extfieldsofstudy: Medicine Biology

text:
Ultrahigh-activity immune inducer from Endophytic Fungi induces tobacco resistance to virus by SA pathway and RNA silencing

Background Plant viruses cause severe economic losses in agricultural production. An ultrahigh activity plant immune inducer (i.e., ZhiNengCong, ZNC) was extracted from endophytic fungi, and it could promote plant growth and enhance resistance to bacteria. However, the antiviral function has not been studied. Our study aims to evaluate the antiviral molecular mechanisms of ZNC in tobacco. Results Here, we used Potato X virus (PVX), wild-type tobacco and NahG transgenic tobacco as materials to study the resistance of ZNC to virus. ZNC exhibited a high activity in enhancing resistance to viruses and showed optimal use concentration at 100–150 ng/mL. ZNC also induced reactive oxygen species accumulation, increased salicylic acid (SA) content by upregulating the expression of phenylalanine ammonia lyase (PAL) gene and activated SA signaling pathway. We generated transcriptome profiles from ZNC-treated seedlings using RNA sequencing. The first GO term in biological process was positive regulation of post-transcriptional gene silencing, and the subsequent results showed that ZNC promoted RNA silencing. ZNC-sprayed wild-type leaves showed decreased infection areas, whereas ZNC failed to induce a protective effect against PVX in NahG leaves. Conclusion All results indicate that ZNC is an ultrahigh-activity immune inducer, and it could enhance tobacco resistance to PVX at low concentration by positively regulating the RNA silencing via SA pathway. The antiviral mechanism of ZNC was first revealed in this study, and this study provides a new antiviral bioagent.

Backgroud
Viruses are biotrophic parasites and important pathogens of plants and cause a range of severe plant diseases and immense annual losses of yield. To date, chemical reagents are widely used to prevent or control plant diseases in agriculture [1]. However, the extensive use of agrochemicals has caused pernicious pesticide residues and environmental pollution, declined crop quality, and threatened human health. Innovative and ground-breaking strategies are therefore urgently required to control the plant viral pathogens and improve grain output because of the growing world population [2,3].
Biological control with endophytic strains is a promising measure to control plant diseases and eliminate pollution at the same time [4,5]. In most cases, endophytes can produce bioactive secondary metabolites to inhibit pathogens, such as bacteria, fungi, and insects, or improve the resistance to pathogens [3,6,7]. The application of endophytes in biocontrol is limited because of unclear mechanism and exceptional growth condition [8]. The biocontrol agent ZhiNengCong (ZNC), which is an extract of an endophytic fungus, has been widely used in China; it is a new, efficient, environment-friendly extract. ZNC functions as an elicitor not only protecting crops from Pseudomonas syringae pv. tomato (Pst) DC3000 but also promoting crop growth [9]. In this study, we found that ZNC induces resistance to viruses at very low concentration, and the antivirus mechanism was studied.
RNA silencing is a conserved surveillance mechanism that plays a key role in defending plants against invasive nucleic acids [10][11][12][13], such as viruses, by sequence-specific degradation of complementary mRNA transcripts (post-transcriptional gene silencing, PTGS) [14,15]. Double-stranded RNA, which is a replication intermediate generated by viral RNAdependent RNA polymerases (RDRPs) of plantinfecting RNA viruses, triggers RNA silencing [16]. This intermediate is then cleaved into short 21-24nucleotide small interfering RNA (siRNA) by an RNaseIII-like enzyme called dicer-like proteins (DCL) [17][18][19][20]. Double-stranded siRNAs are incorporated into RNA-induced silencing complex (RISC) containing an argonaute (AGO) protein that has an small RNA-binding domain and an endonucleolytic activity for the cleavage of target RNAs and then follows the sequence-specific cleavage of target RNAs [21].
Salicylic acid (SA) is a key plant hormone that mediates host responses against microbial pathogens. This plant hormone induces important plant defense response against pathogens [22][23][24][25][26]. Previous studies have shown a possible overlap between RNA-silencing pathway and signal transduction pathways governed by SA [27][28][29][30]; for example, the NtRDRP activity has been increased in tobacco plants after SA treatment [29].
In this study, Potato X virus (PVX) and tobacco were used as materials to study the antiviral function of the plant immune inducer ZNC. We found that ZNC induced a serious defense response in plants, including H 2 O 2 accumulation, SA accumulation, and RNA silencing. SA biosynthesis and signaling transduction pathways are required for the ZNC-mediated defense response. This study is the first to reveal the antiviral mechanism of ZNC. The results will provide a new antiviral bioreagent and help increase the application of the plant immune inducer ZNC crop biological control in the future.

