. Changes in internal organelle morphology of trophozoites following exposure to crude extracts were assessed using transmission electron microscopy (TEM). In subsequent experiments, efficacy of the most active essential oils from crude extracts [half maximal inhibitory concentrations (IC50)???100??g/mL] against G. duodenalis trophozoites was tested. In vitro anti-Giardia assays using essential oils were performed in the same way as those performed using crude extracts. RESULTS:LW-P and FP extracts showed high activity (IC50???100??g/mL) against G. duodenalis trophozoites, with IC50 ±?SD values of 45.67?±?0.51 and 96.00?±?0.46??g/mL, respectively. In subsequent experiments, IC50 ±?SD values of LW-P and FP essential oils were 35.00?±?0.50 and 89.33?±?0.41??g/mL, respectively. TEM revealed the degeneration of flagella and ventral discs of G. duodenalis trophozoites following exposure to crude extracts. CONCLUSION:Crude LW-P and FP extracts of A. conyzoides showed the highest activity against G. duodenalis. Exposure to crude extract induced changes in the flagella and ventral discs of G. duodenalis trophozoites, which play important roles in attachment to the surface of mucosal cells. Our results suggest that the tested extracts warrant further research in terms of their efficacy and safety as giardiasis treatment.
Project description:Goat weed (Ageratum conyzoides L.), or bandotan in Indonesia, is an herbaceous plant that broadly grows up in both subtropical as well as tropical areas. This herb contains many phytoconstituents which have many benefits in different aspects. The essential oil contains phytochemicals such as phenol, phenolic ester, and coumarin, whereas many compounds can been identified in the whole part such as terpenoid, steroid, chromene, pyrrolizidine alkaloid, and flavonoid. Empirically, this herb has been used as an antihemorrhagic, antiseptic, antileprosy, and wound-healing agent. This article reviews the potency of the herb in medication according to the chemical substances being deposited, which are collected from numerous studies, followed by its in silico bioactivity prediction as well as its pharmaceutical dosage form formulation.
Project description:The whitefly-transmitted geminiviruses induce severe developmental abnormalities in plants. Geminivirus-encoded C4 protein functions as one of viral symptom determinants that could induce abnormal cell division. However, the molecular mechanism by which C4 contributes to cell division induction remains unclear. Here we report that tomato leaf curl Yunnan virus (TLCYnV) C4 interacts with a glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase, designed NbSK?, in Nicotiana benthamiana. Pro32, Asn34 and Thr35 of TLCYnV C4 are critical for its interaction with NbSK? and required for C4-induced typical symptoms. Interestingly, TLCYnV C4 directs NbSK? to the membrane and reduces the nuclear-accumulation of NbSK?. The relocalization of NbSK? impairs phosphorylation dependent degradation on its substrate-Cyclin D1.1 (NbCycD1;1), thereby increasing the accumulation level of NbCycD1;1 and inducing the cell division. Moreover, NbSK?-RNAi, 35S::NbCycD1;1 transgenic N. benthamiana plants have the similar phenotype as 35S::C4 transgenic N. benthamiana plants on callus-like tissue formation resulted from abnormal cell division induction. Thus, this study provides new insights into mechanism of how a viral protein hijacks NbSK? to induce abnormal cell division in plants.
Project description:Monopartite begomoviruses and their associated satellites form unique disease complexes that have emerged as a serious threat to agriculture worldwide. It is well known that frequent recombination contributes to the diversification and evolution of geminiviruses. In this study, we identified a novel defective satellite molecule (RecSat) in association with Tobacco leaf curl Yunnan virus (TbLCYNV) in a naturally infected tobacco plant. Sequence analysis showed that Recsat comprises 754 nucleotides in size and is a chimera involving alphasatellite and betasatellite sequences, containing both betasatellite-conserved region and alphasatellite stem-loop structure. Recombination analysis revealed that RecSat has arisen from three independent recombination events likely involving Tomato yellow leaf curl China betasatellite, Ageratum yellow vein China betasatellite and Tobacco curly shoot alphasatellite. Co-inoculation of RecSat with TbLCYNV induced symptoms indistinguishable from those induced by TbLCYNV alone in Nicotiana benthamiana. Southern blot hybridization showed that RecSat could be trans-replicated stably in N. benthamiana plants by TbLCYNV, and impaired the accumulation of helper virus and co-inoculated alphasatellite. Our results provide the first evidence for recombination between two distinct types of satellites among geminivirus complex and highlight recombination as a driving force for geminivirus evolution.
