Strains Of Papaya Ringspot Virus Research Articles

Mapping the CP-Transgene Insert in the Papaya Genome and Developing a Hermaphrodite Transgenic Hybrid with Broad-Spectrum Resistance to Papaya Ringspot Virus.

Papaya ringspot virus (PRSV) limits papaya production worldwide. Previously, we generated transgenic lines of hybrid Tainung No.2 (TN-2) carrying the coat protein (CP) gene of PRSV with broad resistance to PRSV strains. Unfortunately, all of them were female, unacceptable for growers and consumers in practical applications. With our reported flanking sequences and the newly released papaya genomic information, the CP-transgene insert was identified at a non-coding region in chromosome 3 of the papaya genome, and the flanking sequences were verified and extended. The female transgenic line 16-0-1 was first used for backcrossing with the parental Sunrise cultivar six times and then followed by selfing three times. With multi-level molecular markers developed from the PRSV CP transgene and the genomic flanking sequences, the presence and zygosity of the CP transgene were characterized at the seedling stage. Meanwhile, hermaphrodite genotype was identified by a sex-linked marker. With homozygotic transgene and horticultural properties of Sunrise, a selected hermaphrodite individual was propagated by tissue culture (TC) and used as maternal progenitor to cross with non-transgenic parental cultivar Thailand to generate a new hybrid cultivar TN-2 with a hemizygotic CP-transgene. Three selected hermaphrodite individuals of transgenic TN were micropropagated by TC, and they showed broad-spectrum resistance to different PRSV strains from Taiwan, Hawaii, Thailand, and Mexico under greenhouse conditions. The selected clone TN-2 #1, with excellent horticultural traits, also showed complete resistance to PRSV under field conditions. These selected TC clones of hermaphrodite transgenic TN-2 provide a novel cultivation system in Taiwan and elsewhere.

Host range of three isolates of papaya ringspot virus (PRSV) found in North Sumatera and West Java

Papaya ringspot virus (PRSV) isolates collectd from North Sumatera and West Java were evaluated for host range using mechanical inoculation. PRSV is one of the dangerous diseases in papaya plants in Indonesia reported to have a limited host range and caused symptom variation on plants. The study aim to determine the host range of three PRSV isolates from North Sumatera and West Java to several crops. Incidence of PRSV symptoms on plants was observed by morphological characterization. The experimental results showed that two PRSV isolates from Medan Ampelas and Deli Serdang North Sumatera, and Cibinong West Java isolates were pathogenic and caused systemic symptoms to Carica papaya cv. MD (Caricacea), melon (C. melo) and bitter melon (Momordica charantia). The symptom of infected papaya by the three isolates are vein clearing, mosaic on leaves, leaf blanching, wilt, and mottle on leaf lamina. The isolates caused asymptomatic to eggplant (Solanum melongena) and choy sum (Brassica rapa), asparagus bean (Vigna unguiculata), tobacco (Nicotiana tabacum), and tomato (Solanum lycopersicum syn. lycopersicum esculentum). Melon plants showed symptoms of vein clearing and lamina blanching by Medan Ampelas, and Cibinong isolates respectively, and bitter melon showed symptoms of vein clearing and leaf malformation, and mottle by isolate of Medan Ampelas and Cibinong respectively. The two plant species display asymptomatic by isolate of Deli Serdang. Isolate of Deli serdang has the potential to be a mild strain of PRSV. Complete characterization of mild strains of plant virus through molecular technology approaches can enrich understanding of the potential of these organisms for cross-protection.

First report of mosaic disease associated with zucchini tigre mosaic virus in wax gourd (Benincasa hispida) in Taiwan.