ZNC protected plants against virus infection
To examine the antiviral activity of ZNC on plant, we performed an infection assay with N. benthamiana and PVX, which has GFP-YFP tag (Fig. S1). The H 2 O-PVX leaves (the plant was inoculated with PVX after water treatment) showed high fluorescence intensity (Fig. 1a) and large PVX quantity (Fig. 1b). The typical symptoms of virus disease occurred 5 days post inoculation (dpi). On the contrary, the symptoms of plants treated with 100 ng/mL of ZNC displayed a significant reduction in disease severity, and the relative quantity of PVX from the ZNC -PVX group was only 31.77% compared with that from the H 2 O-PVX group ( Fig. 1a and b). To further confirm the function of ZNC-induced resistance against virus, leaves was inoculated with PVX before spraying ZNC and H 2 O. The results showed that ZNC could prevent virus infection to some extent (Fig. S2), and the relative quantity of PVX in ZNC treatment leaves was lower than that in the H 2 O group (74.42%). However, the difference was insignificant in comparison with the ZNC treatment (31.77%) (Fig. 1). These results suggest that ZNC prevents virus by enhancing the plant resistance to virus but does not cure the plant. Besides, ZNC also improve plant resistance to Tobacco mosaic virus (TMV), but have lower resistance to TMV than that to PVX on tobacco (Fig. S3).
The optimum concentration of ZNC was determined using a gradient concentration aqueous solution of ZNC at 0, 50, 100, 150, and 200 ng/mL. The results showed that the antivirus function of ZNC was gradually enhanced with the increase in its concentration. Nonetheless, when the concentration of ZNC was 200 ng/mL, the plant presented wilting. We hence used 150 ng/mL of ZNC in the next experiment.

ZNC promoted hydrogen peroxide accumulation
Abiotic stresses generally enhance the production of cellular ROS and cause oxidative damage. To investigate whether ZNC regulates ROS accumulation, DAB (3,3-diaminobenzidine) and NBT staining were applied to evaluate the levels, namely, H 2 O 2 and O 2− , in leaves that were detached from the water-spraying group and 150 ng/mL ZNC-spraying group. Figure 2a and b, show that DAB staining was first strengthened and then weakened with the extension of time. The DAB staining was the deepest after 2 h of spraying ZNC, which indicated the accumulation of H 2 O 2 was elevated.
The mRNA levels of several important ROSscavenging enzymes encoding genes, namely, catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and ROS-generating-related genes, including respiratory burst oxidase homolog genes (RbohA and RbohB), were determined by qRT-PCR analysis and monitored before and after treatment. After ZNC treatments, the expression levels of CAT, SOD, and APX dramatically decreased at 2 h post-treatment (hpt) compared with those in control group plants (Fig. 2c). By contrast, the expression levels of RbohA and RbohB were markedly increased (Fig. 2d). This phenomenon indicates that the accumulation of hydrogen peroxide is regulated by reducing CAT expression and increasing RbohB expression. These results suggest that ZNC may improve H 2 O 2 production to relieve ROS toxicity but not ROS scavenging.
ZNC promoted the SA biosynthesis and activated the SA signaling pathway The SA sample extraction was determined with sufficient intensity by using HPLC and gave satisfactory results. The standard addition method was used for validation because the presence of SA was detected in almost every sample (Fig. 3). The relative concentration of SA was determined by Empower software and standard SA. The changes in amount of SA induced by ZNC at different time (2, 4, 6, 8 h) in Nicotiana. benthamiana leaves were detected, and the results showed that the SA concentration was first increased and then decreased with the extension of time, and reached its concentration maximum at 6 h in Fig. 3. The results suggest that ZNC promotes SA accumulation to activate the defense response. Wild-type N. benthamiana was inoculated with PVX (GFP and YFP tag) after ZNC (100 ng/mL) treatment for 2 h, the phenotype (a) was obtained under long-wave ultraviolet lamp, and PVX relative expression quantity (b) was calculated using ELISA method at 5 dpi. The emerging areas were measured using a gradient concentration (0, 50, 100, 150, and 200 ng/mL) ZNC (c). Error bars show the mean ± SD of three replicates (at least 20 plants per replicate). ** indicates extremely significant differences determined using the Student's t-test (p < 0.01) Plants synthesize SA mainly by ICS and phenylalanine ammonia lyase (PAL) pathway. Thus, we detected the ICS and PAL genes using qRT-PCR. The results showed that PAL gene was upregulated by ZNC, whereas ICS gene basically remained unchanged (Fig. 4a). These results imply that ZNC promotes SA synthesis by improving PAL transcription level.
Pathogenesis-related (PR) proteins play an important role in plant defense. They can improve plant disease resistance by inhibiting virus reproduction and mainly Data are shown as the mean (n = 5) ± SD. CAT, SOD, and APX were detected using qRT-PCR at various time intervals. Data are shown as the mean (n = 5) ± SD.qRT-PCR analysis of RbohA and RbohB expression at various time intervals. Data are shown as the mean (n = 5) ± SD. * indicates significant differences determined using the Student's t-test (p < 0.05), ** indicates extremely significant differences determined using the Student's t-test (p < 0.01). involved in plant-acquired systemic resistance. They can also be highly expressed in plants with the induction of SA [22,31,32]. PR-1A and nonexpressor of PR (NPR) are the key factors in the SA pathway, involved in N. benthamiana resistance to viruses [33] or other pathogens, such as phytophthora infestations [34]. WRKYs can bind the promoter region of NPR and have functions in the SA signaling pathway [35][36][37]. The high expression of WRKY40 and WRKY70 in tobacco can increase resistance to pathogens [38]; the overexpression of WRKY70 can enhance the expression of SA-responsive PR genes [39]; SA is increased in Arabidopsis mutants by the positive regulation of WRKY genes, including WRKY51 [40]. WRKY transcription factor genes are involved in hormone signaling pathways, such as SA pathway [41]. Furthermore, the expression patterns of several WRKY genes are significantly increased in tobacco after virus infection [42,43]. To confirm the results, we detected the relative genes PR-1A (belonging to the PR1 family), NPR1, WRKY40, WRKY51, and WRKY70, by using qRT-PCR. The results showed that the expression level of the above genes was upregulated (Fig. 4b). ZNC can promote SA synthesis and activate the SA signaling pathway to enhance the defense response.

