Detection of eight different tospovirus species by a monoclonal antibody against the common epitope of NSs protein

Rabbit antisera against the nucleocapsid protein (NP) have been commonly used for detection of tospoviruses and classification into serogroups or serotypes. Mouse monoclonal antibodies (MAbs) with high specificity to the NPs have also been widely used to identify tospovirus species. Recently, a serogroup-specific MAb against the NSs protein of Watermelon silver mottle virus (WSMoV) was produced by our laboratory to react with five members of WSMoV serogroup, i.e., WSMoV, Capsicum chlorosis virus (CaCV), Calla lily chlorotic spot virus (CCSV), Peanut bud necrosis virus (PBNV) and Watermelon bud necrosis virus (WBNV). The epitope recognized by the NSs MAb was determined and the comparison with the reported sequences of tospoviral NSs proteins revealed that the epitope is highly conserved at the N-terminal region of NSs proteins among members of WSMoV and Iris yellow spot virus (IYSV) serogroups, and Melon yellow spot virus (MYSV) serotype. When the NSs MAb was further used to react with the crude antigens of MYSV serotype, IYSV and Tomato yellow ring virus (TYRV) of IYSV serogroup, strong serological reactions, both in ELISA and western blotting, were observed. Thus, our results indicated that the NSs MAb is a useful and convenient tool for detection of the eight tospovirus species. It is also suggested that these eight Asian-type tospoviruses, i.e., WSMoV, CaCV, CCSV, PBNV, WBNV, MYSV, IYSV and TYRV, may share a common evolutionary ancesto

Comparison of the effects of kanamycin and geneticin on regeneration of papaya from root tissue

Kanamycin and geneticin are commonly used for the selection of neomycin phosphotransferase II (npt II) transformed plants. Since papaya tissue is sensitive to both antibiotics, it is difficult to explore their effects on the regeneration process solely based on using non-transformed tissues. Adventitious roots derived from npt II-transgenic and non-transgenic papaya shoots in vitro were used as explants in this investigation. The effects of kanamycin and geneticin on callus formation, embryogenesis, and conversion of somatic embryos to shoots were compared. Callus growth derived from npt II-transformed root explants was apparently enhanced on kanmycin within 50-200 mg l(-1) or on geneticin within 12.5-50 mg l(-1) as compared to those on antibiotic-free controls. The percentages of npt II-transformed somatic embryo-forming callus were not significantly different (16.3-18.3%) on geneticin less than 6.25 mg l(-1) and only slightly reduced (11.2-15.7%) on geneticin within 12.5-50 mg l(-1), whereas, formation of somatic embryos was strongly suppressed on kanamycin media. Conversion rates of npt II-transformed somatic embryos to shoots were not significantly different among all kanamycin or geneticin treatments. Percentages of the callus derived from non-transformed root explants were greatly reduced on the medium containing more than 25 mg l(-1) kanamycin or geneticin, and no somatic embryos formed from untransformed callus on any kanamycin or geneticin media. Our results indicated that somatic embryogenesis of callus derived from npt II-transformed root explants of papaya was strongly inhibited by kanamycin. Thus, to regenerate npt II-transformed cells from papaya root tissue, we recommend using the lower concentration geneticin (12.5-25 mg l(-1)) to avoid the adverse effects of kanamycin on embryogenesis

Enhancement of in vitro growth of papaya multishoots by aeration

Efficient micropropagation of papaya (Carica papaya L.) has become crucial for multiplication of specific sex types of papaya or transgenic lines resistant to virus infection. In this study, aeration at different intervals with a 0.02 mu m filter disc in the closure of culture flasks ensured exchange of gas components. The effect of aeration on development of multibuds to multishoots was investigated. Multibuds grown, in culture flasks after one-week without aeration followed by a two-week aeration treatment caused a 41% increase in the number of shoots greater than or equal to 0.5 cm, 42% increase in leaf expansion, and 17% increase in leaf numbers in comparison with unaerated materials. Ethylene and oxygen concentrations in the culture flasks were measured by gas chromatography and oxygen electrode at weekly intervals during the culture period. Oxygen concentrations were slightly different between aerated and unaerated culture flasks. Ethylene in the unaerated flask reached the highest level (0.11 ppm) 2 weeks after the treatment, while accumulation of ethylene in the aerated flasks was not detected. The multishoots grown for 3 weeks without aeration showed growth retardation on leaves and epinasty on petioles