Wax gourd (Benincasa hispida) is a popular cucurbitaceous summer crop used for culinary purposes, confectionery, and refreshing drinks in Asia. During a field survey for cucurbit viral diseases at Beidou, Changhua in May 2017, B. hispida plants with symptoms of mosaic, chlorosis and leaf curl were found. Because more than 90% of plants showed leaf curl symptoms, samples were first examined by PCR with degenerate primers for begomoviruses (Forward: 5'-TGTGAAGGNCCDTGTAARGT-3' and Reverse: 5'-GCRTDGGTACADGCCATATA-3'). Sequence analysis of amplicons revealed that all of tested plants were infected by squash leaf curl Philippine virus (SqLCPhV) (Liao et al., 2007). However, electron microscopic examination revealed potyvirus-like particles and pinwheel-like inclusion bodies on some of begomovirus-infected leaves showing mild curling and mosaic symptoms. RT-PCR with potyvirusdegenerate primers (PNIbF1: 5'-GGBAAYAATAGTGGNCAACC-3' and PCPR1: 5'-GGGGAGGTGCCGTTCTCDATRCACC-3') were used to detect the potyvirus. Among 8 samples examined, all were infected by SqLCPhV (100%) and 5 samples were mixed infection of SqLCPhV and a potyvirus (62.5%). The filamentous virus was isolated from field samples by sap inoculating plants of B. hispida to separate from nonmechanically-transmissible SqLCPhV. Inoculated B. hispida plants showed severe mosaic and deformation. Total RNA was extracted from inoculated B. hispida and used for RT-PCR with a degenerate potyvirus primer PNIbF1 (5'- GGBAAYAATAGTGGNCAACC) pairing with oligo-dT. A 1.8-kb DNA fragment containing partial NIb gene, full-length coat protein (CP) gene, and 3'-UTR was amplified, cloned, and sequenced. The CP gene (293 aa) shared 82.1% nucleotide and 81% amino acid sequence identity to that of zucchini tigre mosaic virus (ZTMV) isolate E11045 (KC345608). Two degenerate primers (fZTMV-F: 5'- AGCRTGTGGYAHCC-3' and gourd-R: 5'-TCCCACCAYTTYTCRAAHGT-3') target the flanking region between the middle of P3 and 5' of CIP genes of ZTMV strains were designed and used to detect ZTMV. An expected 0.7 kb fragment could be amplified from 2 of 5 field-collected samples and all 6 mechanically inoculated B. hispida plants. Upon sap inoculation, ZTMV was also able to infect pumpkin (Cucurbita pepo var. pepo), zucchini (C. pepo var. cylindrica), squash (C. moschata), bottle gourd (Lagenaria siceraria) and Cucumis metuliferus, but not bitter gourd (Momordica charantia), cucumber (Cucumis sativus), watermelon (Citrullus lanatus), luffa (Luffa cylindrica), tobacco (including Nicotiana benthamiana, N. tabacum, and N. glutinoa), Chenopdium quinoa and C. amaranticolor. The infection of ZTMV to all mechanically inoculated plants were tested by RT-PCR using the fZTMV-F and gourd-R primers and/or TEM examinations. To the best of our knowledge, this is the first report of ZTMV infecting B. hispida in Taiwan. ZTMV was originally reported as a T strain of papaya ringspot virus (PRSV-T) (Quiot-Doine et al., 1986), and was later reclassified as a distinct species in the genus Potyvirus (Romay et al., 2014).

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.

A new strain of Papaya ring spot virus infecting ivy gourd (Coccinia grandis L.) in India

Ivy gourd (Coccinia grandis L.) plants showing virus likesymptoms such as mosaic mottling and thickening of leaflamina were collected from Tindivanam, Tamil Nadu. ThroughReverse transcription polymerase chain reaction, infectionof potyvirus was confirmed with the yield of desiredamplicon (~1.0 kb) using universal primer pair. Sequenceanalysis of amplified product showed the infection of Papayaring spot virus (PRSV). Further association of PRSV wasconfirmed with PRSV specific primer pair (GK PRSV F/R).The nucleotide sequence analysis showed identity of coatprotein region shared only 84% with PRSV isolate fromThailand in BLAST analysis. In phylogenetic analysis, PRSVinfecting ivy gourd formed a separate cluster from otherPRSV isolates. This confirms the association of distinct strainof PRSV on Ivy gourd in India.

First Report of Papaya ring spot virus (PRSV) Infecting Jute (Corchorus olitorius) in India.