Quantitative differences in gene expression in N. benthamiana after ZNC treatment
To further reveal the role of ZNC in inducing plant resistance, a nonparametric transcriptome sequencing analysis was performed on leaves treated with ZNC. The leaves were harvested after 2 h of 150 ng/mL ZNC treatment. More than 47 million clean reads were generated (Table S2). A total of 2801 genes were upregulated twofold, and 1983 genes were downregulated (P < 0.05) in N. benthamiana plants treated with 150 ng/mL of ZNC compared with those treated with H 2 O at 2 hpt ( Fig. 5a and b). Most of the ZNC-regulated genes at 2 hpt belong to a very wide range of GO categories, such as those related to biological processes, cellular component, and molecular function (Fig. S4). The upregulated GO term (Table 1) of biological process included positive regulation of PTGS, defense response, and response to SA (Fig. 5c). Within KEGG classification, plant hormone signal transduction genes showed the highest expression changes, and 95 differentially expressed genes (DEG) were upregulated ( Fig. 5d and Fig. S5). Thus, we speculate that the resistance to PVX was induced by RNA silencing via SA pathway.

ZNC enhanced RNA silencing
The results of transcriptome sequencing showed that the ZNC could positively regulate the PTGS. Thirty N. benthamiana were sprayed with ZNC and deionized water to study the effect of ZNC on RNA silencing. After 2 h, the Agrobacterium culture (OD 600 = 1.0) harboring pBI121-GFP was injected into leaves, and RNA silencing was determined by observing the GFP fluorescence intensity in the infiltrated and newly emerging leaves under long-wavelength ultraviolet light for 7 days. Then, GFP emerging leaves were collected, the total RNA was extracted, and the expression levels of GFP were detected by qRT-PCR. As shown in Fig. 6, the GFP fluorescence intensity was strongest at 3 dpi, and the GFP fluorescence intensity and expression level of the ZNC treatment group were significantly lower than those of the control group at 3 dpi ( Fig. 6a and b). Moreover, siRNA was extracted, and Northern blot was performed. The GFP siRNA accumulation of the ZNC treatment group was higher than that of the control group (Fig. 6c). The above-mentioned results suggest that ZNC promotes RNA silencing.
DCL and AGO are the key proteins in the RNAsilencing pathway. DCL is responsible for cutting the Fig. 4 ZNC promoted SA signal transduction and SA biosynthesis. a SA biosynthesis-associated gene ICS1 and PAL were measured at 2 hpt using qRT-PCR (means ±SD, n ≥ 3). b SA signaling-associated genes NPR1, WRKY40, WRKY51, and WRKY70 were measured at 2 hpt using qRT-PCR (means ±SD, n ≥ 3). ** indicates extremely significant differences determined using the Student's t-test (p < 0.01).
dsRNA produced by cells, and AGO acts as a catalytic component of RNA-induced silencing complex (RISC), which promotes amplified silencing signal. The genes related to RNA-silencing pathway were detected by qRT-PCR. NbDCL1, NbDCL2, NbDCL3, NbDCL4, NbRDR6, NbAGO7, and NbAGO10 were upregulated; among these genes, NbDCL3 and NbAGO10 were significantly upregulated (Fig. 6d). Hence, ZNC plays a role of silencing Volcano plot showing fold change and adjusted p-value of normalized read counts of the transcriptome sequencing data. The criteria of log 2 | (fold change) | ≥ 1 and padj ≤0.05 were used to identify the DEGs. Green dots indicate the downregulated DEGs (1983 genes), and red dots indicate the upregulated DEGs (2801 genes). c GO enrichment top 10 (upregulated) term of biological process. The numbers of -log 10 P value of differential genes between H 2 O and ZNC groups in every term > 2 were screened, and the top 10 terms were sorted by the -log 10 P value corresponding to each term. d Partial KEGG pathway classification. Abscissa axis is the ratio of DEG in a pathway: all DEG in KEGG level 2 pathway (%), ordinate axis is the name of pathway. The numbers above the column represent the quantity of DEG in the pathway.  The relative expression level of GFP was detected by qRT-PCR. Data are shown as the mean (n = 3) ± SD, and ** indicates extremely significant differences determined using the Student's t-test (p < 0.01). c Northern blot analysis of GFP siRNA extracted at 3 dpi from patches, the cropped gel and blot images were shown in Fig. 6c, and tRNA was used as loading control for siRNA. d The relative expression of genes related to RNA silencing pathway was detected using qRT-PCR after ZNC treatment for 2 h. Data are shown as the mean (n = 3) ± SD. * indicates significant differences determined using the Student's t-test (p < 0.05), and ** indicates extremely significant differences determined using the Student's t-test (p < 0.01). enhancement by regulating the upstream pathway of RNA silencing.

