Identification of a promoter motif involved in Curtovirus sense-gene expression in transgenic Arabidopsis

Expression of the seven open reading frames (ORFs) of single-stranded DNA Curtoviruses such as Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV) is driven by a bi-directional promoter. To investigate this bi-directional promoter activity with respect to viral late gene expression, transgenic Arabidopsis plants expressing a GUS reporter gene under the control of either the BCTV or BSCTV bi-directional promoter were constructed. Transgenic plants harboring constructs showed higher expression levels when the promoter of the less virulent BCTV was used than when the promoter of the more virulent BSCTV was used. In transgenic seedlings, the reporter gene constructs were expressed primarily in actively dividing tissues such as root tips and apical meristems. As the transgenic plants matured, reporter gene expression diminished but viral infection of mature transgenic plants restored reporter gene expression, particularly in transgenic plants containing BCTV virion-sense gene promoter constructs. A 30 base pair conserved late element (CLE) motif was identified that was present three times in tandem in the BCTV promoter and once in that of BSCTV. Progressive deletion of these repeats from the BCTV promoter resulted in decreased reporter gene expression, but BSCTV promoters in which one or two extra copies of this motif were inserted did not exhibit increased late gene promoter activity. These results demonstrate that Curtovirus late gene expression by virion-sense promoters depends on the developmental stage of the host plant as well as on the number of CLE motifs present in the promoter.     

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.     

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.     

Characterization of the electrical penetration graphs of the psyllid Bactericera trigonica on carrots

The psyllid Bactericera trigonica Hodkinson (Hemiptera: Triozidae) is a carrot and celery pest recently described as a vector of the plant pathogenic bacterium Candidatus Liberibacter solanacearum (Lso) on Apiaceae. Detailed information on vector stylet penetration activities is essential in the study of Lso transmission. In this study we used the electrical penetration graph (EPG) technique, characterized waveforms produced during the various stylet penetration activities in carrot leaves, and correlated them with stylet tracks and salivary sheath termini on plant tissues as well as with Lso inoculation. In addition, the effect of Lso in B. trigonica on the stylet penetration activities was tested. The EPG waveforms identified were: waveforms C1 and C2 detected in the mesophyll, waveforms D, E1, and E2 near or in the phloem sieve elements, and waveform G in the xylem vessels. A waveform pattern not previously reported for psyllids was the ‘pseudo‐potential drop’ (pseudo‐pd), characterized by sudden voltage dips similar to potential drops. However, the lowered voltage appeared to be inverted when the plant voltage is negative, indicating that it is caused by an increased resistance period and not due to a cell puncture. A direct correlation is shown between the waveform E1 and salivation into phloem sieve elements by B. trigonica as the inoculation of Lso occurred in a period as short as 30 s of E1; Lso transmission occurred in 17 of 35 plants (48%). Stylet activities during waveforms C or D had no consequences on the inoculation of Lso. In conclusion, Lso infection directly affects the probing behaviour of B. trigonica by increasing the total duration of C and D waveforms, but not variables related to phloem salivation (Lso inoculation) or ingestion (Lso acquisition). The reported information here is fundamental for identifying the psyllid vector traits of behaviour associated with transmission of Lso to Apiaceae.     

Beet leafhopper (Hemiptera: Cicadellidae) settling behavior, survival, and reproduction on selected host plants

Experiments were conducted to determine the settling behavior, survival, and reproduction of the beet leafhopper, Circulifer tenellus (Baker), when maintained on selected host plants. This leafhopper was recently identified in the Columbia Basin of Washington and Oregon as the probable vector of the beet leafhopper-transmitted virescence agent phytoplasma, causal agent of several vegetable crop diseases, including potato purple top. Plants selected for study were sugar beet, Beta vulgaris L.; radish, Raphanus sativus L.; dry bean, Phaseolus vulgaris L.; potato, Solanum tuberosum L.; carrot, Daucus carota L.; and tomato, Lycopersicon esculentum Mill. Leafhopper adults were confined on caged plants, and settling behavior was observed during a 72-h period and survival was monitored for 40 d. Also, oviposition and nymphal production were investigated by maintaining leafhoppers for approximately 90 d on each of the selected plants. Sixty to 100% of leafhoppers settled on all studied plants during the first 5 h, but settling on bean and tomato declined sharply thereafter. Leafhopper mortality was very high on bean and tomato, with 95 and 65% of the leafhoppers, respectively, dying in about a week. In contrast, 77, 90, and 95% of leafhoppers maintained on potato, sugar beet, and radish, respectively, survived until the end of the 40-d experimental period. Beet leafhopper oviposition and nymphal production and development only occurred on sugar beet, radish, and potato; reproduction was lower on potato.     

