Development of Attenuated Viruses for Effective Protection against Pepper Veinal Mottle Virus in Tomato Crops.

Tomato (Solanum lycopersicum) is the most important vegetable and fruit crop in the family Solanaceae worldwide. Numerous pests and pathogens, especially viruses, severely affect tomato production, causing immeasurable market losses. In Taiwan, the cultivation of tomato crops is mainly threatened by insect-borne viruses, among which pepper veinal mottle virus (PVMV) is one of the most prevalent. PVMV is a member of the genus Potyvirus of the family Potyviridae and is non-persistently transmitted by aphids. Its infection significantly reduces tomato fruit yield and quality. So far, no PVMV-resistant tomato lines are available. In this study, we performed nitrite-induced mutagenesis of the PVMV tomato isolate Tn to generate attenuated PVMV mutants. PVMV Tn causes necrotic lesions in Chenopodium quinoa leaves and severe mosaic and wilting in Nicotiana benthamiana plants. After nitrite treatment, three attenuated PVMV mutants, m4-8, m10-1, and m10-11, were selected while inducing milder responses to C. quinoa and N. benthamiana with lower accumulation in tomato plants. In greenhouse tests, the three mutants showed different degrees of cross-protection against wild-type PVMV Tn. m4-8 showed the highest protective efficacy against PVMV Tn in N. benthamiana and tomato plants, 100% and 97.9%, respectively. A whole-genome sequence comparison of PVMV Tn and m4-8 revealed that 20 nucleotide substitutions occurred in the m4-8 genome, resulting in 18 amino acid changes. Our results suggest that m4-8 has excellent potential to protect tomato crops from PVMV. The application of m4-8 in protecting other Solanaceae crops, such as peppers, will be studied in the future.

Leucine 127 of Cucurbit Chlorotic Yellows Virus P22 Is Crucial for Its RNA Silencing Suppression Activity and Pathogenicity.

Plant viruses produce particular suppressors to antagonize the host defense response of RNA silencing to establish infection. Cucurbit chlorotic yellows virus (CCYV), a member of the genus Crinivirus of the family Closteroviridae, severely damages the production of economically essential cucurbits worldwide. Here, we used the attenuated zucchini yellow mosaic virus (ZYMV) vector ZAC to express individual coding sequences, including CP, CPm, P25, and P22, of a Taiwan CCYV isolate (CCYV-TW) to identify their possible roles as pathogenicity determinants. ZAC is an HC-Pro function mutant that lacks the ability of local lesion induction on Chenopodium quinoa leaves and induces mild mottling followed by recovery on its natural host zucchini squash plants. Only the recombinant expressing CCYV-TW P22 complemented the effect of ZAC HC-Pro dysfunction, causing more severe symptoms on zucchini squash plants and restoring lesion formation on C. quinoa leaves, with lesions forming faster than those generated by the wild-type ZYMV. This suggests that CCYV-TW P22 is a virulence enhancer. Sequence analysis of criniviral P22s revealed the presence of four conserved leucine residues (L10, L17, L84, and L127) and one conserved lysine residue (K185). The five P22 residues conserved among the CCYV isolates and the P22 orthologs of two other criniviruses were each substituted with alanine in CCYV-TW P22 to investigate its ability to suppress RNA silencing and pathogenicity. The results provide new insights into CCYV-P22, showing that the L127 residue of P22 is indispensable for maintaining its stability in RNA silencing suppression and essential for virulence enhancement.

Identification of an emerging cucumber virus in Taiwan using Oxford nanopore sequencing technology.