Jute is the most important bast fibre crop of the world, which is mainly cultivated in India, Nepal, Bangladesh, China, Indonesia and South American countries. The fibre is utilized for making apparels, ropes, bags, carpets etc (Biswas et al. 2014). This bio-fibre is gaining importance due to growing environmental consciousness worldwide. In June 2019, we noticed jute plants (less than 2%) showing virus like symptoms viz., downward curling, puckering, angular brownish to yellowish spots etc in a farmer's field at Amdanga Block, North 24 Paraganas, West Bengal, India. To identify the virus, five symptomatic leaves from five different plants were used for high throughput sequencing (HTS). We extracted total RNA from each leaf which was subjected to construction of cDNA libraries. Sequencing was done on Illumina Hiseq 4000 (CytoScan, Thermo Fisher). Approximately 46 million 105 nt paired end reads were generated. Raw reads were trimmed and filtered to perform de novo assembly as described previously by (Grabherr et al. 2013). The obtained contig was 10,326 bp nucleotides (nt) long and in BLASTn against GenBank showed highest identity with papaya ring spot virus (PRSV) with the contig covering 99.6% of the viral genome. The obtained contig shared 99.33% sequence similarity with PRSV strain P (Accession No. MT470188). The selected leaf samples were also tested by double-antibody sandwich (DAS)- enzyme linked immunosorbent assay (ELISA) for papaya ring spot virus (PRSV) along with some common viruses, viz., Potato leaf roll virus (PLRV), Watermelon mosaic virus, Cowpea mosaic virus and Cucumber mosaic virus with the help of commercial diagnostic kits (Agdia). However, only the test with PRSV gave positive reaction for the symptomatic samples. The major symptoms of PRSV on papaya are severe mosaic, chlorosis, reduced lamina with curling and puckering (Gonsalves et al. 2010). To confirm PRSV infection, five symptomatic leaf samples (used for HTS) were collected and whole RNA was extracted from the samples using RNeasy plant minikit (Qiagen, USA). Reverse transcriptase polymerase chain reaction (RT-PCR) was conducted by using isolated RNA. One pair of PRSV specific primer (PSRV1F: 5' TTAAATCTGATTCGTC 3' PRSV 1R: 5'GAAATTCACGCAAAGTCGA3') was developed by using primer BLAST software and was used in RT-PCR assays. Amplified fragments were cloned and sequenced and all the fragments shared 98% sequence identity with PRSV. One of the amplicons was deposited in NCBI (Accession No. MN615832). Crude sap was prepared by homogenizing PRSV-infected jute leaf tissues in 0.1 M sodium phosphate buffer and 2% carborundum dust was added as abrasive (Holkar et al. 2018). The sap was then gently rubbed on to the healthy papaya leaves for inoculation. Typical PRSV like symptoms appeared in inoculated leaves 10 days post inoculation which confirmed the presence of PRSV-P. PRSV was detected by RT-PCR as well as (DAS)-ELISA from all inoculated infected papaya leaf tissues, but could not be detected from uninoculated healthy papaya tissues. To the best of our knowledge, this is the first report of PRSV-P infecting jute in India.

Detection of the papaya strain of papaya ringspot virus (PRSV-P) in Paraguay

Detection of the papaya strain of papaya ringspot virus (PRSV-P) in Paraguay

Virus Quantification, Flowering, Yield, and Fruit Quality in Tropical Pumpkin (Cucurbita moschata Duchesne) Genotypes Susceptible or Resistant to Two Potyviruses

