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.     

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.     

Inhibition of virus DNA replication by artificial zinc finger proteins

Prevention of virus infections is a major objective in agriculture and human health. One attractive approach to the prevention is inhibition of virus replication. To demonstrate this concept in vivo, an artificial zinc finger protein (AZP) targeting the replication origin of the Beet severe curly top virus (BSCTV), a model DNA virus, was created. In vitro DNA binding assays indicated that the AZP efficiently blocked binding of the viral replication protein (Rep), which initiates virus replication, to the replication origin. All of the transgenic Arabidopsis plants expressing the AZP showed phenotypes strongly resistant to virus infection, and 84% of the transgenic plants showed no symptom. Southern blot analysis demonstrated that BSCTV replication was completely suppressed in the transgenic plants. Since the mechanism of viral DNA replication is well conserved among plants and mammals, this approach could be applied not only to agricultural crop protection but also to the prevention of virus infections in humans.     

Trade-off between foliage and tuber resistance to Phthorimaea operculella in wild potatoes

Beet leafhopper, Circulifer tenellus (Baker) (Homoptera: Cicadellidae), is the only known North American vector of beet curly top virus (Geminiviridae), which causes major economic losses in a number of crops including sugar beet, tomato, beans, and peppers. Beet curly top virus is a phloem-limited, persistently transmitted, circulative geminivirus. The strain/species of curly top virus used in this study is the CFH strain, also referred to as beet severe curly top virus (BSCTV). The direct current (DC) electrical penetration graph technique was used to determine the specific stylet penetration behavior associated with inoculation of BSCTV. Viruliferous leafhoppers were allowed to feed on healthy 3–4-week-old sugar beet plants until specific electrical penetration graph waveforms were produced, at which point feeding was artificially terminated. A series of comparisons between leafhoppers that produced different combinations of waveforms clearly implicated waveform D1 as the only waveform correlated with inoculation of BSCTV. All successful inoculations contained waveform D1, and 56 out of 64 leafhoppers that produced waveform D1 successfully inoculated test plants. Eighty-five leafhoppers did not produce waveform D1 and none of these inoculated BSCTV. While the occurrence of waveform D1 appears to be necessary for BSCTV inoculation, there was no correlation between duration of waveform D1 and inoculation success rate. The correlation of waveform D1 and BSCTV inoculation found in this study implies that waveform D1 is associated with phloem salivation.     

A nucleic acid hydrolyzing recombinant antibody confers resistance to curtovirus infection in tobacco

A catalytic single chain variable antibody (scFv), 3D8 scFv, which has DNase activities, was functionally expressed in Nicotiana tabacum. The subcellular localization of the GFP-fused 3D8 indicated that the 3D8 protein was expressed in the cytosol of the N. tabacum protoplasts. Progenies of the transgenic tobacco plants exhibited complete resistance against two single stranded (ss) DNA geminiviruses, including the Beet curly top virus and the Beet severe curly top virus, without viral accumulation or disease symptoms. We presented a novel strategy for targeting the viral DNA itself in a sequence non-specific manner, rather than the viral proteins or RNAs, in order to generate virus-resistant transgenic plants. No noticeable adverse effects on the growth and reproduction of the transgenic plants were observed. Our results demonstrated that targeting viral DNA is an effective strategy for protecting plants from ssDNA viruses.     

BSCTV C2 attenuates the degradation of SAMDC1 to suppress DNA methylation-mediated gene silencing in Arabidopsis

Plant viruses are excellent tools for studying microbial-plant interactions as well as the complexities of host activities. Our study focuses on the role of C2 encoded by Beet severe curly top virus (BSCTV) in the virus-plant interaction. Using BSCTV C2 as bait in a yeast two-hybrid screen, a C2-interacting protein, S-adenosyl-methionine decarboxylase 1 (SAMDC1), was identified from an Arabidopsis thaliana cDNA library. The interaction was confirmed by an in vitro pull-down assay and a firefly luciferase complemention imaging assay in planta. Biochemical analysis further showed that the degradation of the SAMDC1 protein was inhibited by MG132, a 26S proteasome inhibitor, and that C2 could attenuate the degradation of the SAMDC1 protein. Genetic analysis showed that loss of function of SAMDC1 resulted in reduced susceptibility to BSCTV infection and reduced viral DNA accumulation, similar to the effect of BSCTV C2 deficiency. Bisulfite sequencing analysis further showed that C2 deficiency caused enhanced DNA methylation of the viral genome in infected plants. We also showed that C2 can suppress de novo methylation in the FWA transgenic assay in the C2 transgene background. Overexpression of SAMDC1 can mimic the suppressive activity of C2 against green fluorescent protein-directed silencing. These results suggest that C2 interferes with the host defense mechanism of DNA methylation-mediated gene silencing by attenuating the 26S proteasome-mediated degradation of SAMDC1.     