BACKGROUND: In June 2020, severe symptoms of leaf mosaic and fruit malformation were observed on greenhouse-grown cucumber plants in Xizhou Township of Changhua County, Taiwan. An unknown virus, designated CX-2, was isolated from a diseased cucumber sample by single lesion isolation on Chenopodium quinoa leaves. Identification of CX-2 was performed. Moreover, the incidence of cucumber viruses in Taiwan was also investigated. METHODS: Transmission electron microscopy was performed to examine virion morphology. The portable MinION sequencer released by Oxford Nanopore Technologies was used to detect viral sequences in dsRNA of CX-2-infected leaf tissue. The whole genome sequence of CX-2 was completed by Sanger sequencing and analyzed. Reverse transcription-polymerase chain reaction (RT-PCR) with species-specific primers and indirect enzyme-linked immunosorbent assay (ELISA) with anti-coat protein antisera were developed for virus detection in the field [see Additional file 1]. RESULTS: Icosahedral particles about 30 nm in diameter were observed in the crud leaf sap of CX-2-infected C. quinoa plant. The complete genome sequence of CX-2 was determined as 4577 nt long and shared 97.0-97.2% of nucleotide identity with that of two cucumber Bulgarian latent virus (CBLV) isolates in Iran and Bulgaria. Therefore, CX-2 was renamed CBLV-TW. In 2020-2022 field surveys, melon yellow spot virus (MYSV) had the highest detection rate of 74.7%, followed by cucurbit chlorotic yellows virus (CCYV) (32.0%), papaya ringspot virus virus watermelon type (PRSV-W) (10.7%), squash leaf curl Philippines virus (SLCuPV) (9.3%), CBLV (8.0%) and watermelon silver mottle virus (WSMoV) (4.0%). Co-infection of CBLV and MYSV could be detected in field cucumbers. CONCLUSION: The emerging CBLV-TW was identified by nanopore sequencing. Whole genome sequence analysis revealed that CBLV-TW is closely related, but phylogenetically distinct, to two known CBLV isolates in Bulgaria and Iran. Detection methods including RT-PCR and indirect ELISA have been developed to detect CBLV and to investigate cucumber viruses in central Taiwan. The 2020-2022 field survey results showed that MYSV and CCYV were the main threats to cucumbers, with CBLV, SLCuPV and WSMoV were occasionally occurring.

Rose Virome Analysis and Identification of a Novel Ilarvirus in Taiwan.

Rose (Rosa spp.), especially R. hybrida, is one of the most popular ornamental plants in the world and the third largest cut flower crop in Taiwan. Rose mosaic disease (RMD), showing mosaic, line patterns and ringspots on leaves, is a common rose disease caused by the complex infection of various viruses. Due to pests and diseases, the rose planting area in Taiwan has been decreasing since 2008; however, no rose virus disease has been reported in the past five decades. In the spring of 2020, rose samples showing RMD-like symptoms were observed at an organic farm in Chiayi, central Taiwan. The virome in the farm was analyzed by RNA-seq. Rose genomic sequences were filtered from the obtained reads. The remaining reads were de novo assembled to generate 294 contigs, 50 of which were annotated as viral sequences corresponding to 10 viruses. Through reverse transcription-polymerase chain reaction validation, a total of seven viruses were detected, including six known rose viruses, namely apple mosaic virus, prunus necrotic ringspot virus, rose partitivirus, apple stem grooving virus, rose spring dwarf-associated virus and rose cryptic virus 1, and a novel ilarvirus. After completing the whole genome sequencing and sequence analysis, the unknown ilarvirus was demonstrated as a putative new species, tentatively named rose ilarvirus 2. This is the first report of the rose virus disease in Taiwan.

IbTLD modulates reactive oxygen species scavenging and DNA protection to confer salinity stress tolerance in tobacco.

Plants accumulate reactive oxygen species (ROS) that may damage the cells under prolonged stress conditions. Reduction of the excessive ROS production can alleviate oxidative damage and enhance the survival rates under stress. TLDc-containing protein (TLD) was reported to confer tolerance to oxidative stress, but the regulatory mechanism of TLD remains unclear. In this study, we ectopically overexpressed the Ipomoea batatas TLDc gene (IbTLD) in tobacco and characterized its functions. RNA-sequencing analysis and Gene Ontology term enrichment analysis revealed that IbTLD up-regulates auxin-responsive genes in response to oxidative stress. Under salinity stress, the IbTLD transgenic lines showed higher germination rates, chlorophyll contents, and root lengths than wild type (W38). In addition, the IbTLD transgenic lines showed higher expression of ROS scavenging genes, nudix hydrolases, ROS scavenging enzyme activity, and lesser DNA damage compared to W38 under salinity stress. Therefore, our results suggest that IbTLD activates the expression of ROS scavenging genes and confers tolerance to salinity stress in planta.