Characterization and correlation of DC electrical penetration graph waveforms with feeding behavior of beet leafhopper, Circulifer tenellus

Feeding behavior of beet leafhopper, Circulifer tenellus (Baker) (Homoptera: Cicadellidae), was studied with a DC electrical penetration graph. Nine different electrical penetration graph waveforms associated with feeding were identified and characterized. Waveforms were correlated with specific feeding behaviors using a number of techniques, including high magnification video recording, honeydew analysis, stylectomy, and histological processing. Waveforms were grouped into three phases based on feeding behavior: pathway phase (waveforms A, B1, B2, and C), non-phloem ingestion phase (waveform G), and phloem phase (waveforms D1, D2, D3, and D4). No ingestion was found to occur during waveforms A, B1, B2, and C. Waveform G was associated primarily with ingestion of xylem sap and occasionally with ingestion of mesophyll sap. Stylet tips were located in phloem during waveforms D1, D2, and D3, and waveforms D2 and D3 were correlated with ingestion of phloem sap. Waveform D4 probably also plays a role in phloem ingestion, because D4 is very brief and always occurs embedded in either waveform D2 or D3. In contrast to most other homopteran insects, rate of honeydew production (and hence rate of ingestion) was much lower on phloem than on xylem. More rapid rates of ingestion are expected on phloem, because its high turgor pressure drives sap into the feeding insect whereas the negative pressure of xylem sap is expected to cause a slow rate of ingestion. The very slow ingestion rate of beet leafhopper feeding on phloem suggests that it is not able to exploit the high turgor pressure of phloem to achieve the high rate of ingestion that is typical of phloem ingestion by other insects.     

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.     

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.     

Electrical penetration graphs from Cicadulina mbila on maize, the fine structure of its stylet pathways and consequences for virus transmission efficiency

Five distinct electrical penetration graph waveforms characterising the feeding behaviour of the leafhopper Cicadulina mbila Naudé (Homoptera: Cicadellidae) on maize (Zea mays L.) were obtained using a DC based system. The waveforms were distinguished by spectral features and by statistical analysis of their median voltages, durations and time to first waveform recording. By changing the polarity of the system voltage and the level of the input resistor it was shown that the waveforms are mainly determined by the electromotive force (emf) component. Based on the correlation between waveforms and the fine structure of the stylet pathways observed by transmission electron microscopy, insect's activities have been associated with five waveforms: stylet pathway formation (waveform 1), active ingestion (waveform 2), putative stylet work (waveform 3), salivation (waveform 4) and passive ingestion (waveform 5). Like waveform E1 and E2 of aphids, waveforms 4 and 5 of C. mbila correspond to feeding activities in sieve tubes. However, unlike aphids which probe briefly in non-vascular cells, waveform 2 corresponds to active ingestion in cells, where the cell content is partially ingested and hence the organelles' integrity severely affected. These observations suggest that this specific feeding feature, typical of leafhoppers, determines their ability to acquire geminivirus virions located in the plant cell nucleus.     

EPG monitoring of the probing behaviour of the common brown leafhopper Orosius orientalis on artificial diet and selected host plants

The common brown leafhopper Orosius orientalis (Hemiptera: Cicadellidae) is a polyphagous vector of a range of economically important pathogens, including phytoplasmas and viruses, which infect a diverse range of crops. Studies on the plant penetration behaviour by O. orientalis were conducted using the electrical penetration graph (EPG) technique to assist in the characterisation of pathogen acquisition and transmission. EPG waveforms representing different probing activities were acquired from adult O. orientalis probing in planta, using two host species, tobacco Nicotiana tabacum and bean Phaseolus vulgaris, and in vitro using a simple sucrose-based artificial diet. Five waveforms (O1?CO5) were evident when O. orientalis fed on bean, whereas only four waveforms (O1?CO4) and three waveforms (O1?CO3) were observed when the leafhopper fed on tobacco and on the artificial diet, respectively. Both the mean duration of each waveform and waveform type differed markedly depending on the food substrate. Waveform O4 was not observed on the artificial diet and occurred relatively rarely on tobacco plants when compared with bean plants. Waveform O5 was only observed with leafhoppers probing on beans. The attributes of the waveforms and comparative analyses with previously published Hemipteran data are presented and discussed, but further characterisation studies will be needed to confirm our suggestions.     

