GM Crop Database

Database Product Description

X17-2 (UFL-X17CP-6)
Host Organism
Carica papaya (Papaya)
Trait
Resistance to viral infection, papaya ringspot virus (PRSV).
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption.

Product Developer
University of Florida

Summary of Regulatory Approvals

Country Food Feed Environment Notes
United States 2008 2008 2009

Introduction Expand

Papaya ringspot virus (PRSV) is the most important papaya pathogen and a major limiting factor in commercial papaya production in the world. All major production areas I the Western Hemisphere [Brazil, the Caribbean region, Mexico, and USA (Florida and Hawaii)] and Eastern Hemisphere (the Philippines, Taiwan, Thailand, and China) are affected. Once introduced to a locality, PRSV becomes established in weeds or perennial plants and has never been successfully eradicated from any region. The aphid vectors involved in dissemination of PRSV are found worldwide. Thus, even if new PRSV-free papaya trees are planted in an area where PRSV occurs, they soon become infected.

Transgenic papaya expressing the coat protein (CP) gene of Hawaiian PRSV isolate HA5-1 (see description of events 55-1/63-1) is highly resistant to PRSV isolates from Hawaii but more susceptible to a number of PRSV isolates from other geographic locations. In general, resistance conferred by the CP gene of a potyvirus (such as PRSV) is RNA-mediated and sequence-specific. Resistance is only effective when the transgene has a high similarity to the CP gene of the infecting virus and appears to be due to post-transcriptional gene silencing (PTGS). PTGS can take place when the antisense RNA, either directly from transcription of an antisense CP transgene or indirectly from sense transgene mRNA via RNA-dependent RNA polymerases, binds to the complementary regions of viral RNA transcripts in the cytoplasm to form duplexes, which are subsequently cleaved by dsRNA-specific nucleases into 21-23 nt RNAs that interfere with transcription of homologous RNAs. This requirement for sequence homology points to a need to utilize local PRSV isolates as sources of the CP gene in order to obtain effective PRSV resistance in transgenic papaya.
In addition to translatable CP genes, transgene constructs with non-translatable CP genes (e.g., antisense constructs, or frame-shift mutants, or three-in-frame stop-codon mutations) have been shown to be effective in conferring resistance to potyviruses. Papaya line X17-2 was developed to contain a frame-shift mutant of the CP gene from PRSV isolate H1K (a thymidine residue inserted after the initiation codon to yield a frame-shift). Subsequent analysis of the fifth generation progeny of the original transformant revealed that the mutation was repaired and that the CP transgene was both translatable and expressed in later generations.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
CP Papaya ringspot virus (PRSV) VR

CaMV 35S

uidA leader sequence.

A. tumefaciens nopaline synthase (nos) 3'-untranslated region

1

Frameshift mutant, repaired

nptII neomycin phosphotransferase II SM Promoter for transcription of the nptII gene from plasmid pTiT37 A. tumefaciens nopaline synthase (nos) 3'-untranslated region 1 Native

Characteristics of Carica papaya (Papaya) Expand

Center of Origin Reproduction Toxins Allergenicity

Believed to be native to tropical America, possibly southern Mexico or northwestern South America.

Commercial papaya lines are mostly gynodioecious as the hermaphroditic plants are generally self-pollinating. A low rate of out-crossing still occurs since the plants produce copious pollen during most of the year.

Green tissues contain benzyl isothiocyanate (BITC), which has been linked to incidents of spontaneous abortions in pregnant women and with a higher incidence of prostate cancer in Japanese men over age 70. Ripe fruit has no latex and virtually no BITC.

The latex is either hyperallergenic or an irritant.

Modification Method Expand

Papaya line X17-2 was developed via Agrobacterium-mediated transformation using vector pBI121fs, which contained two gene expression cassettes located within the T-DNA region. The prsv-cp expression cassette consisted of the 35S promoter from cauliflower mosaic virus (CaMV), a frame-shift mutant of the coat protein encoding gene from PRSV isolate H1K, and the nopaline synthase (NOS) 3’ untranslated region from Agrobacterium tumefaciens. In order to enhance transcription of the prsv-cp gene, the uidA sequence with an initiation codon was placed at the 5’ terminus of the prsv-cp gene. The second cassette contained the NOS promoter region from A. tumefaciens, the neomycin phosphotransferase II (NPTII) encoding gene from the Tn5 transposon of Escherichia coli, and the NOS 3’ untranslated region from A. tumefaciens.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot hybridization analysis was used to confirm the insertion of a single copy of the T-DNA region from plasmid pBI121fs and to confirm the integrity of the prsv-cp gene. Southern analysis also confirmed the lack of any plasmid backbone sequences within the X17-2 genome. The 5' and 3' genomic flanking sequences were cloned and sequenced, as was the T-DNA insert (less approx. 336 bp). The expected and observed nucleotide sequence of the T-DNA insert (from R5 generation) was 99.7% identical, with the differences occurring where the prsv-cp gene was ligated into the T-DNA. These differences appeared to have no consequence, however, the thymidine base originally incorporated into the leader sequence for the CP gene to create a frame-shift mutation was missing. Consequently, the prsv-cp gene is translatable and not non-translatable as intended, and western analyses indicated that the gene was expressed.

Stability and Inheritance

The insert in line X17-2 has persisted as a single copy through six generations of progeny following the initial transformation and segregation analysis using kanamycin treatment of seedlings indicated that the inserted T-DNA segregated as a single locus in a Mendelian fashion.