Complete genome sequence of Amazon lily mosaic virus isolated from amaryllis (Hippeastrum hybridum Hort.).

In April 2011, a virus was isolated by single-lesion isolation on Chenopodium quinoa leaves from an amaryllis plant with chlorotic ringspots in a private garden in Changhua County, Taiwan. An Illumina MiSeq sequencing system was used to determine the genomic nucleotide (nt) sequence of the virus. A de novo-assembled contig with 9377 nt, containing an open reading frame encoding a putative potyviral polyprotein, was annotated as the potyvirus Amazon lily mosaic virus (ALiMV), sharing 95.5% nt sequence identity with a partial genomic sequence of ALiMV available in the GenBank database. Therefore, the amaryllis virus was designated as ALiMV-TW. Through 5´ and 3´ rapid amplification of cDNA ends (RACE), the complete 9618-nt genome sequence of ALiMV-TW was determined. Sequence comparisons indicated that the genome and polyprotein of ALiMV-TW share 52.3-65.1% nt and 30.1-64.2% aa sequence identity, respectively, with those of other potyviruses. This is the first report of a complete genome sequence of ALiMV.

Paladin, a tyrosine phosphatase-like protein, is required for XA21-mediated immunity in rice.

XA21 encodes a rice immune receptor that confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo). XA21-mediated immunity is triggered by recognition of a small protein called RaxX-sY (required for activation of XA21-mediated immunity X, tyrosine-sulfated) secreted by Xoo. To identify components regulating XA21-mediated immunity, we generated and screened a mutant population of fast-neutron-mutagenized rice expressing Ubi:Myc-XA21 for those susceptible to Xoo. Here, we report the characterization of one of these rice mutants, named sxi2 (suppressor of XA21-mediated immunity-2). Whole-genome sequencing revealed that sxi2 carries a deletion of the PALADIN (PALD) gene encoding a protein with three putative protein tyrosine phosphatase-like domains (PTP-A, -B, and -C). Expression of PALD in the sxi2 genetic background was sufficient to complement the susceptible phenotype, which requires the catalytic cysteine of the PTP-A active site to restore resistance. PALD co-immunoprecipitated with the full-length XA21 protein, whose levels are positively regulated by the presence of the PALD transgene. Furthermore, we foundd that sxi2 retains many hallmarks of XA21-mediated immunity, similar to the wild type. These results reveal that PALD, a previously uncharacterized class of phosphatase, functions in rice innate immunity, and suggest that the conserved cysteine in the PTP-A domain of PALD is required for its immune function.

Transmission mode of watermelon silver mottle virus by Thrips palmi.

Thrips and thrips-transmitted tospoviruses cause significant losses in crop yields worldwide. The melon thrips (Thrips palmi) is not only a pest of cucurbit crops, but also a vector that transmits tospoviruses, such as the watermelon silver mottle virus (WSMoV). Vector transmission of tospoviruses has been well studied in the tomato spotted wilt virus (TSWV)-Frankliniella occidentalis model system; however, until now the transmission mode of WSMoV by T. palmi has not been sufficiently examined. The results of the transmission assays suggest that T. palmi transmits WSMoV in a persistent manner, and that the virus is mainly transmitted by adults, having been ingested at the first-instar larval stage. Complementary RNAs corresponding to the NSm and NSs genes of WSMoV were detected in viruliferous thrips by reverse transcription-polymerase chain reaction; NSs protein was also detected in viruliferous thrips by western blotting, verifying the replication of WSMoV in T. palmi. Furthermore, we demonstrated that in thrips infected with WSMoV at the first-instar larval stage, the virus eventually infected various tissues of the adult thrips, including the primary salivary glands. Taken together, these results suggest that T. palmi transmits WSMoV in a persistent-propagative mode. The results of this study make a significant contribution to the understanding of the transmission biology of tospoviruses in general.

Characterization of a New Orthotospovirus from Chilli Pepper in Yunnan Province, China.

