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.

The Fifth Residue of the Coat Protein of Turnip Mosaic Virus Is Responsible for Long-Distance Movement in a Local-Lesion Host and Aphid Transmissibility in a Systemic Host.

HC-Pro and coat protein (CP) genes of a potyvirus facilitate cell-to-cell movement and are involved in the systemic movement of the viruses. The interaction between HC-Pro and CP is mandatory for aphid transmission. Two turnip mosaic virus (TuMV) isolates, RC4 and YC5, were collected from calla lily plants in Taiwan. The virus derived from the infectious clone pYC5 cannot move systemically in Chenopodium quinoa plants and loses aphid transmissibility in Nicotiana benthamiana plants, like the initially isolated virus. Sequence analysis revealed that two amino acids, P5 and A206, of YC5 CP uniquely differ from RC4 and other TuMV strains. Recombination assay and site-directed mutagenesis revealed that the fifth residue of leucine (L) at the N-terminal region of the CP (TuMV-RC4), rather than proline (P) (TuMV-YC5), is critical to permit the systemic spread in C. quinoa plants. Moreover, the single substitution mutant YC5-CPP5L became aphid transmissible, similar to RC4. Fluorescence microscopy revealed that YC5-GFP was restricted in the petioles of inoculated leaves, whereas YC5-CPP5L-GFP translocated through the petioles of inoculated leaves, the main stem, and the petioles of the upper uninoculated leaves of C. quinoa plants. In addition, YC5-GUS was blocked at the basal part of the petiole connecting to the main stem of the inoculated C. quinoa plants, whereas YC5-CPP5L-GFP translocated to the upper leaves. Thus, a single amino acid, the residue L5 at the N-terminal region right before the 6DAG8 motif, is critical for the systemic translocation ability of TuMV in a local lesion host and for aphid transmissibility in a systemic host.

Generation of Attenuated Mutants of East Asian Passiflora Virus for Disease Management by Cross Protection.

East Asian passiflora virus (EAPV) seriously affects passionfruit production in Taiwan and Vietnam. In this study, an infectious clone of the EAPV Taiwan strain (EAPV-TW) was constructed, and EAPV-TWnss, with an nss tag attached to its helper component-protease (HC-Pro), was generated for monitoring the virus. Four conserved motifs of EAPV-TW HC-Pro were manipulated to create single mutations of F8I (simplified as I8), R181I (I181), F206L (L206), and E397N (N397) and double mutations of I8I181, I8L206, I8N397, I181L206, I181N397, and L206N397. Four mutants, EAPV I8I181, I8N397, I181L206, and I181N397, infected Nicotiana benthamiana and yellow passionfruit plants without conspicuous symptoms. Mutants EAPV I181N397 and I8N397 were stable after six passages in yellow passionfruit plants and expressed a zigzag pattern of accumulation dynamic, typical of beneficial protective viruses. An agroinfiltration assay indicated that the RNA silencing suppression capabilities of the four double mutated HC-Pros are significantly reduced. Mutant EAPV I181N397 accumulated the highest level of the small interfering RNA at 10 days postinoculation (dpi) in N. benthamiana plants, then dropped to background levels after 15 dpi. In both N. benthamiana and yellow passionfruit plants, EAPV I181N397 conferred complete cross protection (100%) against severe EAPV-TWnss, as defined by no severe symptoms and absence of the challenge virus, checked by Western blotting and reverse transcription PCR. Mutant EAPV I8N397 provided high degrees of complete protection against EAPV-TWnss in yellow passionfruit plants (90%) but not in N. benthamiana plants (0%). Both mutants showed complete protection (100%) against the Vietnam severe strain EAPV-GL1 in passionfruit plants. Thus, the mutants EAPV I181N397 and I8N397 have excellent potential for controlling EAPV in Taiwan and Vietnam. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Modification of the Helper Component Proteinase of Papaya Ringspot Virus Vietnam Isolate to Generate Attenuated Mutants for Disease Management by Cross Protection.