Infecting cucurbits around the world, Papaya ringspot virus (PRSV) and Zucchini yellow mosaic virus (ZYMV) are members of the genus Potyvirus and family Potyviridae. Tropical pumpkin is grown globally in the lowland humid tropics. In Puerto Rico, tropical pumpkin is the second most important vegetable crop in economic value. In trials in Puerto Rico in 2016 and 2017, susceptible genotypes ‘Waltham’, Mos166, ‘Taína Dorada’ (2016 only), ‘Soler’ with moderate resistance to ZYMV, and resistant ‘Menina’ and ‘Nigerian Local’ were inoculated with PRSV and ZYMV and evaluated in the greenhouse and field. Mock-inoculated (buffer) controls were included. Puerto Rico strains of PRSV and ZYMV were originally collected from plants of Cucurbita moschata in Puerto Rico. Presence of virus was determined by Double Antibody Sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) and symptom severity was evaluated on a 0 to 5 scale in both trials. Days to anthesis of first staminate and pistillate flower were recorded for each plot. Number of fruits, fruit weight per plant, average fruit weight, fruit and mesocarp diameter, mesocarp color, °Brix, and percentage dry matter were measured in 2017. ‘Waltham’, Mos166, ‘Taína Dorada’, and ‘Soler’ tested positive for PRSV when inoculated with PRSV and positive for ZYMV when inoculated with ZYMV. For both PRSV and ZYMV, symptom severity was less (severity = 0) in resistant genotypes ‘Menina’ and ‘Nigerian Local’ than in all other genotypes. ‘Soler’ inoculated with ZYMV exhibited less symptom severity than that of susceptible genotypes. The degree of symptom severity of ‘Soler’ inoculated with PRSV was similar to susceptible genotypes. Symptom severity in plants inoculated with ZYMV was generally greater than when inoculated with PRSV. Compared with controls, yield per plant was unaffected by inoculation with potyvirus in resistant cultivar ‘Menina’. Unexpectedly, yield in resistant ‘Nigerian Local’ was reduced an average of 45% over control plots. Yield loss was 100% in inoculated plots of susceptible ‘Waltham’. Yield reduction ranged from 35% to 80% for susceptible Mos166 and moderately resistant ‘Soler’. There was little evidence that days to anthesis, average fruit weight, fruit diameter, and fruit quality (mesocarp thickness, chroma, hue angle, °Brix and dry matter) of plants inoculated with virus were different from that of uninoculated control plants. The exception was moderately resistant ‘Soler’ where plants inoculated with ZYMV produced fruits with a 32% reduction in average weight, as well as reductions in diameter, mesocarp thickness, and color saturation (chroma) compared with controls. This was unexpected given that ‘Soler’ has some resistance to ZYMV. Greenhouse evaluations by ELISA or symptom severity were generally useful in predicting field resistance to PRSV and ZYMV. In summary, yield reductions of up to 100% can be expected in C. moschata genotypes susceptible to PRSV or ZYMV, but fruit quality traits are usually unaffected. Moderate resistance to ZYMV in ‘Soler’ was observed to reduced symptom severity but not negative effects on yield and other traits. ‘Soler’ was not resistant to PRSV. ‘Menina’ rather than ‘Nigerian Local’ appears to be the best source of resistance because yield of the former was not impacted by inoculation with either potyvirus.

Papaya Ringspot Virus Isolates From Papaya in Bangladesh: Detection, Characterization, and Distribution.

Papaya ringspot virus (PRSV) is the major constraint to papaya (Carica papaya) production in Bangladesh. Disease symptoms occurred in 90 to 100% of the plants surveyed. Full-length genomes of PRSV strains from severely infected papaya plants were determined using the Illumina NextSeq 500 platform, followed by Sanger DNA sequencing of viral genomes obtained by reverse-transcription PCR(RT-PCR). The genome sequences of two distinct PRSV strains, PRSV BD-1 (10,300 bp) and PRSV BD-2 (10,325 bp) were 74 and 83% identical to each other, respectively, at the nucleotide and amino acid levels. PRSV BD-1 and PRSV BD-2 were 74 to 75% and 79 to 88% identical, respectively, to other full-length PRSV sequences at the nucleotide level. Based on phylogenetic analysis, PRSV BD-2 was most closely related to PRSV-Meghalaya (MF356497) from papaya in India. PRSV BD-1 formed a branch distinct from the other PRSV sequences based on nucleotide and amino acid sequence comparisons. Comparisons of the genome sequences of these two strains with other sequenced PRSV genomes indicated two putative recombination events in PRSV BD-2. One recombinant event contained a 2,766-nucleotide fragment highly identical to PRSV-Meghalaya (MF356497). The other recombinant event contained a 5,105-nucleotide fragment highly identical to PRSV-China (KY933061). The occurrence rates of PRSV BD-1 and PRSV BD-2 in the sampled areas of Bangladesh were approximately 19 and 69%, respectively. Plants infected with both strains (11%) exhibited more severe symptoms than plants infected with either strain alone. The full-length genome sequences of these new PRSV strains and their distribution provide important information regarding the dynamics of papaya ringspot virus infections in papaya in Bangladesh.

Cross protection against the watermelon strain of Papaya ringspot virus through modification of viral RNA silencing suppressor

Papaya ringspot virus watermelon strain (PRSV-W) causes huge economic losses to cucurbits production. Here, we constructed an infectious clone of PRSV-W, pCamPRSV-W, which can induce similar symptoms and accumulate to same levels as wild type virus in plants of Cucurbita pepo, Cucumis melo, Citrullus lanatus and Cucumis sativus. The green fluorescence protein gene gfp was cloned into pCamPRSV-W to produce pCamPRSV-W-GFP, which produced strong green fluorescence in systemic leaves of inoculated Cucurbita pepo, Cucumis melo, Citrullus lanatus and Cucumis sativus plants, indicating that pCamPRSV-W can be used to express foreign genes. Ten mutants of PRSV-W, obtained by site-directed mutagenesis in the RNA silencing suppressor helper-component proteinase encoding region, produced dramatically attenuated symptoms in plants of Cucumis melo. The Cucumis melo plants pre-infected with mutants K125D and G317 K showed effective protection against the challenge inoculation of wild type PRSV-W. The attenuated mutants generated in this study will be helpful for the eco-friendly control of PRSV-W.