Chilli pepper (Capsicum annuum L.) is one of the most important crops in Yunnan Province, China. An orthotospovirus isolate 14YV855 was isolated from a diseased chilli pepper plant exhibiting yellow ringspots and necrosis on leaves in Shiping County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province in 2014. The complete genome sequence of 14YV855 was determined. The small, medium, and large RNAs are 3,428, 4,781, and 8,917 nucleotides long, respectively. The complete nucleocapsid (N) protein of 14YV855 shares a high amino acid identity of 84.8 to 89.9% to that of Capsicum chlorosis virus (CaCV), Groundnut bud necrosis virus (GBNV), Watermelon bud necrosis virus (WBNV), and Watermelon silver mottle virus (WSMoV), which is slightly less than the 90% identity threshold for the demarcation of new Orthotospovirus sp. Phylogenetic analyses revealed that the N protein and RNA-dependent RNA polymerase of 14YV855 are the most related to WSMoV, while the NSs, NSm, and Gn/Gc proteins are similar to those of GBNV. As expected, 14YV855 is serologically related to CaCV, GBNV, WBNV, and WSMoV when the monoclonal antibody against the N protein of WSMoV was used; however, 14YV855 can be distinguished from other orthotospoviruses by reverse-transcription PCR using the specific primers. Our results indicate that 14YV855 is a new Orthotospovirus sp. belonging to the WSMoV serogroup and is provisionally named Chilli yellow ringspot virus.

Molecular Characterization and Detection of a Genetically Distinct Tomato Chlorosis Virus Strain in Taiwan.

The whitefly-transmitted tomato chlorosis virus (ToCV) belonging to the genus Crinivirus (family Closteroviridae) affects tomato production worldwide. ToCV was first recorded in Taiwan in 1998 affecting tomato production. In this study, a local virus isolate XS was obtained, after serial whitefly transmissions from a diseased tomato plant displaying general chlorosis were collected in central Taiwan. The whole genome sequence of XS was determined from cDNA fragments amplified by reverse transcription (RT)-PCR, first using the degenerate primers for viruses of Closteroviridae and followed by degenerate and specific primers designed on available sequences of the ToCV isolates. The nucleotide (nt) sequences of RNA-1 and RNA-2 of the XS shared low identities of 77.8 to 78% and 78 to 78.1%, respectively, with genome segments of other ToCV isolates. Nevertheless, the viral RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (Hsp70h), and major capsid protein (CP) shared 88.3 to 96.2% amino acid (aa) identities with other ToCV isolates, indicating that XS is a new strain of this virus. Phylogenetic analyses of these three proteins indicated that all ToCV isolates from different counties outside Taiwan are closely related and clustered in the same clade, whereas the XS isolate is distinct and forms a unique branch. A one tube RT-PCR assay using primers designed from the genomic sequence of the XS was able to detect the ToCV-XS in infected tomato plants and in individual whiteflies. A field survey during 2013 to 2016 revealed a high ToCV-XS prevalence of 60.5% in 172 tested tomato samples, demonstrating that ToCV-XS is becoming an emerging threat for tomato production in Taiwan.

The antioxidant activity and nitric oxide production of extracts obtained from the leaves of Chenopodium quinoa Willd.

BACKGROUND: Most reports have indicated the antioxidant capacity of quinoa seeds. However, the leaves of Quinoa (Chenopodium quinoa Willd.) are usually worthless and little known about their biological activities. In this study, the antioxidant and immunomodulatory potential of the quinoa leaf extracts were explored. METHODS: The crude leaf extracts of quinoa were extracted using water, 50% ethanol or 95% ethanol as solvent, denoted WQL, 50% EQL and 95% EQL, respectively. The antioxidant activities of quinoa leaf extracts were assessed by the ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging and iron chelating. The total phenolic content was determined. Inhibition of nitric oxide (NO) production in the lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells was examined to gauge the anti-inflammatory activity. RESULTS: The 95% EQL showed a higher level of total phenolic content (569.5 mg GAE/g extract) and better DPPH scavenging activity. The WQL exhibited a better iron chelating capacity (28.9% at 10 mg/ml). The iron chelating activity of the 95% EQL increased in a concentration-dependent manner, which ranged from 10.9% up to 53.9%. The 50% EQL and 95% EQL significantly inhibited NO production in the LPSstimulated RAW 264.7 cells. CONCLUSION: We demonstrate that the extracts of quinoa leaves possess the biological activities of antioxidant and anti-inflammatory. Our finding suggests that the leaf extract of quinoa has potential to be utilized for natural health products.