ZNC promoted RNA silencing through SA pathway
Previous studies have shown SA induce RNA silencingrelated genes and plant resistance to RNA pathogens [28]. So we speculate that ZNC induced virus resistance by RNA silencing through SA pathway. To test this speculation, NahG transgenic N. tabacum cv Samsun NN was used as plant material. SA could not accumulate because SA was broken down into catechins. In a transient expression experiment, we could see that the fluorescence intensity of GFP was the strongest at 3 dpi emerging area and then gradually decreased. However, the GFP fluorescence intensity showed no significant change between ZNC treatment leaves and water treatment group, and the expression levels of GFP from the two treatment areas were consistent with the phenomenon (Fig. 7a and b). On the contrary, the GFP fluorescence intensity of the nontransgenic plant treated with ZNC was significantly decreased compared with that of the nontransgenic plant sprayed with water, and the GFP expression level was consistent with the fluorescence intensity.
To further confirm the results, NahG transgenic N. tabacum cv Samsun NN and wild-type N. tabacum cv Samsun NN plants were inoculated with PVX by rubbing leaves with sap. The PVX accumulation (Fig. 7d) was more and the emerging area was larger in the water treatment group than in the ZNC treatment group in the wild-type tobacco at 15 dpi. For NahG transgenic N. tabacum cv Samsun NN, the PVX accumulation and emerging area showed no significant difference (Fig. 7c).
The results indicate that ZNC cannot induce antivirus ability for NahG transgenic N. tabacum cv Samsun NN and SA-deficient plants. All the results mentioned above suggest that ZNC promotes RNA silencing through SA pathway and then induces virus resistance.