Transgene-mediated resistance to Papaya ringspot virus: challenges and solutions

Global papaya production is severely affected by papaya ringspot disease caused by Papaya ringspot virus (PRSV). Management of this potyvirus is challenging, due to 1) its non-persistent transmission by numerous aphid species and 2) the diversity of PRSV strains that exists within a country or between different geographical regions. Papaya cultivars with transgenic resistance have reduced the impact of the disease. There are no effective alternatives to transgenic resistance available in areas where disease pressure is high. In Hawaii, transgenic papayas such as “SunUp” and “Rainbow” have remained resistant to PRSV more than two decades saving the commercial papaya industry. Following the success in Hawaii, researchers from other countries have focused on developing PRSV-resistant transgenic papaya. These transgenic cultivars often demonstrated an initial transitory resistance that was ultimately overcome by the virus. For other cases, resistance was inconsistent. That is, some transgenic lines were resistant while others were not. Transgenic cultivars are now losing PRSV-resistance for various reasons in China and Taiwan. In this review, we present an update on work with transgenic papaya with resistance to PRSV. The focus is on factors affecting transgenic resistance in papaya and our attempt to explain why the Hawaiian scenario of complete and durable resistance has not been replicated in other regions. The utilization of more recent technologies to the development of virus resistance in papaya is also discussed.

Characterization of Vasconcellea cauliflora for morpho-horticultural traits under climatic conditions of Pune, India

Vasconcellea cauliflora is one of the few species in the family Caricaceae that have shown resistance against Papaya ringspot virus strain papaya (PRSV-P) which is a global limiting factor in papaya (Carica papayaL.) cultivation. V. cauliflora has a potential role in introgression of gene(s) of PRSV-P resistance in C. papaya. It was introduced in India for academic and research purposes. Therefore, very little information is availableabout its morpho-horticultural traits under Indian climate and its reaction against local isolates of PRSV-P. V. cauliflora plantation maintained at ICAR-Indian Agricultural Research Institute, Regional Station, Pune since2011 provided valuable information on morpho-horticultural traits under climatic conditions of Pune and itsreaction against local isolate of PRSV-P. Plants gained a height of 0.89 m and collar diameter of 6.94 cm in oneseason which increased in subsequent years. Yellowish white and waxy female flowers were 3.9 cm long. Pollenviability was 86%. Average fruit yield was 4.3 kg/plant in the first year, which increased to 8.4 kg/plant in thethird year. berry type fruits were long, round or intermediate in shape with smooth skin texture. The averagefruit length and width was 6.8 cm and 3.7 cm, respectively. Ridges and grooves were prominent in long fruitshape as compared to intermediate and round ones. Seedlings showed resistance against PRSV-P under fieldconditions and when infected by challenge inoculation in glasshouse. The plants remained disease-free evenwhen exposed to severe disease pressure under field conditions for more than three years. There is a renewedinterest in V. cauliflora as a source of gene(s) for resistance against PRSV-P infection, especially after overcomingthe crossing barrier with C. papaya in India.

Update on gene transfer biotechnology of papaya

Papaya ringspot virus (PRSV) is the most devastating disease of papayas worldwide. For 15 years, since 1998, genetically engineered, PRSV resistant Hawaii papayas, 'Rainbow', 'SunUp', and 'Laie Gold' have been sold locally and 'Rainbow' has been exported to the western US. Following deregulation, it has been exported for eight years to Canada and for three years, on a very small scale, to Japan. Markets like Hong Kong that do not differentiate between non-transgenic products and genetically engineered organisms (GMOs) also import Hawaii GMO papayas. At least fifteen research groups attempted to develop GMO papayas, mostly for PRSV resistance. A majority of the papayas were resistant to their specific PRSV strains; however at least initially China is the only other country to have commercialized transgenic virus resistant papaya. Activity in GMO projects is reduced because of negative publicity and lack of funding, but several projects continue: delayed ripening in Malaysia and the Philippines (field testing); PRSV and Papaya leaf distortion mosaic virus (PLDMV) broad resistance with single, double, and super-constructs in Taiwan; PRSV resistance construct development in Pune, India; multiplication of transgenic plants for PRSV resistance testing in Hainan, China; transformation of papaya for PRSV resistance with a gene from a papaya relative, Vasconcellea pubescens; and the final stages of deregulation of a second PRSV resistant papaya for Florida in the US.