Characterization of the genome of a phylogenetically distinct tospovirus and its interactions with the local lesion-induced host Chenopodium quinoa by whole-transcriptome analyses.

Chenopodium quinoa is a natural local lesion host of numerous plant viruses, including tospoviruses (family Bunyaviridae). Groundnut chlorotic fan-spot tospovirus (GCFSV) has been shown to consistently induce local lesions on the leaves of C. quinoa 4 days post-inoculation (dpi). To reveal the whole genome of GCFSV and its interactions with C. quinoa, RNA-seq was performed to determine the transcriptome profiles of C. quinoa leaves. The high-throughput reads from infected C. quinoa leaves were used to identify the whole genome sequence of GCFSV and its single nucleotide polymorphisms. Our results indicated that GCFSV is a phylogenetically distinct tospovirus. Moreover, 27,170 coding and 29,563 non-coding sequences of C. quinoa were identified through de novo assembly, mixing reads from mock and infected samples. Several key genes involved in the modulation of hypersensitive response (HR) were identified. The expression levels of 4,893 deduced complete genes annotated using the Arabidopsis genome indicated that several HR-related orthologues of pathogenesis-related proteins, transcription factors, mitogen-activated protein kinases, and defense proteins were significantly expressed in leaves that formed local lesions. Here, we also provide new insights into the replication progression of a tospovirus and the molecular regulation of the C. quinoa response to virus infection.

Characterization of a new isolate of pepper chlorotic spot virus from Yunnan province, China.

A new isolate, 14YV733, of pepper chlorotic spot virus (PCSV) from chili peppers in Yunnan province of China was identified. The typical tospoviral particles of 80-120 nm in diameter were observed by electron microscopy. The virus caused systemic symptoms in several solanaceous plants and the Brassica rapa L. Chinensis group with mechanical inoculation. The sap from infected leaves reacted positively to a rabbit antibody to the N protein of watermelon silver mottle virus (WSMoV) in immunoblotting. The S, M, and L RNAs of PCSV-14YV733 are 3310 nts, 4711 nts, and 8913 nts long, respectively. This is the first report of complete sequences of PCSV in mainland China. Phylogenetic analysis of all tospoviral proteins indicated that PCSV-14YV733 is closely related to members of the WSMoV serogroup.

Complete nucleotide sequences of M and L RNAs from a new pepper-infecting tospovirus, Pepper chlorotic spot virus.

Pepper chlorotic spot virus (PCSV), newly found in Taiwan, was identified as a new tospovirus based on the molecular characterization of its S RNA. In this study, the complete M and L RNA sequences of PCSV were determined. The M RNA has 4795 nucleotides (nts), encoding the NSm protein of 311 aa (34.5 kDa) in the viral (v) strand and the glycoprotein precursor (Gn/Gc) of 1122 aa (127.6 kDa) in the viral complementary (vc) strand. The L RNA has 8859 nts, encoding the RNA-dependent RNA polymerase (RdRp) of 2873 aa (330.8 kDa) in the vc strand. Analyses of the NSm, Gn/Gc and RdRp of PCSV revealed that PCSV is phylogenetically clustered within the watermelon silver mottle virus-related clade. Based on the whole genome sequence, PCSV is closely related to Tomato necrotic ringspot virus and should be classified as a new tospovirus species.

Rapid and Delayed Effects of Pulsed Radiofrequency on Neuropathic Pain: Electrophysiological, Molecular, and Behavioral Evidence Supporting Long-Term Depression.