Regulation of tomato leaf curl viral gene expression in host tissues

The regulation of expression of the two virion-sense (V1 and V2) and four complementary-sense (C1, C2, C3, and C4) open reading frames (ORFs) of Tomato leaf curl virus (TLCV) was studied in both stably and transiently transformed Nicotiana tabacum tissues with fusions with the beta-glucuronidase (GUS) reporter gene. GUS-expressing transgenic lines were obtained with each of the four complementary-sense gene-GUS fusion constructs and with truncated versions of the virion-sense gene-GUS fusion constructs (V1GUSdeltaC and V2GUSdeltaC) lacking complementary-sense sequences encoding the C1, C2, and C3 ORFs. However, little or no GUS expression was observed in kanamycin-resistant plants transformed with full-length, virion-sense gene constructs (V1GUS and V2GUS) constituting the complete viral genome. In contrast, V1GUS and V2GUS were found to direct high-level GUS expression in transient assays with tobacco protoplasts, suggesting that integration of viral constructs containing functional, complementary-sense genes may lead to repression or deletion of the introduced constructs in transgenic tissues. V2GUS expression in the transient protoplast assay was found to be severely curtailed by specific mutation of the C2 ORF, supporting a role for the C2 protein in transactivation of TLCV virion-sense gene expression. TLCV ORF-GUS constructs displayed distinctive tissue expression patterns in transgenic tobacco plants that could be divided into constitutive (C1, C4, and V2GUSdeltaC), predominantly vascular (C2, C3), or reduced expression in cells associated with the vascular bundles (V1GUSdeltaC). The significance of these results is discussed in terms of current models of gene function and regulation in geminiviruses.     

Involvement of C4 Protein of Beet Severe Curly Top Virus (Family Geminiviridae) in Virus Movement

Background

Beet severe curly top virus (BSCTV) is a leafhopper transmitted geminivirus with a monopartite genome. C4 proteins encoded by geminivirus play an important role in virus/plant interaction.

Methods and Findings

To understand the function of C4 encoded by BSCTV, two BSCTV mutants were constructed by introducing termination codons in ORF C4 without affecting the amino acids encoded by overlapping ORF Rep. BSCTV mutants containing disrupted ORF C4 retained the ability to replicate in Arabidopsis protoplasts and in the agro-inoculated leaf discs of N. benthamiana, suggesting C4 is not required for virus DNA replication. However, both mutants did not accumulate viral DNA in newly emerged leaves of inoculated N. benthamiana and Arabidopsis, and the inoculated plants were asymptomatic. We also showed that C4 expression in plant could help C4 deficient BSCTV mutants to move systemically. C4 was localized in the cytosol and the nucleus in both Arabidopsis protoplasts and N. benthamiana leaves and the protein appeared to bind viral DNA and ds/ssDNA nonspecifically, displaying novel DNA binding properties.

Conclusions

Our results suggest that C4 protein in BSCTV is involved in symptom production and may facilitate virus movement instead of virus replication.     

Identification of a tolerant locus on Arabidopsis thaliana to hypervirulent beet curly top virus CFH strain

The infection of hosts by the geminivirus depends on the interactions between host and viral factors for viral DNA replication, viral gene expression, and the movement of virus throughout the hosts. This work reports that a hypervirulent strain of Beet curly top virus (BCTV) is different in its ability to infect several ecotypes of Arabidopsis thaliana. Symptoms appeared on Arabidopsis ecotypes around 7 to 10 d after inoculation with BCTV-CFH. Symptoms were more severe in ecotype SKKU including severe leaf curling and development of severely deformed and stunted boting compared to Col-O as a lab standard ecotype. One ecotype Cen-O was asymptomatic to BCTV-CFH infection. Studies of viral DNA replication and virus movement in three excised organs of asymptomatic Cen-O demonstrated that BCTV-CFH could replicate viral DNA and move systemically in this ecotype, suggesting that tolerance was due to the blocks of interactions between host and viral factors on symptom development. This asymptomatic phenotype is similar to the mutation of leftward ORFs, especially ORF R2. Genetic analysis of this ecotype Cen-O indicated that tolerance is due to a single recessive locus.     