Morphological traits, growth and yield performance, andPapaya ringspot virusstrain papaya reaction inVasconcellea cauliflorain Pune, India

Vasconcellea cauliflora is one of the few Vasconcellea species that have shown resistance against Papaya ringspot virus strain papaya (PRSV-P), a major limiting factor in papaya (Carica papaya L.) cultivation. It has a potential role in breeding PRSV-P resistance in papaya. It is neither cultivated nor found in the wild in India where only a few plantations are maintained for research purposes. Therefore, very little information is available about morphological traits related to growth and yield in India as well as its reaction against Indian isolates of PRSV-P. A V. cauliflora plantation maintained at Indian Agricultural Research Institute, Regional Station, Pune India since 2010 provided valuable information on morphological traits under Pune climate and reaction against Indian isolate of PRSV-P. Plants attained a height of 1.04 m and stem diameter of 9.49 cm in one season. Average fruit yield was 4.3 kg plant-1 in the first year, which rose to 8.4 kg plant-1 in the third year. Yellowish white, waxy female flowers were 3.9 cm long. Berry type fruits were long, round or intermediate in shape with smooth skin texture. The average fruit length and width was 6.76 and 3.68 cm, respectively. Ridges on fruits were deep in long fruits and intermediate in round ones. V. cauliflora seedlings showed resistance against a Pune isolate of PRSV-P when infected by challenge inoculation. There were no symptoms of PRSV-P infections even with exposure to severe disease pressure under field conditions for more than three years. Pollen from grafted plants (V. cauliflora on C. papaya) showed slightly different characteristics when compared with V. cauliflora. There is a renewed interest in V. cauliflora as the resistant parent in a PRSV-P resistant breeding programme, especially after overcoming the crossing barrier with C. papaya at some institutes in India.

Nucleotide sequence-homology-independent breakdown of transgenic resistance by more virulent virus strains and a potential solution.

Controlling plant viruses by genetic engineering, including the globally important Papaya ringspot virus (PRSV), mainly involves coat protein (CP) gene mediated resistance via post-transcriptional gene silencing (PTGS). However, the breakdown of single- or double-virus resistance in CP-gene-transgenic papaya by more virulent PRSV strains has been noted in repeated field trials. Recombination analysis revealed that the gene silencing suppressor HC-Pro or CP of the virulent PRSV strain 5-19 is responsible for overcoming CP-transgenic resistance in a sequence-homology-independent manner. Transient expression assays using agro-infiltration in Nicotiana benthamiana plants indicated that 5-19 HC-Pro exhibits stronger PTGS suppression than the transgene donor strain. To disarm the suppressor from the virulent strain, transgenic papaya lines were generated carrying untranslatable 5-19 HC-Pro, which conferred complete resistance to 5-19 and other geographic PRSV strains. Our study suggested the potential risk of the emergence of more virulent virus strains, spurred by the deployment of CP-gene-transgenic crops, and provides a strategy to combat such strains.

Screening Cucumber Plant Introduction Accession Lines for Resistance against Cucumber Strain of Papaya ringspot virus (PRSV)

Screening Cucumber Plant Introduction Accession Lines for Resistance against Cucumber Strain of Papaya ringspot virus (PRSV)

Identification and Detection of Genetically Modified Papaya Resistant to Papaya Ringspot Virus Strains in Thailand

Many lines of genetically modified (GM) papaya (Carica papaya Linnaeus) have been developed worldwide to resist infection from various strains of papaya ringspot virus (PRSV). We found an unidentified and unauthorized GM papaya in imported processed papaya food. Transgenic vector construct that provides resistance to the PRSV strains isolated in Thailand was detected. An original and specific real-time polymerase chain reaction method was generated to qualitatively detect the PRSV-Thailand-resistant GM papaya.