Discussion
Most plants possess endophytes (mostly bacteria and fungi), and endophytes can promote growth and plant health in most cases. During long-term coevolution with plants, endophytic bacteria and fungi have developed many factors that help plants resist stresses and promote their growth. For instance, endophytic microbes are often functional in that they may increase the stress tolerance of plants, enhance disease resistance in of plants, suppress virulence in pathogens, restrain the development of competitor plant species, and carry nutrients from soil into plants. Endophytic microbes may significantly reduce the use of agrochemicals (fertilizers, fungicides, insecticides, and herbicides) in the cultivation of crop plants because of the effective functions of endophytic microbes [44][45][46]. However, the number of biocontrol agents used in agriculture is very small. ZNC, which is a commercialized product, is the crude extract of a P. variotii strain, which is isolated from the roots of plants [9]. ZNC has been used on crops, vegetables and fruit trees widely, and the molecular mechanisms of plant growth promotion and bacterial disease protection have been evaluated recently. To further understand the antiviral mechanism of ZNC, we investigated ZNCmediated virus resistance in tobacco. In our study, we found that ZNC cannot cure virus-infected plants, but it has prophylactic function, enhances the resistance to virus in plants ( Fig. 1 and S3). ZNC can promote H 2 O 2 (Fig. 2), SA accumulation (Fig. 3), and activate RNA silencing ( Fig. 6a and b). These data confirm that ZNC acts as an elicitor of active defense responses in plants. The antiviral mechanism of ZNC was first revealed in this study.
Many elicitors, which can induce plant defense response and protect them from pathogen infection have been identified [47]; they include flagellin [48], elongation factor [49], chitin [50,51], and other oligosaccharide (e.g., oligogalacturonide acid, OGs) [52]. They are pathogen-associated molecular patterns (PAMPs) and interact with plant receptors that active the PAMPtriggered immunity [53][54][55]. However, the abovementioned elicitor's effective concentration is between μg/mL and mg/mL in order of magnitude, which is considerably higher than the working concentration of ZNC (ng/mL), such as Flg22 (1 μM, approximately 2.232 μg/ mL) and OGs (50 μg/mL) [48]. Moreover, several plant hormones' working concentration is higher than that of ZNC; for example, the antiviral concentration of SA is 10 μM, approximately 1.38 μg/mL [26]. ZNC is the fermentation extract from p. variotii, mainly containing nucleoside, saccharides, protein and other compounds (data not shown). We do not know which molecular is an effective elicitor that induces plant resistance to virus because we cannot acquire single compound. Hence, further research is required to separate, extract, and identify effective compounds.
Abiotic stress generally enhances the production of cellular reactive oxygen species (ROS) in plants [56]. Previously studies have shown that SA and hydrogen peroxide levels are closely correlated, and they mutually induce each other's accumulation during many biological processes [57][58][59]. Virus invasion causes plant hypersensitivity reaction and generates abundant hydrogen peroxide, which accelerate the cells into programmed cell death, inhibit the reproduction and spread of the virus, and enhance the resistance to virus of plant. So we believe that hydrogen peroxide plays an integral role as signaling molecules in the regulation of numerous biological processes such as responses to abiotic stimuli in plants [60,61]. Plants have ROS scavenging activity to prevent plants from its toxic effects. SOD and Rbohs are mainly responsible for generating hydrogen peroxide, which can convert harmful superoxide radicals into hydrogen peroxide, and then ROS scavenging enzymes APX and/or CAT hydrolyze hydrogen peroxide. In this study, we found that DAB staining showed a significant difference after ZNC treatment, whereas NBT staining showed no significant difference. NbCAT gene related to the hydrogen peroxide-scavenging process was significantly downregulated, whereas RbohB gene was upregulated. ZNC may promote the accumulation of hydrogen peroxide by promoting the accumulation of SA; this finding needs further study.
SA biosynthesis induced by pathogen infection mainly depends on the ICS1 gene and partly depends on PAL gene. The ICS1 protein has isochorismate synthase activity, whereas PAL is a phenylalanine ammonia lyase [62][63][64]. Interestingly, in our study the SA biosynthesis mainly depends on PAL gene (Fig. 4a). The reason is unclear and requires further research.
The phytohormone SA plays important roles in regulating disease resistance. Systemic acquired resistance (SAR) depends on SA signaling in plants, such as the chemical benzothiadiazole induces SAR by increasing endogenous SA levels [65,66]. Previous studies have shown that exogenous SA also increases PR gene expression [67]. Thus, SAR requires signal molecule SA and is associated with the accumulation of PR proteins, which are supposed to contribute to resistance [22]. In our study, the SA content in plants increased after spraying ZNC, and the related marker genes PR1A, NPR1, WRKY40, WRKY51 and WRKY 70 in SA pathway were upregulated by qRT-PCR (Fig. 4b). These results confirm that the biosynthesis of SA dependent on PAL is required in the ZNC-mediated defense response.
Transcriptome sequencing showed that many different genes were upregulated; such genes are involved in various aspects, such as positive regulation of PTGS, SA catabolic process, response to SA, and defense response (Fig. 5, Fig. S4, S5). The aforementioned biological process is closely related to plant defense and induction of resistance. Previous studies have shown that SA induces RNA-silencing-related genes [27,28]. Moreover, the RNA-silencing pathways modulate responses to certain stresses and can be partially tuned by SA [68], and SA-related transcription factors are coexpressed with several AGO and DCL [69]. In our study, we found that NbDCL1, NbDCL2, NbDCL3, NbRDR6, NbAGO7, and NbAGO10 were upregulated with SA accumulation in the marker genes of RNA-silencing pathway (Fig. 6). The results suggest that preinduction of RNA-silencingrelated genes by SA inhibits PVX accumulation.
Previous studies showed that SA induces RNA silencing-related genes and plant resistance to virus [28], and SA-deficient N. benthamiana attenuates virusinduced gene silencing but does not affect transgeneinduced PTGS [70]. Our study showed that ZNC could promote the accumulation of SA (Fig. 4), and transcriptome sequencing results showed that ZNC upregulated the expression of RNA silencing-related genes (Fig. 5).
Silencing efficiency analysis showed that the fluorescence intensity of wild-type plants treated with ZNC was significantly weaker than that of the water group. However, no significant difference in fluorescence intensity in NahG transgenic plants was observed ( Fig. 7a and b). Furthermore, ZNC-sprayed wild-type leaves showed decreased infection areas, whereas ZNC failed to induce a protective effect against PVX in NahG leaves. These results indicated that virus resistance can be achieved by RNA silencing via the SA pathway for ZNC. However, RNA silencing is dispensable for SA-mediated resistance against plant RNA viruses in Arabidopsis thaliana [71]. These findings suggest that SA may increase plant resistance to viruses in various of parallel resistance mechanisms, such as inducing systemic acquired resistance (SAS) [72] or acting as an enhancer of the RNA silencing antiviral defense in tobacco. SA and RNA silencing defense mechanisms may also cooperate to prevent virus invasion [73][74][75]. Thus, we presumed that ZNC increases plant viral resistance by enhancing RNA silencing via the SA pathway, which is a mechanism to fight diseases. Whether ZNC can improve resistance to the virus through other SA pathways remains unclear.
Furthermore, SA was found in numerous plants (Nicotiana tabacum, tomato, Arabidopsis thaliana, Orange), and could induce RNA silencing-related genes and plant resistance to RNA pathogens [28,75]. The previous report showed that ZNC activated the salicylic acid biosynthesis and signaling pathways in Arabidopsis thaliana [9]. In the support information, we found that ZNC also enhance N.benthamiana resistance to TMV. Therefore, we speculated that ZNC can induce the resistance to various virus through SA and RNA silencing pathway in different plants. The antiviral phenomenon of ZNC is nonspecific.

Conclusions
We firstly demonstrated that ultrahigh-activity ZNC could induce virus resistance at low concentration, and the antiviral mechanism was preliminary revealed. All results indicated that ZNC mediated resistance to viruses by positively regulating RNA silencing via salicylic acid SA pathway. This study provides a new antiviral bioagent, namely, ZNC. This bioagent will be very useful bioagent for plant antiviral research and application.

