Novel Food Information: Virus Resistant C5 Plum

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• Background
1. Introduction
2. Development of the Modified Plant
3. Characterization of the Modified Plant
4. Dietary Exposure
5. Nutrition
6. Chemistry/Toxicology
• Conclusion

Background:

Health Canada has notified the United States Department of Agriculture that it has no objection to the food use of virus resistant C5 plum. Health Canada conducted a comprehensive assessment of this variety according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.

The following provides a summary of the notification from the United States Department of Agriculture and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

The United States Department of Agriculture developed C5 plum to resist plum pox virus infection.

The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflect international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: how C5 plum was developed; how the composition and nutritional quality of C5 plum compared to non-modified plum varieties; and the potential for C5 plum to be toxic or cause allergic reactions. The United States Department of Agriculture provided data that demonstrates that C5 plum is as safe and of the same nutritional quality as traditional plum varieties used as food in Canada.

The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in the Food and Drug Regulations (Division B.28). Food use of C5 plum is considered a novel food under the following part of the definition of novel foods:

• "c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that
  • (i) the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism."

2. Development of the Modified Plant

C5 plum was developed through Agrobacterium-mediated transformation of plum variety 'Bluebyrd' with the pGA482GG/PPV-CP-33 transformation vector (Mante et al. 1991¹ ; Scorza et al., 1994²).

The pGA482GG/PPV-CP-33 transformation vector was also used in the development of Papaya Line 55-1 – the precursor to Rainbow and SunUp papaya cultivars – for which Health Canada issued a letter of no objection for food use in 2003, following a pre-market safety assessment as a novel food.

The pGA482GG/PPV-CP-33 transformation vector contains a transfer DNA (T-DNA) encoding a gene for the plum pox virus coat protein (PPV-CP) from plum pox virus D-strain isolate. This protein is intended to be overexpressed, and it protects against plum pox virus infection by inhibiting viral particle dissolution.

The pGA482GG/PPV-CP-33 transformation vector also encodes a neomycin phosphotransferase type II (NPTII) protein. This protein functions as a selectable marker by inactivating neomycin and kanamycin antibiotics and conferring an antibiotic resistance characteristic to transformed plant cells. The NPTII protein has been introduced into 22 products³ that have been previously reviewed by Health Canada and that have all received letters of no objection.

The pGA482GG/PPV-CP-33 transformation vector also encodes a beta-glucuronidase protein (GUS). This protein catalyzes the breakdown of complex carbohydrates. In transgenic plants, it is used as a reporter gene to identify transformed plant cells through the appearance of blue colour. This protein has been introduced into 3 products⁴ that have been previously reviewed by Health Canada that have all received letters of no objection.

Information was provided to support the safety and long history of use of the donor organisms (Plum pox virus D-strain isolate, and E. coli K12 strain) and the recipient organism (Prunus domestica). None of these organisms pose a safety concern from a food perspective.

3. Characterization of the Modified Plant

The number of T-DNA inserts in C5 plum was characterized by using a combination of Southern blot analysis (Scorza et al., 1994; Scorza et al., 2001⁵ ), bacterial artificial chromosome (BAC) library construction and direct sequencing of BAC library clones (Georgi et al., 2002⁶; Scorza et al., 2001; Scorza et al., 2010⁷), whole genome sequencing (WGS), as well as direct sequencing of junction sequences identified by WGS (Callahan et al., 2021⁸). The results of the analyses demonstrated that there are multiple integration sites in C5 plum.

C5 plum possesses two complex T-DNA insertion sites ("Insert-1" and "Insert-2"). Insert-1 is made up of a duplicated and rearranged T-DNA insert. Insert-1 contains two copies of the ppv-cp expression cassette, three copies of the nptII expression cassette, and three copies of the uidA expression cassette. Insert-2 is also made up of a duplicated and rearranged T-DNA. However, in this case the result is a tail-to-tail arrangement of two copies of the ppv-cp expression cassette. Functionally, Insert-2 is an RNAi gene-suppression cassette which confers plum pox virus resistance through an RNA interference (RNAi) mechanisms, rather than the intended protein based viral suppression mechanism (Scorza et al. 1994²); Scorza et al. 2001⁵); Georgi et al. 2002⁶); Scorza et al. 2010⁷); Callahan et al., 2021⁷).