The ability of Papaya ringspot virus strains overcoming the transgenic resistance of papaya conferred by the coat protein gene is not correlated with higher degrees of sequence divergence from the transgene

The coat protein (CP) gene mediated transgenic resistance is found to be the best approach for protecting papaya plants against the destructive disease caused by Papaya ringspot viruses (PRSV). In order to study the variability of PRSV and the potential threat to the CP-transgenic resistance, five virus isolates were collected from transgenic plants of papaya line 16-0-1, which carry the CP gene of the typical mosaic strain of Taiwan PRSV YK, in an approved test field and fourteen from untransformed papaya plants in different areas of Taiwan. The results of biological, serological, and molecular characterization indicated that all isolates are related to PRSV YK. Among them, the isolate 5-19 from the transgenic line and the isolates CS and TD2 from untransformed papaya were able to overcome the YK CP gene-mediated resistance of papaya lines 18-2-4, 17-0-5, and 16-0-1, which provide high degrees of resistance to different geographic PRSV strains of Hawaii (HA), Mexico (MX), and Thailand (TH). These three isolates were also able to cause symptoms on untransformed papaya plants more severe than those induced by YK. In addition to the host reactions, the variability of the collected 19 isolates was also analyzed and compared with YK and other geographic strains by heteroduplex mobility assay (HMA) and sequence analyses. The results of HMA indicated that the CP genes of isolates 5-19 and TD2 are more divergent than those of other isolates when compared with YK. However, sequence analyses of the transgenic-resistance overcoming isolates 5-19, CS, and TD2 revealed that their CP coding regions and the 3' untranslated regions (UTRs) share nucleotide identities of 93.9-96.6% and 94.2-97.9% with those of YK, respectively; whereas the other geographic strains of HA, MX, and TH that could not overcome the transgenic resistance share lower nucleotide identities of 89.8-92.6% and 92.3-95.3% with those of YK, respectively. Our results indicate that the ability for overcoming the transgenic resistance is not solely correlated with higher degrees of sequence divergence from the transgene. The possible mechanism for overcoming the transgenic resistance and the potential threat of these PRSV strains to the application of the transgenic papaya lines carrying PRSV YK CP gene are discussed

Efficient rooting for establishment of papaya plantlets by micropropagation

A low cost micropropagation protocol to produce high quality root systems which are easy and economical to acclimatize is essential for large-scale micropropagation of papaya (Carica papaya L.). In this study, individual shoots (> 0.5 cm) with 2 similar to 3 leaves from in vitro papaya multiple shoots were cultured on MS agar medium containing 2.5 mu M IBA under dark conditions for 1 week for root induction. They were then transferred to agar or vermiculite media, containing half strength MS medium, under aerated or non-aerated conditions, for root development. Rooting percentage of shoots cultured for 2 weeks in aerated vermiculite was 94.5%, compared with 90.0% in non-aerated vermiculite, 71.1% in aerated agar, and 62.2% in non-aerated agar. Shoots with roots were acclimated in vermiculite under 100% RH for 1 week and then under ambient conditions for 2 weeks in a temperature-controlled growth chamber (28 degrees C). The survival rates of the plantlets were 94.5% from aerated vermiculite, 87.8% from non-aerated vermiculite, 42.2% from aerated agar, and 35.6% from non-aerated agar. Thus, root induction in low-concentration IBA agar medium followed by root development in vermiculite containing half strength MS medium under aerated conditions results in efficient rooting of in vitro papaya shoots

Emerging threat of thrips-borne Melon yellow spot virus on melon and watermelon in Taiwan