Materials and growth conditions
ZNC was provided by Shandong Pengbo Biotechnology Co., Ltd.; it is an ethanol crude extract of Paecilomyces variotii. The pCaPVX440-GFP-YFP plasmids were presented by professor Xiangdong Li (Shandong agricultural university, china) [76] (Fig. S1). For RNA-silencing analysis, the empty vector pBI121 with GFP tag was transformed into Agrobacterium tumefaciens GV3101 in this study. Wild-type Nicotiana benthamiana and N. tabacum cv Samsun NN were propagated and stored in this study. NahG (which encodes a salicylate hydroxylase converting SA into catechol) transgenic N. tabacum cv Samsun NN (from Dr. Josefa M. Allimino, campus universidad atonoma de madrid, Spain) [74] were raised in a greenhouse at 24°C under a 16-h light/8-h dark photoperiod. The DNA polymerase, and other genetic engineering enzymes were purchased from Takara Inc. (Dalian, China).
All other chemicals and reagents were of the highest quality available.

Antivirus study of ZNC
The wild-type N. benthamiana was inoculated at five or six leaf stage with Potato Virus X (PVX)-GFP-YFP before or after spraying 100 ng/mL of ZNC to confirm the resistance of ZNC to the infection of PVX. For virus inoculation, 1 g of PVX-infected N. tabacum leaves was ground in 1 mL of 5 mM phosphate buffer with pH 7.2. Control plants at six-leaf stage were inoculated by rubbing leaves with freshly prepared sap. Inoculated plants were grown in an insect-free greenhouse at 24°C, and the viral symptom was monitored. Each experiment was replicated three times and included 10 independent plants.
A gradient concentration aqueous solution of ZNC at 0, 50, 100, 150, and 200 ng/mL was sprayed on N. benthamiana for 2 h before inoculating PVX to further determine the optimal working concentration for the antivirus of ZNC; all other conditions were the same as above. The infection of PVX was determined by observing GFP-YFP fluorescence under long-wavelength (365 nm) UV light (Spectroline Model SB-100P/A; Spectronics Corporation, Lexington, KY, USA) and photographed with a Fujifilm FinePix S8000fd digital camera (Fujifilm Holdings Corporation) [77] . The virus content was detected by enzyme-linked immunosorbent assay (ELISA) [78,79]. The plates were followed by colorimetric readout using an EnSpire Multilabel Reader at 490 nm wavelength. Control groups were N. benthamiana leaves without inoculating PVX.

SA extraction and content analysis
After N. benthamiana were sprayed with 150 ng /mL ZNC, the free SA was extracted at different time points (0, 2, 4, 6, 8 h) using the previous methods. Briefly, 1 mL of 70-% methanol was added in a mortar after 0.1 g of seeding samples was grinded and then left overnight at 4°C. The samples were transferred into polypropylene centrifuge tubes. Extract was obtained by centrifugation (8000×g, 10 min). The supernatant was then placed in another tube for evaporation (N 2 , 45°C). The dry residues were reconstituted with 500 μL ddH 2 O. A total of 300 μL of extract was reconstituted with 40 μL 0.5 mg/ mL Trifluoroacetic Acid (TFA), Mix and shake for 1 min. 1 ml of ethyl acetate and cyclohexane was added for removing organic phase, the aqueous phase was then placed in another tube for evaporation (N 2 , 45°C). The dry residues were reconstituted with 500 μL mobile phase. Finally, an aliquot of 500 μL of extracting solution was transferred to an EP tube after 0.22 μm filtration (0), [81]. SA extracting solution (10 μL) and SA standard were analyzed using HPLC by monitoring the absorbance at 306 nm. All the samples were loaded on the C18 reversephase chromatographic column (250 mm × 4.6 mm, 5 μm), respectively. Separation was eluted methanol and 0.1% acetic acid water (the ratio is 65:35) and then eluted at 35°C over 30 min with a flow rate of 0.8 mL/min.

RNA extraction and quantitative real-time PCR
Total RNAs were extracted from leaves using Trizol reagent (TaKaRa, Shiga, Japan) in accordance with the instructions of the manufacturer and treated with DNase I at 37°C for 30 min prior to reverse transcription. cDNA was synthesized from 1 μg of total RNA using FastKing gDNA Dispelling RT SuperMix kit (Tiangen, Beijing, China). Quantitative real-time (qRT-PCR) was performed using Talent SYBR Green Kit (Tiangen, Beijing, China). Each reaction was conducted in triplicate and repeated three times. The results were analyzed by Bio-Rad CFX Manager software (Bio-Rad, California, USA). The relative expression of SOD, CAT, APX, RbohA, and RbohB were measured using qRT-PCR. Each assay was repeated three times. Primer sequences for the qRT-PCR experiment are presented in Table S1.
Data analysis of RNA-sequencing RNA samples were taken from five or six leaf stage leaves, which were treated with 0 (H 2 O) or 150 ng/mL of ZNC for 2 h. Each treatment contain three samples (plants). The samples were selected depending on quality (RIN score ≥ 7). They were pooled and sequenced by Shanghai OE Biotech. Co., Ltd. (Shanghai, China). Previous articles have provided a detailed description of how to analyze RNA-sequening data [82,83]. All differential gene expressions were based on the following standard: the absolute value of log2 ratio ≥ 1 and FDR ≤ 0.001.