As only four insert-genome junctions were identified in the WGS study, and the junctions corresponded to Insert 1 and Insert 2, it was concluded that no vector backbone sequences were integrated in to C5 plum (Callahan et al., 2021⁸).

The WGS study, combined with an expanded analysis of the BAC clone library (Scorza et al. 2010⁷); Callahan et al. 2021⁸) evaluated whether endogenous plum genes were disrupted by Insert 1 and Insert 2. This analysis determined that no endogenous plum genes were disrupted (Callahan et al., 2021⁸).

To determine if genes close to the insertions had their expression profiles influenced by the inserts, RNA-seq was used (Callahan et al., 2021⁸). The results of this study suggest that any changes to the expression of genes that are proximal to Insert 1 and Insert 2 were unlikely to be caused by the inserts, but rather by environmental influences.

The possibility that new genes could potentially be encoded in the complex inserts and expressed in C5 plum was also investigated (Callahan et al., 2021⁸). The study also used RNA-seq technology, and determined that only reads corresponding to the mRNA transcripts for nptII, uidA and ppv-cp were expressed.

Given the complex and rearranged copies of the T-DNA inserts in C5 plum, the petitioner also conducted a sequence level analysis from the 5' to the 3' genome-insert regions of Insert-1 and Insert-2. This approach was used to look for open reading frames (ORFs) that could encode proteins of at least 30 amino acids. Based on this analysis, three possible ORFs were identified (ORF-1, 41 amino acids; ORF-2, 130 amino acids; ORF-3, 157 amino acids).

The predicted ORFs were further screened for similarity to known allergens and toxins. To screen for possible toxins, the amino acid sequence of ORF-1, ORF-2 and ORF-3 were used to search the UniProt Knowledgebase toxin database. The search used the FAST All sequence alignment tool (FASTA) using the BLOcks Substitution Matrix (BLOSUM) 50 and an acceptance criteria (E score) of 1x10-5. These parameters, and the curation of the toxin database were deemed acceptable. No significant hits for toxins were returned in the search.

To screen for possible allergens, the same amino acid sequences from ORF-1, ORF-2, ORF-3 were used to search the Food Allergy Research and Resource Program (FARRP) dataset of putative allergens and celiac proteins. The search used cut-offs of 35 percent identity over 80 amino acids. These parameters, and the curation of the database were acceptable. No hits for allergens were returned in the search. To further verify that the ORF-1, ORF-2, and ORF-3 amino acid sequences were not likely to be allergenic, a search using all possible 8 amino acid windows was performed between the predicted sequences and the sequences contained in the database for perfect matches. No matches were observed for the amino acid sequences predicted from ORF-1, ORF-2, or ORF-3 that would suggest these could be potential allergens.

Generational stability was not assessed through multi-generational analysis as plum trees are not grown from seed each growing generation. Stability of the PPV resistance characteristic was demonstrated at the phenotypic level by assessing C5 plum plant populations in field trials for plum pox virus resistance over 4 consecutive years. Plum tree populations of PPV resistant C5 plum, PPV susceptible C3 plum and non-transgenic parents were inoculated with PPV. The results of this study determined that C5 plum viral resistance characteristic is stable over the 4 year trial (Hily et al., 2004⁹).

To characterize the genetic mode of inheritance of the C5 plum inserts, interspecific crosses were performed using heterozygous C5 plum as the male parent, and non-transgenic plum varieties "Prunier d'Ente', 'Quetsche' and P. spinose as the female parents (Ravelonandro et al. 2001¹⁰; Ravelonandro et al. 2010¹¹). Fruit yielded from these crosses were harvested, mature seeds isolated from fruit, seeds germinated to generate plants, and resulting plants were grown for genotyping and phenotyping. Genotyping was performed by PCR by amplifying ppv-cp as well as nptII genes from genomic DNA isolated from the leaves of hybrid seedlings, as well as from parents. Phenotyping was performed by harvesting leaf material and evaluating for GUS marker staining using X-glucuronide substrate. The staining assay functions by visual detection of an insoluble, indigo-blue precipitate that forms in plant tissues expressing GUS when X-glucuronide substrate is provided. It is expected that seedlings containing the C5 plum transgenic inserts would demonstrate GUS marker activity, while those lacking the transgenic inserts would not. Based on the genotyping and phenotyping studies, and the resulting Chi-Square analysis of the results, it was determined that inheritance of the C5 plum inserts followed the expected Mendelian inheritance pattern.

