Cloning of the papaya ringspot virus (PRSV) replicase gene and generation of PRSV-resistant papayas through the introduction of the PRSV replicase gene

Papaya ringspot virus (PRSV) can cause a destructive disease in papaya (Carica papaya L.). Based on observations that viral replicase (RP) gene confers resistance to virus in other plants, we designed a pair of primers and cloned the RP gene from PRSV by RT-PCR. The 3'-truncated and 5'-extended RP gene fragment was then oriented under the control of the CaMV35 S promoter and nos termination sequence in the mini Ti plasmid vector pRok to construct a plant expression vector, designated pRPTW. Papaya (C. papaya L.) cv. Tai-nong-2 embryogenic calli were transformed by Agrobacterium tumefaciens LBA4404 harboring the pRPTW vector. After selection on 100 mg/ml kanamycin, 20 putative transgenic papayas were regenerated and confirmed by PCR-Southern blot and Southern blot analyses. PRSV inoculation tests showed that the RP gene conferred resistance to PRSV in transgenic papayas and those offspring carrying the RP gene. The consistency of the presence of the RP gene and PRSV resistance indicates that replicase-mediated resistance against PRSV was attained in papaya. Possible mechanisms include RNA-mediated resistance and protein-mediated resistance, as well as others, although further studies are required.     

Genomics of Helper Component Proteinase Reveals Effective Strategy for <Emphasis Type="Italic">Papaya Ringspot Virus</Emphasis> Resistance

Papaya ringspot virus (PRSV) causes severe economic losses in both cucurbits and papaya throughout the tropics and subtropics. Development of PRSV-resistant transgenic plants faces a major hurdle in achieving resistance against geographically distinct isolates. One of the major reasons of failing to achieve the broad-spectrum PRSV resistance is the involvement of silencing suppressor proteins of viral origin. Here, based on sequence profile of silencing suppressor protein, HcPro, we show that PRSV-HcPro, acts as a suppressor of RNA silencing through micro RNA binding in a dose- dependent manner. In planta expression of PRSV-HcPro affects developmental biology of plants, suggesting the interference of suppressor protein in micro RNA-directed regulatory pathways of plants. Besides facilitating the establishment of PRSV, it showed strong positive synergism with other heterologous viruses as well. This study provides a strategy to develop effective and stable PRSV-resistant transgenic plants.     

Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus

The commercially valuable transgenic papaya lines carrying the coat protein (CP) gene of Papaya ringspot virus (PRSV) and conferring virus resistance have been developed in Hawaii and Taiwan in the past decade. Prompt and sensitive protocols for transgene-specific and event-specific detections are essential for traceability of these lines to fulfill regulatory requirement in EU and some Asian countries. Here, based on polymerase chain reaction (PCR) approaches, we demonstrated different detection protocols for characterization of PRSV CP-transgenic papaya lines. Transgene-specific products were amplified using different specific primer pairs targeting the sequences of the promoter, the terminator, the selection marker, and the transgene, and the region across the promoter and transgene. Moreover, after cloning and sequencing the DNA fragments amplified by adaptor ligation-PCR, the junctions between plant genomic DNA and the T-DNA insert were elucidated. The event-specific method targeting the flanking sequences and the transgene was developed for identification of a specific transgenic line. The PCR patterns using primers designed from the left or the right flanking DNA sequence of the transgene insert in three selected transgenic papaya lines were specific and reproducible. Our results also verified that PRSV CP transgene is integrated into transgenic papaya genome in different loci. The copy number of inserted T-DNA was further confirmed by real-time PCR. The event-specific molecular markers developed in this investigation are crucial for regulatory requirement in some countries and intellectual protection. Also, these markers are helpful for prompt screening of a homozygote-transgenic progeny in the breeding program.     

Variation in the Coat Protein Gene of Papaya ringspot Virus Isolates from Multiple Locations of China

The potyvirus Papaya ringspot virus （PRSV） is an important pathogen of papaya that causes severe losses in economic crops for papaya production globally. The coat protein （CP） genes of five PRSV isolates originating from different locations in China were cloned and sequenced. The CP-coding region varied in size from 864-873 nucleotides, encoding proteins of 288-291 amino acids. The five Chinese isolates of PRSV have been characterized as papaya-infecting （PRSV-P）. The CP sequences of the Chinese isolates were compared with those of previously published PRSV isolates originating from different countries at amino acid levels. A number of KE repeat boxes in the N terminus of the PRSV-CP were found in all Chinese isolates. The phylogenetic branching pattern revealed that there was certain extended grouping between geographic locations, and the Asian type probably represents the oldest population of PRSV. The information of CP genes will be useful in designing and developing durable virus resistant-PRSV transgenic papaya in China. Meanwhile broad-spectrum-virus resistant, strongly resistant-PRSV and good safe papaya lines are required.     