RNA-silencing analysis
The wild-type N. benthamiana, N. tabacum cv Samsun NN, and NahG Transgenic N. tabacum cv Samsun NN leaves were infiltrated at five-or six-leaf stage with Agrobacterium GV3101 carrying the GFP gene. Each Agrobacterium culture (OD 600 = 1.0) was incubated for 3 h and then mixed with other culture (s) in a 1:1 (v/v) ratio prior to infiltration. Local and systemic RNA silencing were determined by observing GFP-YFP fluorescence in the infiltrated and newly emerging leaves under long-wavelength (365 nm) UV light as described above. Silencing pathway-related genes, such as (AGO) protein, DCL protein, and RDRP were also detected by qTR-PCR.

Northern blot analysis of siRNA
Low-molecular-weight RNAs were extracted from leaves as described previously [84]. For siRNA detection, 15 μg of Low-molecular-weight RNAs was separated on 15% polyacrylamide-7 M urea gel and transferred to a Hybond-N + membrane in 0.5 X TBE at 0.8 mA cm − 2 for 1 h. After the membrane was UV-crosslinked and incubated at 80°C for 2 h, it was hybridized with DIG labeled probe GFP mRNA. Chemiluminescent detection was conducted using a DIG Northern Starter Kit (Roche, Basel, Switzerland) in accordance with the instructions of the manufacturer.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

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Divergent Approaches Regulating Beta Agonists and Cloning of Animals for Food: USA and European Union

Technologies being used to produce nonhuman animals who are used for meat and dairy products are viewed by some people as meaningful. Two technologies receiving scrutiny in agriculture are beta agonists that are fed to food animals to improve weight gain and cloning animals to secure offspring with specific traits. The technologies enhance the productive capacities of animals so that fewer resources are needed to produce meat and dairy products. Yet consumers are not sure they want food products with beta agonist residues and that are produced from clones. In overseeing the safety of food products and animals, legislators and regulators in the United States (US) and European Union (EU) have developed contrasting provisions regarding the usage of these technologies. An evaluation of heuristics involving information and experiences with bovine spongiform encephalopathy and animal production technologies offers support in explaining the US’s and EU’s divergent provisions.

Introduction
Many members of the public are concerned about what they eat and their exposure to injurious substances in food products they consume. In the United States (US), 3,000 people die per year and one in six people become sick from contaminated foods or beverages (US CDC, 2013). Some believe food products have been altered from their natural states in ways that are unhealthy due to the use of technological advances involving nonhuman animal feed additives, genetically engineered (GE) crops, hormones, irradiation, veterinary drugs, and cloned animals (Bové & Dufour, 2001). Consumers wanting food items that are not associated with certain technologies are offering to pay more for such products. A market for specialized food products can be seen in thriving organic and locally-grown food markets.
This paper looks at governmental oversight of two technologies: beta agonists used as animal feed additives and cloning animals used for food. These two technologies are being employed by producers of animals used for food because they reduce the resources needed to produce meat and dairy products. With fewer resources required, producers may be more competitive and more land would be available to grow crops for human consumption. Simultaneously, the technologies are accompanied by concerns about food safety, animal welfare, and non-scientific factors.
Despite the evaluation of the same science involved with these technologies, the oversight by governments in the US and European Union (EU) has been different. The EU banned the use of beta agonists in the feed of animals used in food production in 1996 (EU, 1996), while the US approved their use in animal feed in the 2000s. With respect to cloning, a report by the EU in 2015 noted the importance of animal health and welfare in responding with restrictions on cloned products (European Commission, 2015). However, regulators in the US found no significant distinctions concerning animal welfare. The two technologies have gained approval for their use in the US but EU countries have thwarted their widespread adoption.
When addressing differences about the regulation of food products by the US and EU, the most common explanation is the application of the precautionary principle. This principle was adopted to apply to food law as part of an EU regulation adopted in 2002. Whenever "the possibility of harmful effects on health is identified but scientific uncertainty persists, provisional risk management measures necessary to ensure the high level of health protection chosen in the Community may be adopted" (European Parliament and the Council of the European Union, 2002). Some Americans maintain that the precautionary principle fails to incorporate "sound science," which should govern the acceptance of food products (US Congress, 2003). The inference of the American concern is that the precautionary principle is a disguised barrier to trade that allows a minority opinion to form the basis for precluding a product (Lupien, 2002).
This includes the use of the principle by the EU as a justification for banning meat products containing beta agonist residues (Lively, 2013). However, research on risks related to other technologies suggests that American regulations impose more precaution for many situations (Weiner et al., 2014). This supports a conclusion that precaution may not form the dividing line for the transatlantic discord between the US and EU on animal production practices involving inputs such as beta agonists and cloning.
The objective of this paper is to analyze why the US and EU approach the regulation of beta agonists and cloning differently. By looking at the availability of information and experiences with technologies already being used in animal production, justifications may be discerned for the conflicting interpretations of similar data. Affect and availability heuristics led decisionmakers in the US and EU to interpret the risks of beta agonists and cloning differently.