Expression of transgenic PPV-CP mRNA in C5 plum was evaluated by northern blot analysis of total RNA isolated from C5 leaf tissues. Northern blot PPV-CP signal was only observable after 40 hours of audioradiograph exposure, consistent with low-level transcript expression (Scorza et al. 1994²). To evaluate whether PPV-CP protein accumulated in C5 plum, western immunoblots were performed on total protein isolated from C5 plum leaf tissues. Western blot PPV-CP signal was not detected from C5 plum leaf total protein, though positive controls demonstrated the PPV polyclonal antibody functioned under experimental conditions (Scorza et al. 1994²). Lastly, as plum leaves are not consumed as food, the petitioner supplied details of a RNA-seq study that was performed to evaluate ppv-cp mRNA transcript in non-transgenic plum 'Stanley' and transgenic C5 plum. The results of this study demonstrated that in fruit tissues, ppv-cp transcript in C5 plum is comparable to non-transgenic Stanley, and therefore represents background signal levels observable only through highly sensitive tools like RNA-seq.

Measurement of NPTII and GUS proteins was not performed as the petitioner has taken a weight-of-evidence approach towards establishing the safety of these proteins in food. The transformation plasmid (pGA482GG) used to produce C5 plum was also used to generate transgenic 55-1 papaya, which has previously undergone pre-market safety assessment by Health Canada (Papaya Line 55-1)¹². Furthermore, the encoded npt and uidA genes in C5 plum are identical to those in 55-1 papaya - the precursor to Rainbow and SunUp papaya cultivars. Papaya event 55-1 has made up 85 % of Hawaiian papaya production for the last 20 years (Gonsalves, 2014)¹³, therefore there has been significant, ongoing exposure to diverse human populations to the identical NPTII and GUS proteins in C5 plum without apparent negative human health effects. Furthermore, there have been many previously reviewed Novel Foods containing NPTII proteins, and GUS proteins that have all undergone rigorous safety assessment where no objections have been raised.

Based on the available data provided, there are no safety concerns regarding C5 plum from a molecular biology perspective.

4. Dietary Exposure

Plums are consumed fresh, canned, as juice or concentrate, frozen, dried as prunes, or used to produce spirits. Fresh plum or dried plum (prune) concentrate is used in baked goods, sauces and marinades, snack foods, and energy bars. In Canada, plums are mainly produced for fresh market. Plums generally account for a very small portion of the daily diet. An estimate of per capita plum consumption in Canada in 2017 was about 0.35 kg/person per year.

Data was presented showing low levels of PPV-CP small RNA (sRNA) in non-infected non-transgenic plum fruit, and high levels of PPV-CP sRNA in infected non-transgenic plum fruit. Levels of PPV-CP sRNA in transgenic C5 plum fruit are six times lower than the infected non-transgenic plum. Therefore, consumption of asymptomatic PPV-infected fruit would result in more exposure to PPV-CP sRNA than consumption of C5 plum fruit.

The petitioner noted that PPV infects many Prunus species including apricots (Prunus armeniaca), peaches (Prunus persica) plums (Prunus domestica, Prunus salicina and Prunus cerasifera) and almonds (Prunus dulcis). These fruits are commonly found in PPV-endemic regions of the world, and contain PPV and PPV-CP derived sRNA to which humans have been exposed for many years.

PPV-CP protein is not produced by the PPV-CP gene fragment that was inserted. PPV-CP protein was not detected (<1 ng/100 µg protein) in a western immunoblot of total protein from leaf material from C5 plum. In addition, PPV-CP protein is already present in the diet due to consumption of asymptomatic or mildly PPV-infected fruits.

5. Nutrition

To evaluate if there were any unanticipated consequences of the genetic modifications to C5 plum, the nutritional composition of the C5 plum was compared to that of commercial cultivars representing a range of diverse plum types. Twenty-two comparators were used, including one non-transgenic PPV-tolerant cultivar, and one non-transgenic PPV-resistant cultivar.