BACKGROUND: Pulsed radiofrequency (PRF) has been widely employed for ameliorating clinical neuropathic pain. How PRF alters electrophysiological transmission and modulates biomolecular functions in neural tissues has yet to be clarified. We previously demonstrated that an early application of low-voltage bipolar PRF adjacent to the dorsal root ganglion (DRG) reduced acute neuropathic pain in animals. By contrast, the present study investigated how PRF alters postsynaptic sensitization to produce early and delayed effects on neuropathic pain. OBJECTIVES: Our objective was to test the hypothesis that a 5-minute session of PRF could rapidly produce selective long-term depression (LTD) on C-fiber-mediated spinal sensitization and sustain the effect through the long-lasting inhibition of injury-induced ERK-MAPK activation. This may explain the prolonged analgesic effect of PRF on chronic neuropathic pain. STUDY DESIGN: Experiments were conducted on both normal rats and neuropathic pain rats that received spinal nerve ligation (SNL) 8 days prior. SETTING: An animal laboratory in a medical center of a university in Taiwan. METHODS: We first compared changes in field potentials in the L5 superficial spinal dorsal horn (SDH) that were evoked by conditioning electrical stimuli in the sciatic nerve in male adult rats before (as the baseline) and after PRF stimulation for at least 2 hours. Bipolar PRF was applied adjacent to the L5 DRG at an intensity of 5 V for 5 minutes, whereas the control rats were treated with sham applications. The electrophysiological findings were tested for any correlation with induction of spinal phospho-ERK (p-ERK) in normal and neuropathic pain rats. We then investigated the delayed effect of PRF on SNL-maintained pain behaviors for 2 weeks as well as p-ERK in SDH among the control, SNL, and PRF groups. Finally, potential injury in the DRGs after PRF stimulation was evaluated through behavioral observations and ATF-3, a neuronal stress marker. RESULTS: In the evoked field-potential study, the recordings mediated through A- and C-afferent fibers were identified as A-component and C-component, respectively. PRF significantly reduced the C-components over 2 hours in both the normal and SNL rats, but it did not affect the A-components. In the SNL rats, the C-component was significantly depressed in the PRF group compared with the sham group. PRF also inhibited acute p-ERK induced by mechanical nociception in both the control and SNL rats. For a longer period, PRF ameliorated SNL-maintained mechanical allodynia for 10 days and thermal analgesia for 14 days, and it significantly reduced late ERK activation within spinal neurons and astrocytes 14 days afterward. Moreover, PRF in the normal rats did not alter basal withdrawal thresholds or increase the expression and distribution of ATF-3 in the DRGs. LIMITATIONS: Several issues should be considered before translating the animal results to clinical applications. CONCLUSIONS: Low-voltage bipolar PRF produces LTD through selective suppression on the C-component, but not on the A-component. It also inhibits ERK activation within neurons and astrocytes in SDHs. The findings suggest that PRF alleviates long-lasting neuropathic pain by selectively and persistently modulating C-fiber-mediated spinal nociceptive hypersensitivity.Key words: Pulsed radiofrequency (PRF), dorsal root ganglion (DRG), neuropathic pain, ERK activation, evoked field potential, ATF-3, long-term depression (LTD), spinal nerve ligation (SNL).

Development of a microarray for simultaneous detection and differentiation of different tospoviruses that are serologically related to Tomato spotted wilt virus.