Project description:A previously unknown disease which causes severe vein thickening and inward leaf curl was observed in a number of opium poppy (Papaver somniferum L.) plants. The sequence analysis of full-length viral genome and associated betasatellite reveals the occurrence of Ageratum enation virus (AEV) and Ageratum leaf curl betasatellite (ALCB), respectively. Co-infiltration of cloned agroinfectious DNAs of AEV and ALCB induces the leaf curl and vein thickening symptoms as were observed naturally. Infectivity assay confirmed this complex as the cause of disease and also satisfied the Koch's postulates. Comprehensive microscopic analysis of infiltrated plants reveals severe structural anomalies in leaf and stem tissues represented by unorganized cell architecture and vascular bundles. Moreover, the characteristic blebs and membranous vesicles formed due to the virus-induced disintegration of the plasma membrane and intracellular organelles were also present. An accelerated nuclear DNA fragmentation was observed by Comet assay and confirmed by TUNEL and Hoechst dye staining assays suggesting virus-induced programmed cell death. Virus-infection altered the biosynthesis of several important metabolites. The biosynthesis potential of morphine, thebaine, codeine, and papaverine alkaloids reduced significantly in infected plants except for noscapine whose biosynthesis was comparatively enhanced. The expression analysis of corresponding alkaloid pathway genes by real time-PCR corroborated well with the results of HPLC analysis for alkaloid perturbations. The changes in the metabolite and alkaloid contents affect the commercial value of the poppy plants.
Project description:Tomato leaf curl Guangdong virus (ToLCGdV) is a begomovirus associated with a Tomato yellow leaf curl disease (TYLCD) epidemic in Guangdong province, China. Being the least conserved protein among geminivirus proteins, the function of C4 during ToLCGdV infection has not been elucidated. In this study, the infectious clones of ToLCGdV and a ToLCGdV mutant (ToLCGdVmC4) with disrupted C4 ORF were constructed. Although ToLCGdV and ToLCGdVmC4 could infect Nicotiana benthamiana and tomato plants, ToLCGdVmC4 elicited much milder symptoms compared with ToLCGdV. To further verify the role of C4 in viral pathogenesis, C4 was expressed in N. benthamiana from Potato virus X (PVX) vector. The results showed that ToLCGdV C4 enhanced the pathogenicity of PVX and induced more severe developmental abnormalities in plants compared with PVX alone or PVX-mC4. In addition, ToLCGdV C4 suppresses systemic gene silencing in the transgenic N. benthamiana line 16c, but not local gene silencing induced by sense GFP in wild-type N. benthamiana plants. Moreover, C4 suppresses transcriptional gene silencing (TGS) by reducing the DNA methylation level of 35S promoter in 16c-TGS N. benthamiana plants. Furthermore, C4 could also interact with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1), suggesting that C4 may suppress gene silencing by interfering with the function of BAM1 in the cell-to-cell spread of RNAi. All these results suggest that C4 is a pathogenic determinant of ToLCGdV, and C4 may suppress post-transcriptional gene silencing (PTGS) by interacting with BAM1.
Project description:A new grapevine geminivirus A (GGVA) isolate (named as GGVA-17YM1) and its associated defective genome (GGVA-D) were identified from a grapevine sample collected in Yuanmou, Yunnan Province, using sRNA high throughput sequencing and traditional Sanger sequencing. To explore the pathogenicity of GGVA and GGVA-D, infectious clones of GGVA-17YM1 and GGVA-D-17YM1 were constructed. Infection assays indicated that Nicotiana benthamiana plants inoculated with GGVA alone or a combination of GGVA and GGVA-D exhibited upward curled apical leaves and dwarfism. Southern blotting and quantitative real-time polymerase chain reaction analysis revealed that GGVA-D increased the accumulation level of GGVA DNA. Transient expression using a PVX-derived recombinant vector indicated that C2 and C4 encoded by GGVA are involved in symptom induction in N. benthamiana. Furthermore, the V2 protein inhibited local RNA silencing in co-infiltration assays in GFP transgenic N. benthamiana plants. Subsequently, full-length genome sequencing resulted in the identification of 11 different isolates of GGVA and 9 associated defective DNA molecules. Phylogenetic analysis based on whole genome sequences showed that all GGVA isolates, including our sequences, clustered into two distinct branches with no geographical grouping. Analyses of molecular variation indicated single nucleotide polymorphisms (SNPs) with more transitions (55.97%) than transversions (44.03%). Furthermore, the main variants for ORF C1, C3, or V1 were synonymous mutations, and non-synonymous mutations for ORF C2, C4, and V2. Genetic selection analysis indicated that negative selection acted on four ORFs (V1, C1, C2, and C3), while V2 and C4 were under positive selection. Our results contribute to the characterization of the genetic diversity of GGVA and provide insights into its pathogenicity.