Occurrence and relative incidence of viruses infecting papaya in Venezuela

A survey of the main papaya (Carica papaya L.) production fields in Venezuela during 1997, indicated that crops were heavily affected with various virus‐like symptoms. A total of 745 samples from papaya plants showing symptoms suggestive of virus infection were collected and analysed using electron microscopy and enzyme‐linked immunosorbent assay (ELISA). Papaya ringspot virus (PRSV) and Papaya mild yellowing virus (PMYV) were the most frequently found viruses, which also occurred, in mixed infections. Rhabdovirus‐like particles were found only in samples collected in Distrito Federal (D.F). Papaya mosaic virus (papMV) and Tomato spotted wild virus (TSW V) were not detected during the survey.     

Production of Polyclonal Antibodies Using Recombinant Coat Protein of Papaya ringspot virus and their Use in Immunodiagnosis

Production of polyclonal antibodies requires large amount of purified virus that can be avoided by the use of recombinant coat protein (CP). Recombinant CP of Papaya ringspot virus (PRSV) was thus used for the production of polyclonal antibodies as the virus purification from papaya tissues provides low virus yields. CP was expressed as a fusion protein (～72 kD) containing a fragment of E. coli maltose binding protein. Polyclonal antibodies from rabbits immunized with the fusion protein, successfully detected natural infection of PRSV in papaya and cucurbits samples collected from different locations at 1:4000 dilution in direct antigen-coated enzyme-linked immunosorbent assay.     

Papaya ringspot virus-P: characteristics, pathogenicity, sequence variability and control

TAXONOMY: Papaya ringspot virus (PRSV) is an aphid-transmitted plant virus belonging to the genus Potyvirus, family Potyviridae, with a positive sense RNA genome. PRSV isolates belong to either one of two major strains, P or W. The P strains infect both papaya and cucurbits whereas the W strains infect only cucurbits. GEOGRAPHICAL DISTRIBUTION: PRSV-P is found in all major papaya-growing areas. PHYSICAL PROPERTIES: Virions are filamentous, non-enveloped and flexuous measuring 760-800 x 12 nm. Virus particles contain 94.5% protein and 5.5% nucleic acid. The protein component consists of the virus coat protein (CP), which has a molecular weight of about 36 kDa as estimated by Western blot analysis. Density of the sedimenting component in purified PRSV preparations is 1.32 g/cm(3) in CsCl. GENOME: The PRSV genome consists of a unipartite linear single-stranded positive sense RNA of 10 326 nucleotides with a 5' terminus, genome-linked protein, VPg. TRANSMISSION: The virus is naturally transmitted via aphids in a non-persistent manner. Both the CP and helper component (HC-Pro) are required for vector transmission. This virus can also be transmitted mechanically, and is typically not seed-transmitted. HOSTS: PRSV has a limited number of hosts belonging to the families Caricaceae, Chenopodiaceae and Cucurbitaceae. Propagation hosts are: Carica papaya, Cucurbita pepo and Cucumis metuliferus cv. accession 2459. Local lesion assay hosts are: Chenopodium quinoa and Chenopodium amaranticolor. CONTROL: Two transgenic papaya varieties, Rainbow and SunUp, with engineered resistance to PRSV have been commercially grown in Hawaii since 1998. Besides transgenic resistance, tolerant varieties, cross-protection and other cultural practices such as isolation and rogueing of infected plants are used to manage the disease. VIRUS CODE: 00.057.0.01.045. VIRUS ACCESSION NUMBER: 57010045. USEFUL LINK: http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/57010045.htm.     

Efficient transformation of papaya by coat protein gene of papaya ringspot virus mediated byAgrobacterium following liquid-phase wounding of embryogenic tissues with caborundum

Summary Generation of transgenic papaya (Carica papaya L.) has been hampered by the low rates of transformation achieved by conventionalAgrobacterium infection or microprojectile bombardment. We describe an efficientAgrobacterium-mediated transformation method based on wounding of cultured embryogenic tissues with carborundum in liquid phase. Embryogenic tissues were obtained from cultured immature zygotic embryos collected 75–90 days after pollination. The expressible coat protein (CP) gene of a Taiwan strain of papaya ringspot virus (PRSV) was constructed in a Ti binary vector pBGCP, which contained the NPT-II gene as a selection marker. The embryogenic tissues were vortexed with 600 mesh carborundum in sterile distilled water for 1 min before treating with the disarmedA. tumefaciens containing the pBGCP. Transformed cells were cultured on kanamycin-free medium containing 2,4-D and carbenicillin for 2–3 weeks and then on the kanamycin medium for 3–4 months. The developed somatic embryos were transferred to the medium containing NAA, BA and kanamycin and subsequently regenerated into normal-appearing plants. Presence of the PRSV CP gene in the putative transgenic lines was detected by PCR and the expression of the CP was verified by Western blotting. The transgene was nuclearly inherited as revealed by segregation analysis in the backcrossed R₁ progeny. From five independent experiments, the average successful rate of transformation was 15.9% of the zygotic embryos treated (52 transgenic somatic embryo clusters out of 327 zygotic embryos treated), about 10–100 times higher than the available methods previously reported. Thus, wounding highly regenerable differentiating tissues by carborundum vortexing provides a simple and efficient way for papaya transformation mediated byAgrobacterium.     