Papaya (Carica papaya) production is seriously limited by papaya ringspot virus (PRSV) worldwide, including in Vietnam. Control of PRSV by cross protection is dependent on the availability of effective mild strains. Here, an infectious cDNA clone was constructed from PRSV isolate TG5 from South Vietnam. Site-directed mutagenesis with point mutations on the essential motifs of the helper component proteinase (HC-Pro) was performed, with or without deleting five amino acids (d5) from its N-terminal region. Mutants TG-d5, TG-d5I7, and TG-d5L206 containing d5, d5 + F7I, and d5 + F206L, respectively, induced mild mottling followed by symptomless recovery on papaya and infected Chenopodium quinoa without lesion formation. Each mutant accumulated in papaya at reduced levels with a zigzag pattern and was stable beyond six monthly passages. The cross-protection effectiveness of the three mutants in papaya against TG5 was investigated, each with 60 plants from three independent trials. The results showed that each mutant provided complete protection (100%) against TG5, 1 month after the challenge inoculation, as verified by the lack of severe symptoms and lack of local lesions in C. quinoa. Further tests revealed that TG-d5I7 also confers high levels of protection against other severe PRSV isolates from South Vietnam, including isolates DN (97%) and ST2 (50%). However, TG-d5I7 is ineffective or less effective (0 to 33%) against seven other severe PRSV strains from different geographic origins, including the isolate HN from North Vietnam. Our results indicate that the protection by the three mutants is highly strain-specific and suitable for the control of PRSV in South Vietnam.

Concurrent Control of Two Aphid-Borne Potyviruses in Cucurbits by Two-in-One Vaccine.

The application of attenuated viruses has been widely practiced for protecting crops from infection by related severe strains of the same species. Papaya ringspot virus W-type (PRSV W) and zucchini yellow mosaic virus (ZYMV) devastate cucurbits worldwide. However, the prevailing of these two viruses in cucurbits cannot be prevented by a single protective virus. In this study, we disclosed that co-infection of horn melon plants by two mild strains, PRSV P-type (PRSV P) HA5-1 and ZYMV-ZAC (a previously developed mild mutant of ZYMV) confers concurrent protection against PRSV P and ZYMV. Consequently, mild mutants of PRSV W were created by site-directed mutagenesis through modifications of the pathogenicity motifs FRNK and PD in helper component-protease (HC-Pro). A stable PRSV W mutant WAC (PRSV-WAC) with R181I and D397N mutations in HC-Pro was generated, inducing mild mottling, followed by symptomless recovery in cucurbits. Horn melon plants pre-infected by PRSV-WAC and ZYMV-ZAC showed no apparent interference on viral accumulation with no synergistic effects on symptoms. An agroinfiltration assay of mixed HC-Pros of WACHC-Pro + ZACHC-Pro revealed no additive effect of RNA silencing suppression. PRSV-WAC or ZYMV-ZAC alone only antagonized a severe strain of homologous virus, while co-infection with these two mild strains provided complete protection against both PRSV W and ZYMV. Similar results were reproduced in muskmelon and watermelon plants, indicating the feasibility of a two-in-one vaccine for concurrent control of PRSV W and ZYMV in cucurbits.

Generation of Attenuated Passiflora Mottle Virus Through Modification of the Helper Component Protease for Cross Protection.

Passiflora mottle virus (PaMoV), an aphid-borne potyvirus, is the primary causal virus of devastating passionfruit woodiness disease in Vietnam. Here we generated a nonpathogenic, attenuated PaMoV strain for disease control by cross protection. A full-length genomic cDNA of PaMoV strain DN4 from Vietnam was constructed to generate an infectious clone. The green fluorescent protein was tagged at the N-terminal region of the coat protein gene to monitor in planta the severe PaMoV-DN4. Two amino acids within the conserved motifs of helper component protease (HC-Pro) of PaMoV-DN4 were mutated individually or in combination as K53E or/and R181I. Mutants PaMoV-E53 and PaMoV-I181 induced local lesions in Chenopodium quinoa plants, while PaMoV-E53I181 caused infection without apparent symptoms. In passionfruit (Passiflora edulis) plants, PaMoV-E53 elicited severe leaf mosaic and PaMoV-I181 induced leaf mottling, while PaMoV-E53I181 caused transient mottling followed by symptomless recovery. PaMoV-E53I181 was stable after six serial passages in yellow passionfruit (Passiflora edulis f. flavicarpa) plants. Its temporal accumulation levels were lower than those of the wild type, with a zigzag accumulation pattern, typical of a beneficial protective virus. An RNA silencing suppression (RSS) assay revealed that all three mutated HC-Pros are defective in RSS. Triplicated cross-protection experiments with a total of 45 plants showed that the attenuated mutant PaMoV-E53I181 provided a high protection rate (91%) against the homologous wild-type virus in passionfruit plants. This work revealed that PaMoV-E53I181 can be used as a protective virus to control PaMoV by cross protection.