Field trials were conducted at various times in 12 locations (one orchard in Canada, 10 in Europe, and one in the United States). Collections in Spain were made at two different harvest times to enable comparisons between different growing seasons and different harvest times from the same trees. Overall, 62 fruit samples were collected from 23 cultivars (14 samples of C5 plum, 19 samples of a PPV-tolerant cultivar, 9 samples of a PPV-resistant cultivar, and one sample from each of 20 other cultivars). Fruits (greater than 10) at ripe stage were collected from one to four trees from 12 different orchards.

Fifty-four constituents were chosen for nutritional composition analysis based on attributes associated with plums as well as a known anti-nutrient constituent, oxalic acid. Key constituents analysed were starch, carbohydrates, calories, fibre, moisture, ash, antioxidants, carotenes, acidity, acids, sugars, sugar alcohols, vitamins, total phenolics, calcium, iron, magnesium, potassium, and sodium. The fruit samples were analysed using the Association of Official Analytical Chemists (AOAC) methods or other acceptable methods.

The nutritional composition data were analysed using the Statistical Analysis System version 9.2. An ANOVA, using the general linear procedure (PROC GLM), was performed at α = 0.05 significance level. The sample means were compared using the Tukey-Kramer multiple comparison test (α = 0.05).

Of the 54 constituents measured, sugars and organic acids were the main nutritive compounds in all plum cultivars, where the total sugar content varied from 8.81 % to 10.58 %, with the C5 plum value being 10.47 %, within the reported range. There were no differences between C5 plum and its comparators except for four constituents, which were higher in C5 plum than the comparators: ash, vitamin C, titratable acidity, and malic acid (Tukey-Kramer test, α = 0.05). While statistically higher in C5 plum, ash content was very low in all plum cultivars. The higher vitamin C reported for C5 plum (9.38 mg/100 g) is similar to the value reported for plum in the Canadian Nutrient File (9.5 mg/100 g; Food code: 1740). Both levels of ash and vitamin C in C5 plum do not present a nutritional concern. With regard to the titratable acidity and malic acid levels, C5 plum and five other cultivars were high in these two related components. Malic acid is usually the prevailing organic acid in plum fruit, and while not a nutritional concern, may impact flavour and consumer acceptance. However, the interplay of acids and sugar on the consumer’s perception of sweetness is complex and varying sugar levels can offset high acidity, such that high acid fruit cultivars are acceptable in the market.

The level of anti-nutrient oxalic acid in C5 plum (0.02 %) as well as in six other cultivars was at or above minimal detectable levels. The level detected in C5 plum is similar to that found in foods with low oxalate content such as sweet corn (0.01 %) and cucumber (0.02 %), compared to foods with high oxalate content such as spinach (0.97 %) or amaranth (1.09 %). There is no nutritional safety concern associated with the anti-nutrient oxalic acid in C5 plum.

By providing the data obtained from a range of geographies and different growing conditions over multiple years, the petitioner demonstrated that overall, the composition of the C5 plum falls within the range determined for a wide variety of commercial plums.

Based on the information provided, the Bureau of Nutritional Sciences has no nutritional concerns with the C5 plum, or foods derived from this plum.

6. Chemistry/Toxicology

Data for toxic trace elements were not provided by the petitioner. However, a compositional assessment comparing samples of C5 plum to commercial cultivars representing several varieties of plums was provided, comparing the levels of various nutrients, including minerals (calcium, iron, magnesium, potassium and sodium). The petitioner concluded that the nutrient composition assessment found no statistically significant differences in the levels of all of the minerals measured in C5 plum and commercial cultivars. This conclusion was supported by the Bureau of Nutritional Sciences. Based on the above information, there is no indication that the modifications would result in significant differences in the uptake of toxic trace elements in C5 plum relative to conventional plum.