Potential for introducing cold tolerance into papaya by transformation with C-repeat binding factor (CBF) genes

Papaya (Carica papaya L.) production is affected by low temperatures that occur periodically in the subtropics. The C-repeat binding factor (CBF) gene family is known to induce the cold acclimation pathway in Arabidopsis thaliana. Embryogenic papaya cultures were induced from hypocotyls of “Sunrise Solo” zygotic embryos on semisolid induction medium. The CBF 1/CBF 3 genes along with the neomycin phosphotransferase (NPT II) gene were placed under the control of the CaMV 35 S promoter and introduced into a binary vector pGA 643. Embryogenic cultures were transformed with Agrobacterium strain GV 3101 harboring pGA 643. After selection of transformed embryogenic cultures for resistance to 300 mg l⁻¹ kanamycin, somatic embryo development was initiated and transgenic plants were regenerated. The presence of the CBF transgenes in regenerated plants was confirmed by Southern blot hybridization. The papaya and the related cold-tolerant Vasconcella genomes were probed for the presence of cold inducible sequences using polymerase chain reaction (PCR). Possible cold inducible sequences were present in the Vasconcella genome but were absent in the Carica genome.     

Papaya Ring Spot Virus: An Understanding of a Severe Positive-Sense Single Stranded RNA Viral Disease and Its Management

Viral diseases have been studied in-depth for reducing quality, yield, health and longevity of the fruit, to highlight the economic losses. Positive-sense single-stranded RNA viruses are more devastating among all viruses that infect fruit trees. One of the best examples is papaya ringspot virus (PRSV). It belongs to the genus Potyvirus and it is limited to cause diseases on the family Chenopodiaceae, Cucurbitaceae and Caricaceae. This virus has a serious threat to the production of papaya, which is famous for its high nutritional and pharmaceutical values. The plant parts such as leaves, latex, seeds, fruits, bark, peel and roots may contain the biological compound that can be isolated and used in pharmaceutical industries as a disease control. Viral disease symptoms consist of vein clearing and yellowing of young leaves. Distinctive ring spot patterns with concentric rings and spots on fruit reduce its quality and taste. The virus has two major strains P and W. The former cause disease in papaya while the later one in papaya. Virion comprises 94.4% protein, including a 36 kDa coat protein which is a component responsible for a non-persistent transmission through aphids, and 5.5% nucleic acid. Cross protection, development of transgenic crops, exploring the resistant sources and induction of pathogen derived resistance have been recorded as effective management of PRSV. Along with these practices reduced aphid population through insecticides and plant extracts have been found ecofriendly approaches to minimize the disease incidence. Adoption of transgenic crops is a big challenge for the success of disease resistant papaya crops. The aim of this review is to understand the genomic nature of PRSV, detection methods and the different advanced control methods. This review article will be helpful in developing the best management strategies for controlling PRSV.     

转基因番木瓜研究进展

番木瓜环斑病毒 (PRSV)使热带亚热带的重要水果番木瓜的生产受到严重影响 ,在众多方法防效不佳的情况下 ,利用病原获得抗性防治PRSV给番木瓜的生产带来了光明。综述了近年来转PRSVCP基因番木瓜中影响番木瓜转化因素和转基因番木瓜的抗性因素。转PRSV外壳蛋白 (CP)基因的番木瓜中多以胚性组织为转化材料 ,被转化材料的生理状态和基因型 ,是影响转化效率和转基因植株质量的主要因素。所获得的转基因番木瓜对PRSV的抗性在很大程度上依赖于接种PRSV与所转化PRSVCP基因的序列同源性、转基因拷贝数和所转基因的位置等。     

A single amino acid of niapro of papaya ringspot virus determines host specificity for infection of papaya

Most strains of Papaya ringspot virus (PRSV) belong to type W, causing severe loss on cucurbits worldwide, or type P, devastating papaya in tropical areas. While the host range of PRSV W is limited to plants of the families Chenopodiaceae and Cucuribitaceae, PRSV P, in addition, infects plants of the family Caricaceae (papaya family). To investigate one or more viral genetic determinants for papaya infection, recombinant viruses were constructed between PRSV P-YK and PRSV W-CI. Host reactions to recombinant viruses indicated that the viral genomic region covering the C-terminal region (142 residues) of NIaVPg, full NIaPro, and N-terminal region (18 residues) of NIb, is critical for papaya infection. Sequence analysis of this region revealed residue variations at position 176 of NIaVPg and positions 27 and 205 of NIaPro between type P and W viruses. Host reactions to the constructed mutants indicated that the amino acid Lys27 of NIaPro determines the host-specificity of PRSV for papaya infection. Predicted three-dimensional structures of NIaPros of parental viruses suggested that Lys27 does not affect the protease activity of NIaPro. Recovery of the infected plants from certain papaya-infecting mutants implied involvement of other viral factors for enhancing virulence and adaptation of PRSV on papaya.     