Modification of Papaya Ringspot Virus HC-Pro to Generate Effective Attenuated Mutants for Overcoming the Problem of Strain-Specific Cross Protection.

Cross protection application of HA5-1, an attenuated mutant of papaya ringspot virus (PRSV) HA strain from Hawaii, was withdrawn from Taiwan due to the narrow geographic strain specificity of HA5-1. Here, to overcome this problem, we created attenuated mutants of PRSV YK, a dominant severe strain from Taiwan, by mutating helper component protease (HC-Pro) at F7, R181, F206, and D397 residues critical for potyviral pathogenicity. PRSV YK HC-Pro R181I, F206L, and D397N single-mutant viruses induced mild symptoms, but their adverse effects on growth of papaya plants disqualified them as useful protective viruses. However, F7I single-mutant and F7I + F206L double-mutant viruses displayed mild symptoms followed by recovery, and they showed a zigzag pattern of accumulation in papaya plants, indicating their potential to trigger RNA silencing and retain partial antagonistic suppression of host defense. Although F7I + R181I and F7I + D397N double-mutant viruses caused symptomless infection, they accumulated barely above mock level and, thus, were not qualified as proper protective viruses. RNA silencing suppression (RSS) analysis by agroinfiltration in Nicotiana benthamiana plants revealed that the HC-Pro F7I and F7I + F206L mutant proteins were weaker in RSS ability than the wild-type protein. Under greenhouse conditions, F7I and F7I + F206L mutant viruses were genetically stable but not aphid transmissible. Compared with the HA5-1 mutant's low degree (10%) of protection to papaya plants, the F7I and F7I + F206L mutants provided complete (100%) protection to papaya and horn melon plants against strain YK. Thus, F7I and F7I + F206L mutants solve the problem of strain-specific protection and have great potential for control of PRSV in Taiwan.

Generation of Mild Recombinants of Papaya Ringspot Virus to Minimize the Problem of Strain-Specific Cross-Protection.

Papaya ringspot virus (PRSV) causes severe damage to papaya (Carica papaya L.) and is the primary limiting factor for papaya production worldwide. A nitrous acid-induced mild strain, PRSV HA 5-1, derived from Hawaii strain HA, has been applied to control PRSV by cross-protection for decades. However, the problem of strain-specific protection hampers its application in Taiwan and other geographic regions outside Hawaii. Here, sequence comparison of the genomic sequence of HA 5-1 with that of HA revealed 69 nucleotide changes, resulting in 31 aa changes, of which 16 aa are structurally different. The multiple mutations of HA 5-1 are considered to result from nitrous acid induction because 86% of nucleotide changes are transition mutations. The stable HA 5-1 was used as a backbone to generate recombinants carrying individual 3' fragments of Vietnam severe strain TG5, including NIa, NIb, and CP3' regions, individually or in combination. Our results indicated that the best heterologous fragment for the recombinant is the region of CP3', with which symptom attenuation of the recombinant is like that of HA 5-1. This mild recombinant HA51/TG5-CP3' retained high levels of protection against the homologous HA in papaya plants and significantly increased the protection against the heterologous TG-5. Similarly, HA 5-1 recombinants carrying individual CP3' fragments from Thailand SMK, Taiwan YK, and Vietnam ST2 severe strains also significantly increase protection against the corresponding heterologous strains in papaya plants. Thus, our recombinant approach for mild strain generation is a fast and effective way to minimize the problem of strain-specific protection.

Characterization and Detection of Passiflora Mottle Virus and Two Other Potyviruses Causing Passionfruit Woodiness Disease in Vietnam.