The thrips-borne Melon yellow spot virus (MYSV) has recently been found infecting cucurbits in Taiwan. However, this virus was indistinguishable from another thrips-borne virus species Watermelon silver mottle virus (WSMoV), which has been devastating on cucurbits in Taiwan for decades, when the antisera against their nucleocapsid proteins (NPs) were used for diagnosis. To understand the incidences of WSMoV and MYSV in melon and watermelon fields, a survey was conducted in central and southern Taiwan from July 2007 to December 2009. The samples collected from symptomatic plants were tested by indirect enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (MAbs) specific to the NP of WSMoV or MYSV and the reliability of the results was verified by reverse transcription-polymerase chain reaction (RT-PCR) using species-specific primers. Among a total of 10,480 melon samples collected, 6% and 18.2% of them were found singly infected with WSMoV and MYSV, respectively, and 0.16% infected with both viruses. On the other hand, among 1,811 watermelon samples assayed, 22.4% and 9.2% samples were singly infected with WSMoV and MYSV, respectively, and 0.17% were infected with both viruses. In addition, the aphid-borne viruses Zucchini yellow mosaic virus (ZYMV), Papaya ringspot virus watermelon type (PRSV-W) and Cucumber mosaic virus (CMV) were also detected as prevalent viruses. Our results indicated that mixed infection with the two thrips-borne viruses is rare. Moreover, host preference for both viruses is different; WSMoV prevails on watermelon whereas MYSV is more widespread on melon. We conclude that MYSV has become a serious threat for watermelon and melon production in Taiwan and the possible control measures are discussed

Role of calcineurin in Porphyromonas gingivalis-induced myocardial cell hypertrophy and apoptosis

Background and objective: Periodontal pathogen Porphyromonas gingivalis (P. gingivalis) increased cardiomyocyte hypertrophy and apoptosis whereas Actinobaeillus actinomycetemcomitans and Prevotella intermedia had no effects. The purpose of this study is to clarify the role of calcineurin signaling pathway in P. gingivalis-induced H9c2 myocardial cell hypertrophy and apoptosis. Methods: DNA fragmentation, nuclear condensation, cellular morphology, calcineurin protein, Bcl2- associated death promoter (Bad) and nuclear factor of activated T cell (NFAT)-3 protein products in cultured H9c2 myocardial cell were measured by agarose gel electrophoresis, DAPI, immunofluorescence, and Western blotting following P. gingivalis and/or pre-administration of CsA (calcineurin inhibitors cyclosporin A). Results: P. gingivalis not only increased calcineurin protein, NFAT-3 protein products and cellular hypertrophy, but also increased DNA fragmentation, nuclear condensation and Bad protein products in H9c2 cells. The increased cellular sizes, DNA fragmentation, nuclear condensation, and Bad of H9c2 cells treated with P. gingivalis were all significantly reduced after pre-administration of CsA. Conclusion: Our findings suggest that the activity of calcineurin signal pathway may be initiated by P. gingivalis and further lead to cell hypertrophy and death in culture H9c2 myocardial cells

Evidence for two-quark content of f0(980)f_{0}(980) in exclusive b→cb\to c decays

Inspired by a large decay branching ratio (BR) of $B^{+}\to f_{0}(980)K^{+}$ measured by Belle recently, we propose that a significant evidence of the component of $n\bar{n}=(u\bar{u}+d\bar{d})/\sqrt{2}$ in $f_{0}(980)$ could be demonstrated in exclusive $b\to c$ decays by the observation of $f_{0}(980)$ in the final states $\bar{B}\to D^{0(*)} \pi^{+} \pi^{-}(KK)$ and $\bar{B}\to J/\Psi \pi^{+} \pi^{-}(KK)$. We predict the BRs of $\bar{B}\to D^{0(*)} (J/\Psi) f_{0}(980)$ to be ${\cal {O}}(10^{-4})$ (${\cal {O}}(10^{-5})$) while the unknown wave functions of $D^{(*)0}$ ($J/\Psi$) are chosen to fit the observed decays of $\bar{B}\to D^{(*)0} \pi^{0} (J/\Psi K^{0(*)})$.Comment: 4 pages, 2 figures, Revtex4, version to appear in PR