Expressed Material

Expression of the PRSV CP in papaya line X17-2 was confirmed by enzyme linked immunosorbent assay (ELISA) using polyclonal anti-PRSV serum, and by western immunoblotting. The 35 kDa size of the expressed PRSV CP in line X17-2 was the same as predicted from the nucleotide sequence of PRSV RNA, although the natural CP is one of at least seven proteins that are post-translationally cleaved from the expressed poly-protein of PRSV in infected plants.

Environmental Safety Considerations Expand

Growth Habit and Ability to Overwinter

Leaf, stem, and fruit morphologies of X17-2 plants were normal throughout the year. X17-2 plants continuously grow throughout the year; more vigorously when ambient temperatures are above 65F and more slowly during cool weather (i.e., temperatures 50-65F). Once warm temperatures commence in the spring X17-2 papayas resume vigorous growth whereas PRSV-infected non-transgenic papaya only slowly resume growth; not growing vigorously until high temperatures in mid-summer.

Life-Span and Vegetative Vigor

Papaya plants may live up to 20 years, however, typically, viral infection, other diseases, or natural disasters reduce their life-span to 4-5 years; commercial plants are replaced after 2-3 years. The fifth generation of the X17-2 has been grown for over two years with some losses due to 4 hurricanes in 2005.

Days to Flowering and Fruit Maturity

The days from planting to first flowering and the rate of fruit development is influenced by genetics, environmental conditions (mostly temperature), and cultural practices. During the summer, there were no observed differences in the days to flowering or rate of fruit maturity between X17-2 papayas and PRSV-tolerant papaya cultivars (e.g., ‘Red Lady’). During spring, fall, and winter, fruit development was slower for non-transgenic plants compared to X17-2 papayas, most likely due to the effect of the PRSV on leaf area and health.

Seed Parameters

No differences among X17-2 line papayas and non-transgenic papayas in seed production, dormancy, percent germination, seedling survival, or seedling emergence.

Pollination and Reproduction

After X17-2 seedling field establishment and once plants begin to flower (usually 4-5 months post planting), female plants are generally eliminated from the field since only hermaphrodites have the ability to self-pollinate (thus reducing the need for male plants) and possess the desired fruit characteristics. The X17-2 line papayas are self-compatible as are all hermaphroditic papaya plants, and there were no observed changes in flower morphology or potential pollinator species in the field plots. There were no observed differences in the fertility of the X17-2 line papayas compared to other commercial papaya cultivars. Papaya seeds do not shatter even if the fruit is damaged.

Weediness

No Carica species is considered a weed, and there is no evidence in the scientific literature to suggest that susceptibility to PRSV is the factor that prevents these plants from being weed pests. Therefore, it seems likely that even if the PRSV-resistance trait could be transferred from line X17-2 to another Carica species, the resultant offspring would not be weed pests.

Secondary and Non-Target Adverse Effects

It was concluded that the genes inserted into the transgenic papaya line X17-2 would not result in any deleterious effects or significant impacts on non-target organisms, including threatened and endangered species or beneficial organisms. The PRSV coat protein expressed in these papaya lines is found in all PRSV-infected plants, and there are no reports of this protein having any toxic effects. In fact, this viral coat protein is routinely ingested by virtually all animals, including humans, when papaya is consumed. Naturally occurring infections of susceptible papaya varieties result in concentrations of coat proteins far higher than those that occur in the tissues of the transgenic papaya line X17-2. No direct pathogenic properties, nor any hypothetical mechanisms for pathogenesis toward beneficial organisms, such as bees and earthworms, were identified for papaya line X17-2.

Food and/or Feed Safety Considerations Expand

Compositional Analysis

Mature fruits from four different specimens of line X17-2 were subjected to compositional analysis. Published information on other transgenic papaya lines (Hawaiian cultivars 55-1 and 63-1) and nontransgenic varieties was used for comparison. The range of analytes included proximates (moisture, ash, total dietary fiber, calories, total fat, carbohydrate, and protein), minerals (sodium, calcium, iron), total sugar, fats (saturated, mono- and poly-unsaturated), benzyl isothiocyanate (BITC), and vitamins (beta-carotene, vitamin A, and vitamin C).

It was concluded that compositional values of the X17-2 line were not meaningfully different from those given for the other varieties. Vitamin C values were comparable to the literature values of other varieties cited by the developer. Although Vitamin A values were somewhat higher on average, the range overlapped with the value reported in the United States Department of Agriculture's National Nutrient Database.

Endogenous Toxicants

The presence of benzyl isothiocyanate (BITC) in the latex of green papaya tissues has been linked to incidents of spontaneous abortions in pregnant women and with a higher incidence of prostate cancer in Japanese men over the age of 70. Because ripe papaya fruits have no latex they also have virtually no BITC content, and, therefore, the consumption of ripe fruit would not be expected to be linked to any abortifactant effects. BITC was assayed in green fruits of seven different X17-2 breeding lines with concentrations ranging from 0.2 milligrams (mg) to 4.8 mg per 100 mg of green fruit. Similar or greater ranges were found with the same assay in three conventional cultivars.

Potential Toxicity and Allergenicity

There is a history of safe consumption of PRSV CP derived from virus-infected papaya fruit, and there is no indication that the form of the CP expressed in transgenic X17-2 papaya is materially different in any way that would affect its potential for toxic or allergenic effect. Additionally, the PRSV CP does not possess any of the physiochemical properties normally associated with protein allergens or toxins, such as heat stability and resistance to digestion by simulated gastric fluids. Also, there were no regions of homology when the deduced amino acid sequence of PRSV CP was compared to the amino acid sequences of known protein allergens or toxins.

Links to Further Information Expand

U.S. Department of Agriculture, Animal and Plant Health Inspection Service United States Food and Drug Administration

This record was last modified on Tuesday, September 15, 2015