Passionfruit plantings in Vietnam increased to 10,000 ha in 2019. However, outbreaks of passionfruit woodiness disease (PWD) have become a serious threat to production. In this study, five virus isolates (DN1, DN4, NA1, GL1, and GL2) were collected from different areas of Vietnam. Their causal roles in PWD were verified by back-inoculation to passionfruit. Analyses of coat protein (CP) and genomic sequences revealed that the GL1 isolate is closely related to East Asia Passiflora virus (EAPV) AO strain of Japan (polyprotein nt and aa identities of 98.1 and 98.2%, respectively), and the GL2 isolate is related to Telosma mosaic virus (TelMV) isolate PasFru, China (polyprotein nt and aa identities of 87.1 and 90.9%, respectively). CP comparison, host range, and cytological characterization indicated that DN1, DN4, and NA1 are potyviruses but are different from EAPV and TelMV. Phylogenic analyses of their CP and genome sequences indicated that these three isolates and the passionfruit severe mottle-associated virus Fujian isolate of China belong to a distinct clade, which does not meet the threshold (76% nt identity of polyprotein) to be regarded as any of potyviral species. Thus, a new species name, Passiflora mottle virus, (PaMoV), has been proposed by the International Committee on Taxonomy of Viruses. A rabbit antiserum was produced against the CP of DN1, and it can distinguish PaMoV from TelMV and EAPV in western blotting and enzyme-linked immunosorbent assay (ELISA) without cross-reactions. Field surveys of 240 samples by ELISA and reverse transcription PCR found that PWD in Vietnam is caused mainly by PaMoV, followed by EAPV, mixed infection of PaMoV and EAPV, and rare cases of TelMV.

Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins.

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein-protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein-protein interactions.

A Conserved Helix in the C-Terminal Region of Watermelon Silver Mottle Virus Nonstructural Protein S Is Imperative For Protein Stability Affecting Self-Interaction, RNA Silencing Suppression, and Pathogenicity.

In orthotospovirus, the nonstructural protein S (NSs) is the RNA-silencing suppressor (RSS) and pathogenicity determinant. Here, we demonstrate that a putative α-helix, designated H8, spanning amino acids 338 to 369 of the C-terminal region of the NSs protein, is crucial for self-interaction of watermelon silver mottle virus NSs protein and that the H8 affects RSS function. Co-immunoprecipitation, yeast two-hybrid, and bimolecular fluorescence complementation analyses revealed that the triple point mutation (TPM) of H8 amino acids Y338A, H350A, and F353A resulted in NSs protein self-interaction dysfunction. Transient expression of H8-deleted (ΔH8) and TPM NSs proteins in Nicotiana benthamiana plants by agroinfitration indicated that these proteins have weaker RSS activity and are far less stable than wild-type (WT) NSs. However, an electrophoretic mobility assay revealed that small interfering RNA (siRNA) binding ability of TPM NSs protein is not compromised. The pathogenicity assay of WT NSs protein expressed by the attenuated turnip mosaic virus vector restored severe symptoms in recombinant-infected N. benthamiana plants but not for ΔH8 or TPM proteins. Taken together, we conclude that the H8 helix in the C-terminal region of NSs protein is crucial for stabilizing NSs protein through self-interaction to maintain normal functions of RSS and pathogenicity, but not for NSs-siRNA binding activity.

ELF18-INDUCED LONG-NONCODING RNA Associates with Mediator to Enhance Expression of Innate Immune Response Genes in Arabidopsis.

The plant immune response is a complex process involving transcriptional and posttranscriptional regulation of gene expression. Responses to plant immunity are initiated upon the perception of pathogen-associated molecular patterns, including peptide fragment of bacterial flagellin (flg22) or translation elongation factor Tu (elf18). Here, we identify an Arabidopsis thaliana long-noncoding RNA, designated ELF18-INDUCED LONG-NONCODING RNA1 (ELENA1), as a factor enhancing resistance against Pseudomonas syringe pv tomato DC3000. ELENA1 knockdown plants show decreased expression of PATHOGENESIS-RELATED GENE1 (PR1) and the plants are susceptible to pathogens. By contrast, plants overexpressing ELENA1 show elevated PR1 expression after elf18 treatment and display a pathogen resistance phenotype. RNA-sequencing analysis of ELENA1-overexpressing plants after elf18 treatment confirms increased expression of defense-related genes compared with the wild type. ELENA1 directly interacts with Mediator subunit 19a (MED19a) and affects enrichment of MED19a on the PR1 promoter. These results show that MED19a regulates PR1 expression through ELENA1. Our findings uncover an additional layer of complexity, implicating long-noncoding RNAs in the transcriptional regulation of plant innate immunity.

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.

Potyviral Gene-Silencing Suppressor HCPro Interacts with Salicylic Acid (SA)-Binding Protein 3 to Weaken SA-Mediated Defense Responses.