Data for mycotoxins were also not provided by the petitioner. However, field trials were conducted for various PPV transgenic plum clones grown in a range of geographies and different growing conditions over multiple years. C5 plums demonstrated high resistance to PPV via natural aphid transmission. Graft inoculation of trees using infected budwood, or trees growing on infected susceptible rootstocks, showed few symptoms, and when present, the symptoms were very mild. This information suggests that C5 plum is not more susceptible to insect and disease stressors in comparison to conventional plum. Since infection from insect pests and disease can increase the susceptibility of a plant to mycotoxin-producing fungi, it can be inferred that the modifications to C5 plum are unlikely to increase susceptibility to mycotoxins compared to conventional varieties.

The C5 plum expresses two marker proteins (i.e., neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) from E.coli K12). The C5 plum also expresses double stranded RNA (dsRNA; i.e., coat protein (CP) 990 base-pair genetic fragment from plum pox virus strain D). The resulting small interfering RNAs (siRNA) confer resistance to plum pox virus (PPV) infection through RNA silencing/interference (RNAi).

The petitioner provided a scientific rationale for the lack of toxicity of the C5 plum based on published information on the safety of the marker proteins NPTII and GUS, and based on a history of consumption of PPV-CP dsRNA/siRNA via the consumption of asymptomatic or mildly PPV-infected fruits containing these RNAs.

The function of NPTII is well known: it is not a toxin, does not have amino acid homology with known toxins, and is not acutely toxic in mice up to 5000 mg/kg bw. GUS has no known toxicity to humans, and GUS from E.coli is naturally present in the digestive tract of humans. No toxicologic safety concerns were noted for the markers NPTII and GUS.

PPV-CP RNAs are present in PPV-infected fruits, which have been widely consumed for over 50 years. PPV-infected fruit was first identified in the 1930s, and PPV infection is now present in many fruit-growing regions around the world. PPV infects a wide variety of fruit, and fruit with mild or no symptoms of infection would be consumed.

The level of PPV-CP small RNA (sRNA), which includes siRNA, in the C5 plum was 6-fold lower than levels present in PPV-infected conventional plum. The release of the C5 plum would therefore result in similar or possibly lower exposure levels to PPV-CP siRNA (and its precursor dsRNA) compared to the levels already present in the human diet from consumption of PPV-infected fruits. The Canadian Food Inspection Agency (CFIA) reports that PPV does not affect human or animal health (CFIA, 2019).

The hypothetical concern regarding RNAi stems from the possibility that intact foreign RNAs from food may enter the ingesting organism, and silence endogenous transcripts. Currently, evidence that this process occurs in mammals is weak. While initial positive findings in mammals were reported, subsequent experiments have not been able to reproduce these findings (Witwer, 2016)¹⁴.

There are significant physical barriers to absorption of PPV-CP dsRNA/siRNA in the human digestive system, and very little, if any, would be expected to enter the cells where they would have any potential to interact with off-target human mRNAs.

No toxicologic safety concerns were noted for the PPV-CP dsRNA/siRNA based on a history of human consumption at levels similar to those expected for the C5 plum, as well as barriers to absorption in the human digestive system.

The petitioner provided a scientific rationale for the lack of allergenicity of the C5 plum based on published information on the marker proteins NPTII and GUS, and known plum allergens.

NPTII does not have amino acid homology with known allergens. NPTII is also rapidly degraded in the simulated gastric fluid assay, unlike some food allergens. GUS from E.coli is naturally present in the digestive tract of humans. GUS is also reported to be rapidly degraded under stomach conditions.

Plum is not a priority food allergen, although some proteins found in plum (e.g., lipid transfer protein) are associated with food allergy. The genetic changes made to the C5 plum are not expected to affect the presence of natural plum allergens, therefore food products derived from C5 plum would be expected to have the same level of food allergy concerns as food derived from conventionally bred plum cultivars.

The RNAi changes do not produce novel proteins, and are therefore unlikely to elicit allergic reactions given that almost all known food allergens are proteins.

It was concluded that the C5 plum would be just as safe as the conventional plum in terms of potential chemical contaminants, toxicants and allergens.

Conclusion:

Health Canada's review of the information presented in support of the food use of C5 plum does not raise concerns related to food safety. Health Canada is of the opinion that food derived from C5 plum is as safe and nutritious as food from current commercial corn varieties.

Health Canada's opinion concerns only the food use of C5 plum.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

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For further information, please contact:

Novel Foods Section
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
bmh-bdm@hc-sc.gc.ca