Characterisation of the feeding behaviour of western flower thrips in terms of electrical penetration graph (EPG) waveforms

Western flower thrips, Frankliniella occidentalis (Pergande) causes damage to plants when they are feeding. Also, this thrips species transmits Tomato spotted wilt virus (TSWV) during stylet penetration. We investigated the penetration behaviour (probing) of thrips on pepper leaves and on liquid diet by electrical penetration graph (EPG, DC-system) recording. In addition, we used high-magnification video observations to correlate EPG waveforms with the insect's posture, head movements, and muscle contractions. Also, EPGs were correlated with probing on liquid diets containing radio-active tracers to distinguish and quantify ingestion waveforms. The previously described waveforms P, Q, and R were distinguished and additionally, a new waveform 'S' was distinguished. Waveform P could be linked with mandibular leaf penetration, waveform Q presumably with insertion of the maxillary stylets, and waveform R with ingestion of cell contents, whereas waveform S could not be correlated with any behavioural activity. Histology of the feeding damage in pepper leaves shows that thrips ingests the contents of multiple cells per probe.     

Beet leafhopper (Hemiptera: Cicadellidae) transmits the Columbia Basin potato purple top phytoplasma to potatoes, beets, and weeds

Experiments were conducted to determine whether the beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), transmits the purple top phytoplasma to potato, Solanum tuberosum L.; beets, Beta vulgaris L.; and selected weed hosts. The beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma was identified as the causal agent of the potato purple top disease outbreaks that recently occurred in the Columbia Basin of Washington and Oregon. The phytoplasma previously was found to be associated almost exclusively with the beet leafhopper, suggesting that this insect is the probable vector of BLTVA in this important potato-growing region. Eight potato cultivars, including 'Russet Burbank', 'Ranger Russet', 'Shepody', 'Umatilla Russet', 'Atlantic', 'FL-1879', 'FL-1867', and 'FL-1833', were exposed for a week to BLTVA-infected beet leafhoppers. After exposure, the plants were maintained outdoors in large cages and then tested for BLTVA by using polymerase chain reaction after 6 to 7 wk. The leafhoppers transmitted BLTVA to seven of the eight exposed potato cultivars. Sixty-four percent of the exposed plants tested positive for the phytoplasma. In addition, 81% of the BLTVA-infected potato plants developed distinct potato purple top disease symptoms. Beet leafhoppers also transmitted BLTVA to beets and several weeds, including groundsel, Senecio vulgaris L.; shepherd's purse, Capsella bursa-pastoris (L.) Medik); kochia, Kochia scoparia (L.) Schrad; and Russian thistle, Salsola kali L. This is the first report of transmission of BLTVA to potatoes, beets, and the above-mentioned four weed species. Results of the current study prove that the beet leafhopper is a vector of the potato purple top disease.     

The effects of Beet mild yellowing virus and Beet chlorosis virus on the yield of UK field-grown sugar beet in 1997,1999 and 2000

The separate effects of the aphid‐transmitted poleroviruses; Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV), on the yield of field‐grown sugar beet were studied following different inoculation dates from May to July in 1997,1999 and 2000. Each sugar beet plant within the appropriate plots was infected with virus using at least 10 wingless viruliferous Myzus persicae per plant. In all 3 years, overall yield losses caused by BMYV were negatively correlated with time of infection with early season (May) inoculations causing 18–27% losses in sugar yield but late season losses only 4–15%. BChV decreased the sugar yield and sugar content of beet following early season inoculations, although the effects on sugar yield were more variable (range 8–24%) and the virus appeared to be less damaging compared to BMYV. However, inoculations with BChV in July of each year caused greater root and sugar losses than inoculations with BMYV at that time. Both poleroviruses increased the sodium content of the roots early in the season, although neither virus had an effect on potassium levels at any stage.     

Beet curly top virus transmission by artificially fed and injected beet leafhoppers (Circulfer tenellus)

The transmission of beet curly top virus (BCTV) by leafhoppers, Circulifer tenellus, fed virus through Parafilm® membranes was compared with their transmission when injected with virus from phloem exudates of Amsinckia douglasiana. Virus uptake from ³²P-labelled test solutions and the resulting virus transmission, as measured by an infectivity index, varied widely. By contrast, insects injected with virus transmitted with similar efficiencies. If insects were fasted for 3, 5, or 7 h before a 6 h acquisition access period on test solutions, their ³²P, and presumably virus uptake, was greater than that of nonfasted insects and their variability in virus transmission decreased. The proportion of insects transmitting curly top virus, after fasting and given a 6 h acquisition access period, was similar to that of insects injected with virus. Maximum liquid uptake by the beet leafhopper occurred with a 12% sucrose solution.     

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.