Effects of the <Emphasis Type="Italic">Papaya meleira virus</Emphasis> on papaya latex structure and composition

Spontaneous latex exudation is the main symptom of papaya sticky (meleira) disease caused by the Papaya meleira virus (PMeV), a double-stranded RNA (dsRNA) virus. This paper describes different effects of PMeV on papaya latex. Latex samples were subjected to different histochemical tests to evaluate their chemical composition. Additionally, the integrity of the latex particles was assessed by transmission and scanning electron microscopy analysis. Biochemical and micro- and macro-element measurements were performed. PMeV dsRNA extraction was performed to evaluate the interaction of the virus with the latex particles. Sticky diseased latex was positive for alkaloid biosynthesis and showed an accumulation of calcium oxalate crystals. PMeV also increased H₂O₂ synthesis within sticky diseased laticifers. The protein, sugar and water levels were altered, probably due to chemical changes. The morphology of the latex particles was further altered; PMeV particles seemed to be bound to the latex particles. The alkaloid and H₂O₂ biosynthesis in the papaya laticifers indicate a papaya defense response against PMeV. However, such efforts failed, as the virus affected the plant latex. The effects described here suggest some advantages of the infection process, including facilitating the movement of the virus within the papaya plant.     

番木瓜感染环斑花叶病毒后蛋白质和还原糖的变化

本文对番木瓜不同抗性的品种感染环斑花叶病毒后,可溶性蛋白含量和电泳谱带以及还原糖含量的变化规律进行了研究,并分析其与抗性的关系。结果表明,接种处理后,感病品种(岭南种)的可溶性蛋白含量变化率的峰值较抗病品种(穗中红48号)出现早且高;前者出现在接种后24h,高达54.6%,而后者出现在接种后48h,为38.2%。在未接种处理时,感病品种叶片可溶性蛋白谱带较抗病品种多1条;但在接种初期(接种后24h),抗病品种的蛋白谱带比感病品种多1条(Rf值为0.602)。不同抗性品种在接种后的还原糖含量变化也有差异,抗病品种的还原糖含量变化率在接种后48h达到高峰,峰值为12.3%;而感病品种的还原糖含量变化率在接种后都为负值。     

Analysis on virus resistance and fruit quality for T4 generation of transgenic papaya

Molecular biological characterization, fruit characters, and nutrients were analyzed for T4 generation of transgenic papaya. All transgenic papaya plants with the mutated replicase (RP) gene from papaya ringspot virus (PRSV) showed high resistance or immunity against PRSV in the field. The RP transgene can be steadily inherited to, and expressed at RNA level, the progenies. The growth characteristics of transgenic papaya were much better than non-transgenic papaya in the field. The non-transgenic papaya seedlings began to show typical symptoms caused by PRSV after being inoculated with PRSV. They died quickly and never grew to produce fruit. The adult trees developed yellow leaves and produced smaller fruits and were doomed to a slow death after some time, while most of transgenic papaya plants (about 91.8%) did not show any symptoms caused by PRSV, and produced more, bigger, and high quality fruits. Compared with non-transgenic plants, the fresh fruit length of T4 generation of transgenic papaya increased 2.6%–5%, and the diameter decreased 0.6%–1.5%. The flesh thickness of fresh fruit increased 12%–15%, which made it fitter for eating. Although the fresh fruit quality changed, there was no significant difference between transgenic and non-transgenic papaya. The quality characteristics of dry fruit including the contents of water, lipid, N, protein, reduced sugar, vitamin A, vitamin C, and carotene in the T4 generation of transgenic papaya were all the same as their non-transgenic parents. This means that transgenic plants and non-transgenic plants are substantially equivalent, and the transgene has no effect on dry fruit quality. In this study, we found that vitamin A and vitamin C in red-fleshed papaya were 1.4–1.8 and 1.78–2.07 times more than the yellow-fleshed ones, respectively, while N and protein were only 84.2%–92.1% and 82.1%–98.9% of the yellow-fleshed ones. Translated from Acta Ecologica Sinica, 2005, 25(12): 3301–3306 [译自: 生态学报]