BACKGROUND: Tospoviruses, the plant-infecting genus in the family Bunyaviridae, are thrips borne and cause severe agricultural losses worldwide. Based on the serological relationships of the structural nucleocapsid protein (NP), the current tospoviruses are divided into six serogroups. The use of NP-antisera is convenient for virus detection, but it is insufficient to identify virus species grouped in a serogroup due to the serological cross-reaction. Alternatively, virus species can be identified by the N gene amplification using specific primers. Tomato spotted wilt virus (TSWV) is the type species of the genus Tospovirus and one of the most destructive plant viruses. Eight known tospoviruses, Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Melon severe mosaic virus (MeSMV), Pepper necrotic spot virus (PNSV), Tomato chlorotic spot virus (TCSV) and Zucchini lethal chlorosis virus (ZLCV), sharing serological relatedness with TSWV in NP, are grouped in the TSWV serogroup. Most of the TSWV-serogroup viruses prevail in Europe and America. An efficient diagnostic method is necessary for inspecting these tospoviruses in Asia, including Taiwan. METHODS: A microarray platform was developed for simultaneous detection and identification of TSWV-serogroup tospoviruses. Total RNAs extracted from Chenopodium quinoa leaves separately inoculated with ANSV, CSNV, GRSV, INSV, TCSV and TSWV were used for testing purposes. The 5'-biotinylated degenerate forward and reverse primers were designed from the consensus sequences of N genes of TSWV-serogroup tospoviruses for reverse transcription-polymerase chain reaction (RT-PCR) amplification. Virus-specific oligonucleotide probes were spotted on the surface of polyvinyl chloride (PVC) chips to hybridize with PCR products. The hybridization signals were visualized by hydrolysis of NBT/BCIP with streptavidine-conjugated alkaline phosphatase. The microarray was further applied to diagnose virus infection in field crop samples. RESULTS: Amplicons of approximately 0.46 kb were amplified from all tested TSWV-serogroup tospoviruses by RT-PCR using the degenerate primer pair Pr-dTS-f/Pr-dTS-r. Virus species were identified on chips by hybridization of PCR products with respective virus-specific probes. The microarray was successfully used to diagnose TSWV infection in field pepper samples. CONCLUSIONS: In this study, a rapid, sensitive and precise microarray method has been developed to simultaneously detect and identify six TSWV-serogroup tospoviruses. The microarray platform provides a great potential to explore tospoviruses that can help researchers and quarantine staff to prevent invasions of tospoviruses.

Mutation of the rice XA21 predicted nuclear localization sequence does not affect resistance to Xanthomonas oryzae pv. oryzae.

BACKGROUND: The rice receptor kinase XA21 confers robust resistance to the bacterial pathogen Xanthomonas oryzaepv. oryzae(Xoo). We previously reported that XA21 is cleaved in transgenic plants overexpressing XA21 with a GFP tag (Ubi-XA21-GFP) and that the released C-terminal domain is localized to the nucleus. XA21 carries a predicted nuclear localization sequence (NLS) that directs the C-terminal domain to the nucleus in transient assays, whereas alanine substitutions in the NLS disrupt the nuclear localization. METHODS: To determine if the predicted NLS is required for XA21-mediated immunity in planta, we generated transgenic plants overexpressing an XA21 variant carrying the NLS with the same alanine substitutions (Ubi-XA21nls-GFP). RESULTS: Ubi-XA21nls-GFP plants displayed slightly longer lesion lengths, higher Xoobacterial populations after inoculation and lower levels of reactive oxygen species production compared with the Ubi-XA21-GFP control plants. However, the Ubi-XA21nls-GFP plants express lower levels of protein than that observed in Ubi-XA21-GFP. DISCUSSION: These results demonstrate that the predicted NLS is not required for XA21-mediated immunity.

Spatial Ion Peak Compression and its Utility in Ion Mobility Spectrometry.

A novel concept for ion spatial peak compression is described, and discussed primarily in the context of ion mobility spectrometry (IMS). Using theoretical and numerical methods, the effects of using non-constant (e.g., linearly varying) electric fields on ion distributions (e.g., an ion mobility peak) is evaluated both in the physical and temporal domains. The application of a linearly decreasing electric field in conjunction with conventional drift field arrangements is shown to lead to a reduction in IMS physical peak width. When multiple ion packets (i.e., peaks) in a selected mobility window are simultaneously subjected to such fields, there is ion packet compression (i.e., a reduction in peak widths for all species). This peak compression occurs with only a modest reduction of resolution, which can be quickly recovered as ions drift in a constant field after the compression event. Compression also yields a significant increase in peak intensities. Ion mobility peak compression can be particularly useful for mitigating diffusion-driven peak broadening over very long path length separations (e.g., in cyclic multi-pass arrangements), and for achieving higher S/N and IMS resolution over a selected mobility range. Graphical Abstract ᅟ.