The viral infection process is a battle between host defense response and pathogen antagonizing action. Several studies have established a tight link between the viral RNA silencing suppressor (RSS) and the repression of salicylic acid (SA)-mediated defense responses, nonetheless host factors directly linking an RSS and the SA pathway remains unidentified. From yeast two-hybrid analysis, we identified an interaction between the potyviral RSS helper-component proteinase (HCPro) and SA-binding protein SABP3. Co-localization and bimolecular fluorescence complementation analyses validated the direct in vivo interaction between Turnip mosaic virus (TuMV) HCPro and the Arabidopsis homologue of SABP3, AtCA1. Additionally, transient expression of TuMV HCPro demonstrated its ability to act as a negative regulator of AtCA1. When the plants of the AtCA1 knockout mutant line were inoculated with TuMV, our results indicated that AtCA1 is essential to restrict viral spreading and accumulation, induce SA accumulation, and trigger the SA pathway. Unexpectedly, the AtCA1 overexpression line also displayed a similar phenotype, suggesting that the constitutive expression of AtCA1 antagonizes the SA pathway. Taken together, our results depict AtCA1 as an essential regulator of SA defense responses. Moreover, the interaction of potyviral HCPro with this regulator compromises the SA pathway to weaken host defense responses and facilitate viral infection.

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.

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.

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.

Untranslatable tospoviral NSs fragment coupled with L conserved region enhances transgenic resistance against the homologous virus and a serologically unrelated tospovirus.

Tospoviruses cause severe damages to important crops worldwide. In this study, Nicotiana benthamiana transgenic lines carrying individual untranslatable constructs comprised of the conserved region of the L gene (denoted as L), the 5' half of NSs coding sequence (NSs) or the antisense fragment of whole N coding sequence (N) of Watermelon silver mottle virus (WSMoV), individually or in combination, were generated. A total of 15-17 transgenic N. benthamiana lines carrying individual transgenes were evaluated against WSMoV and the serologically unrelated Tomato spotted wilt virus (TSWV). Among lines carrying single or chimeric transgenes, the level of resistance ranged from susceptible to completely resistant against WSMoV. From the lines carrying individual transgenes and highly resistant to WSMoV (56-63% of lines assayed), 30% of the L lines (3/10 lines assayed) and 11% of NSs lines (1/9 lines assayed) were highly resistant against TSWV. The chimeric transgenes provided higher degrees of resistance against WSMoV (80-88%), and the NSs fragment showed an additive effect to enhance the resistance to TSWV. Particularly, the chimeric transgenes with the triple combination of fragments, namely L/NSs/N or HpL/NSs/N (a hairpin construct), provided a higher degree of resistance (both 50%, with 7/14 lines assayed) against TSWV. Our results indicate that the untranslatable NSs fragment is able to enhance the transgenic resistance conferred by the L conserved region. The better performance of L/NSs/N and HpL/NSs/N in transgenic N. benthamiana lines suggests their potential usefulness in generating high levels of enhanced transgenic resistance against serologically unrelated tospoviruses in agronomic crops.

Genetic and serological characterization of chrysanthemum stem necrosis virus, a member of the genus Tospovirus.

Chrysanthemum stem necrosis virus (CSNV) is a member of a tentative tospovirus species. In this study, the complete genomic sequence of the Japanese CSNV isolate TcCh07A was determined. The L RNA is 8960 nt long and encodes the 331.0-kDa RNA-dependent RNA polymerase. The M RNA is 4828 nt long and encodes the 34.1-kDa movement protein (NSm) and the 127.7-kDa glycoprotein precursor (Gn/Gc). The S RNA is 2949 nt long and encodes the 52.4-kDa silencing suppressor protein (NSs) and the 29.3-kDa nucleocapsid (N) protein. The N protein of CSNV-TcCh07A was purified from virus-infected plant tissues and used for production of a rabbit polyclonal antiserum (RAs) and a monoclonal antibody (MAb). Results of serological tests by indirect ELISA and western blotting using the prepared RAs and MAb and a previously produced RAs against the N protein of tomato spotted wilt virus (TSWV) indicated that CSNV-TcCh07A, TSWV, tomato chlorotic spot virus, groundnut ringspot virus, alstroemeria necrotic streak virus and impatiens necrotic spot virus are serologically related.