GENETIC DETERMINANT OF PAPAYA RINGSPOT VIRUS FOR INFECTION OF PAPAYA

Papaya ringspot virus (PRSV) is a major limiting factor for large-scale planting of papaya and cucurbits. Based on the difference of host range, most strains of PRSV belong to type P (PRSV P, papaya infecting) devastating on papaya in tropical areas or type W (PRSV W, non-papaya-infecting) causing severe loss on cucurbits worldwide. Viruses of PRSV P infect plants of the families Chenopodiaceae, Cucuribitaceae and Caricaceae (papaya family), whereas, viruses of PRSV W only infect plants of Chenopodiaceae and Cucuribitaceae. Phylogenetic analyses of PRSV isolates revealed that the divergence of PRSV is correlated to geographic distribution and not to the biological difference in host reactions, suggesting that P type viruses may derive from W type viruses. To study the genetic determinant(s) of PRSV for papaya infection, infectious cDNA clones of type P and type W viruses, driven by a bacteriophage promoter to express in vitro infectious transcript or by Cauliflower mosaic virus 35S promoter to express in vivo infectious transcript, were generated in our laboratory and used for recombination analyses. The bioassay of recombinant viruses revealed that the nt 6500-7000 of PRSV P genome is responsible for papaya infection. Sequence comparison showed that amino acid positions at 176 of NIaVPg, and 27 and 205 of NIaPro are significantly different among type P and type W viruses. Site-directed mutagenesis indicated that Lys 27 of NIaPro determines host specificity of PRSV for infection of papaya. Our results indicate that the host range of PRSV can be switched just by a single amino acid mutation. Thus, we have shown experimental evidence that PRSV type P may easily evolve from type W and explained the reason why type P and W viruses are closer to each other in the same geographic zone. Furthermore, the potential risk for planting papaya adjacent to cucurbit fields is also discussed.

Generation of Transgenic Papaya with Double Resistance to Papaya ringspot virus and Papaya leaf-distortion mosaic virus

During the field tests of coat protein (CP)-transgenic papaya lines resistant to Papaya ringspot virus (PRSV), another Potyvirus sp., Papaya leaf-distortion mosaic virus (PLDMV), appeared as an emerging threat to the transgenic papaya. In this investigation, an untranslatable chimeric construct containing the truncated CP coding region of the PLDMV P-TW-WF isolate and the truncated CP coding region with the complete 3' untranslated region of PRSV YK isolate was transferred into papaya (Carica papaya cv. Thailand) via Agrobacterium-mediated transformation to generate transgenic plants with resistance to PLDMV and PRSV. Seventy-five transgenic lines were obtained and challenged with PRSV YK or PLDMV P-TW-WF by mechanical inoculation under greenhouse conditions. Thirty-eight transgenic lines showing no symptoms 1 month after inoculation were regarded as highly resistant lines. Southern and Northern analyses revealed that four weakly resistant lines have one or two inserts of the construct and accumulate detectable amounts of transgene transcript, whereas nine resistant lines contain two or three inserts without significant accumulation of transgene transcript. The results indicated that double virus resistance in transgenic lines resulted from double or more copies of the insert through the mechanism of RNA-mediated posttranscriptional gene silencing. Furthermore, three of nine resistant lines showed high levels of resistance to heterologous PRSV strains originating from Hawaii, Thailand, and Mexico. Our transgenic lines have great potential for controlling a number of PRSV strains and PLDMV in Taiwan and elsewhere.

Yield of varieties of Cucurbita pepo preimmunized with mild strains of Papaya ringspot virus - type W and Zucchini yellow mosaic virus

Papaya ringspot virus - type W (PRSV-W) and Zucchini yellow mosaic virus (ZYMV) are the most prevalent viruses in cucurbit crops in Brazil and responsible for frequent yield losses. Diseases caused by these viruses are difficult to control. The objective of this work was to evaluate the effects of mild strains PRSV-W-1 and ZYMV-M on the yield of Cucurbita pepo L. cvs. Samira, Novita Plus, AF 2847, and Yasmin, under plastic greenhouse and field conditions. Plants infected with ZYMV-M and grown in a plastic greenhouse did not exhibit typical leaf symptoms or significant alterations in quantitative and qualitative fruit yield. However, when infected with PRSV-W-1, or PRSV-W-1 + ZYMV-M, the plants exhibited severe leaf mosaic symptoms and reduced fruit quality, although there were no changes in the number and mean fruit weight harvested from these plants. When these plants were infected with PRSV-W-1 and studied simultaneously in the field and plastic greenhouse, intensification of symptoms in the fruits and leaves was more pronounced under the greenhouse conditions. Quantitative yield did not change. Environmental factors seem to influence symptoms induced by PRSV-W-1.