Monoclonal antibodies for differentiating infections of three serological-related tospoviruses prevalent in Southwestern China.

BACKGROUND: The thrips-borne tospoviruses Calla lily chlorotic spot virus (CCSV), Tomato zonate spot virus (TZSV) and a new species provisionally named Tomato necrotic spot associated virus (TNSaV) infect similar crops in southwestern China. The symptoms exhibiting on virus-infected crops are similar, which is difficult for distinguishing virus species by symptomatology. The sequences of nucleocapsid proteins (NPs) of CCSV, TNSaV and TZSV share high degrees of amino acid identity with each other, and their serological relationship was currently demonstrated from the responses of the previously reported monoclonal antibodies (MAbs) against the NP of CCSV (MAb-CCSV-NP) and the nonstructural NSs protein of Watermelon silver mottle virus (WSMoV) (MAb-WNSs). Therefore, the production of virus-specific antibodies for identification of CCSV, TNSaV and TZSV is demanded to improve field surveys. METHODS: The NP of TZSV-13YV639 isolated from Crinum asiaticum in Yunnan Province, China was bacterially expressed and purified for producing MAbs. Indirect enzyme-linked immunosorbent assay (ELISA) and immunoblotting were conducted to test the serological response of MAbs to 18 tospovirus species. Additionally, the virus-specific primers were designed to verify the identity of CCSV, TNSaV and TZSV in one-step reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Two MAbs, denoted MAb-TZSV-NP(S15) and MAb-TZSV-NP(S18), were screened for test. MAb-TZSV-NP(S15) reacted with CCSV and TZSV while MAb-TZSV-NP(S18) reacted specifically to TZSV in both indirect ELISA and immunoblotting. Both MAbs can be used to detect TZSV in field-collected plant samples. The epitope of MAb-TZSV-NP(S18) was further identified consisting of amino acids 78-86 (HKIVASGAD) of the TZSV-13YV639 NP that is a highly conserved region among known TZSV isolates but is distinct from TNSaV and TZSV. CONCLUSIONS: In this study, two MAbs targeting to different portions of the TZSV NP were obtained. Unlike MAb-CCSV-NP reacted with TNSaV as well as CCSV and TZSV, both TZSV MAbs can be used to differentiate CCSV, TNSaV and TZSV. The identity of CCSV, TNSaV and TZSV was proven by individual virus-specific primer pairs to indicate the correctness of serological responses. We also proposed an serological detection platform using MAb-CCSV-NP, MAb-TZSV-NP(S15) and MAb-TZSV-NP(S18) to allow researchers and quarantine staff to efficiently diagnose the infections of CCSV, TNSaV and TZSV in China and other countries.

Full-length M and L RNA sequences of tospovirus isolate 2009-GZT, which causes necrotic ringspot on tomato in China.

The virus isolate 2009-GZT, collected from tomato in Guizhou province of China, was identified as a new member of the genus Tospovirus based on its S RNA sequence. Because its provisional name, "tomato necrotic spot virus" (TNSV), was identical to an already existing member of the genus Ilarvirus, 2009-GZT was renamed "tomato necrotic spot-associated virus" (TNSaV). In this study, the full-length sequences of the genomic M and L RNAs of TNSaV were determined and analyzed. The M RNA has 4,773 nucleotides (nt), encoding the NSm protein of 309 aa (34.4 kDa) in the viral (v) strand and the glycoprotein precursor (Gn/Gc) of 1123 aa (128 kDa) in the viral complementary (vc) strand. The NSm and Gn/Gc of TNSaV share the highest aa sequence identity (86.2 % and 86.9 %, respectively) with those of tomato zonate spot virus. The L RNA contains 8,908 nt and codes for the putative RNA-dependent RNA polymerase (RdRp) of 2885 aa (332 kDa) in the vc strand. The RdRp of TNSaV shares the highest aa sequence identity (85.2 %) with that of calla lily chlorotic spot virus (CCSV). Serological assays showed that TNSaV cross-reacts with rabbit antisera against watermelon silver mottle virus (WSMoV) NP and CCSV NP, indicating that TNSaV is a member of the WSMoV serogroup.