RKP, a RING finger E3 ligase induced by BSCTV C4 protein, affects geminivirus infection by regulation of the plant cell cycle

The C4 protein from Curtovirus is known as a major symptom determinant, but the mode of action of the C4 protein remains unclear. To understand the mechanism of involvement of C4 protein in virus–plant interactions, we introduced the C4 gene from Beet severe curly top virus (BSCTV) into Arabidopsis under a conditional expression promoter; the resulting overexpression of BSCTV C4 led to abnormal host cell division. RKP, a RING finger protein, which is a homolog of the human cell cycle regulator KPC1, was discovered to be induced by BSCTV C4 protein. Mutation of RKP reduced the susceptibility to BSCTV in Arabidopsis and impaired BSCTV replication in plant cells. Callus formation is impaired in rkp mutants, indicating a role of RKP in the plant cell cycle. RKP was demonstrated to be a functional ubiquitin E3 ligase and is able to interact with cell-cycle inhibitor ICK/KRP proteins in vitro . Accumulation of the protein ICK2/KRP2 was found increased in the rkp mutant. The above results strengthen the possibility that RKP might regulate the degradation of ICK/KRP proteins. In addition, the protein level of ICK2/KRP2 was decreased upon BSCTV infection. Overexpression of ICK1/KRP1 in Arabidopsis could reduce the susceptibility to BSCTV. In conclusion, we found that RKP is induced by BSCTV C4 and may affect BSCTV infection by regulating the host cell cycle.     

Expression of a recombinant chimeric protein of hepatitis A virus VP1-Fc using a replicating vector based on Beet curly top virus in tobacco leaves and its immunogenicity in mice

We describe the expression and immunogenicity of a recombinant chimeric protein (HAV VP1-Fc) consisting of human hepatitis A virus VP1 and an Fc antibody fragment using a replicating vector based on Beet curly top virus (BCTV) in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Recombinant HAV VP1-Fc was expressed with a molecular mass of approximately 68?kDa. Recombinant HAV VP1-Fc, purified using Protein A Sepharose affinity chromatography, elicited production of specific IgG antibodies in the serum after intraperitoneal immunization. Following vaccination with recombinant HAV VP1-Fc protein, expressions of IFN-γ and IL-4 were increased in splenocytes at the time of sacrifice. Recombinant VP1-Fc from infiltrated tobacco plants can be used as an effective experimental immunogen for research into vaccine development.     

Neurons differentially activate the herpes simplex virus type 1 immediate-early gene ICP0 and ICP27 promoters in transgenic mice

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins are required for the expression of viral early and late proteins. It has been hypothesized that host neuronal proteins regulate expression of HSV-1 IE genes that in turn control viral latency and reactivation. We investigated the ability of neuronal proteins in vivo to activate HSV-1 IE gene promoters (ICP0 and ICP27) and a late gene promoter (gC). Transgenic mice containing IE (ICP0 and ICP27) and late (gC) gene promoters of HSV-1 fused to the Escherichia coli beta-galactosidase coding sequence were generated. Expression of the ICP0 and ICP27 reporter transgenes was present in anatomically distinct subsets of neurons in the absence of viral proteins. The anatomic locations of beta-galactosidase-positive neurons in the brains of ICP0 and ICP27 reporter transgenic mice were similar and included cerebral cortex, lateral septal nucleus, cingulum, hippocampus, thalamus, amygdala, and vestibular nucleus. Trigeminal ganglion neurons were positive for beta-galactosidase in adult ICP0 and ICP27 reporter transgenic mice. The ICP0 reporter transgene was differentially regulated in trigeminal ganglion neurons depending upon age. beta-galactosidase-labeled cells in trigeminal ganglia and cerebral cortex of ICP0 and ICP27 reporter transgenic mice were confirmed as neurons by double labeling with antineurofilament antibody. Nearly all nonneuronal cells in ICP0 and ICP27 reporter transgenic mice and all neuronal and nonneuronal cells in gC reporter transgenic mice were negative for beta-galactosidase labeling in the absence of HSV-1. We conclude that factors in neurons are able to differentially regulate the HSV-1 IE gene promoters (ICP0 and ICP27) in transgenic mice in the absence of viral proteins. These findings are important for understanding the regulation of the latent and reactivated stages of HSV-1 infection in neurons.