Beta Agonist Feed Additives
The technology of using of beta-adrenergic agonist drugs (beta agonists) as additives in livestock feed is accompanied by residues in meat products that may pose a risk to human health. The use of beta agonists has been approved in the United States and Canada, and the Codex Alimentarius has adopted international acceptable daily intake limits for these substances. Conversely, the European Union and most other countries feel the residues pose health risks. They have adopted provisions to exclude meat products containing residues of these drugs.
Ractopamine hydrochloride and zilpaterol hydrochloride are beta agonist drugs fed to selected animals used for food to enhance muscle growth, improve feed efficiency, and limit the amount of fat in meat products (Mersmann, 2002). Ractopamine was approved by the US Food and Drug Administration (US FDA) for use in swine in 2000and cattle in 2003(US FDA, 2000, and zilpaterol was approved by the US FDA for use in cattle in 2006(US FDA, 2006. These drugs activate protein synthesis and decrease protein degradation on a cellular level in animals used for food to stimulate skeletal muscle growth society & animals 28 (2020) 613-632 without increasing natural hormone levels (Gonzalez et al., 2007). Thus, they cause protein accretion that increases the animals' hot carcass weight and improve yield grade with corresponding economic benefits for producers (Boyd et al., 2015). American livestock producers are feeding their animals beta agonists because of the economic advantages (Lean et al., 2014).
Yet many important meat-importing countries have claimed that there may be potential human health problems from beta agonists and have adopted policies to preclude the sale of products containing beta agonist residues (Bories et al., 2009). Beta agonist drug residues in food products are known to adversely affect humans, although there are no reported cases of humans being adversely affected by livestock products containing zilpaterol or ractopamine residues (Baynes et al., 2016).
Concern also exists about the welfare of animals (American Veterinary Medical Association, 2014; Loneragan et al., 2014). Evidence suggests that beta agonists lead to fatigued cattle syndrome that are manifested through labored breathing, reluctance to move, and lameness (Grandin, 2013;Thomson et al., 2015). In 2013, the drug firm Merck took its zilpaterol feed additive off the market due to its negative effects on animal health (Hughlett, 2013). Research on pigs suggests that those fed beta agonists exhibited more aggressive behaviors and a greater number of offensive behaviors, particularly bites (Poletto et al., 2010). These data provide justifications for determining that the usage of animals in the production of food is contrary to the acceptable norms.

Cloning of Animals Used for Food
The cloning of animals used for food involves food animals by using the somatic cell nucleus transfer (SCNT) cloning technique (Edwards et al., 2003). The main objectives of reproductive animal cloning for livestock are to increase reproductive efficiency, enhance animal genetic improvement, and to increase resistance to disease (Liu et al., 2016;US FDA, 2014, 2015. In agriculture, cloning generates animals for breeding. The offspring of clones are used for food production. Agency risk assessments. The US FDA and the European Food Safety Authority (EFSA) reviewed similar scientific evidence of the SCNT cloning technique to determine whether animal cloning and its derived foods were safe (US Center for Veterinary Medicine, 2008;EFSA, 2008). Their 2008 risk assessments were science-based and focused on problems with genetic reprogramming, animal health risks, and food consumption risks. The agencies concluded that animal clones and their offspring are similar to their conventional counterparts, and that the safety of the meat and milk of clones and their progeny is similar compared with products from conventionally bred animals (Petetin, 2009). However, a closer examination shows that the agencies reached different conclusions regarding the health of the animals ( Table 1).
The US FDA noted a higher proportion of health risks for cloned animals but concluded that the risks did not qualitatively differ from those of natural breeding (US Center for Veterinary Medicine, 2008). Because the US FDA risk assessment found no science-based reason to require labels to distinguish between products from clones and products from conventionally produced animals, products from cloned animals are not labeled in the United States (US FDA, 2008).
The EFSA (2012) delineated issues of welfare and health of the surrogate dams carrying the cloned offspring. The dams were affected by abnormal pregnancies that included abortions. The agency also noted that the cloning efficiency of cattle, namely the percentage of viable offspring born from transferred embryo clones, is low at about 6 to 15% for bovine animals and about 6% for pigs (EFSA, 2012;European Commission, 2013b). Moreover, the EFSA expressed concerns about animal suffering, as an increased percentage of clones have been found to be adversely affected, often severely and with a fatal outcome, compared to sexually produced animals (EFSA, 2008). This led the EFSA to conclude that the welfare of a significant proportion of clones was negatively impacted, leading the EU to take a protectionist approach to this technology (Petetin, 2012). With respect to the safety of food products from cloned animals, the EFSA did not make any risk management decision because its authority is limited to independent scientific evidence. The policies and regulation of cloned foods. The approach of the US FDA to cloned foods was founded on the 1986 Coordinated Framework of Biotechnology (US Office of Science and Technology Policy, 1986). Because cloned food raised no unique risk, no new laws were needed to respond to challenges posed by this technology (US FDA, 2008). The US FDA declared that cloned foods are basically as safe as traditional foods, so they do not require any specific labeling information on cloning. However, the US FDA (2008) recommended that cloned species other than cows, goats, and pigs should not enter the human food supply.
The EU regulation of cloned foods falls under the scope of the 2015 Novel Foods Regulation (European Parliament and the Council of the European Union, 2015). Cloned foods are considered as novel foods and must be preauthorized. A novel food is labeled to inform consumers whenever it is no longer equivalent to an existing food (Petetin, 2017). Novel foods that give rise to ethical concerns may require additional specific labeling. The 2015 Novel Foods Regulation taking effect on January 1, 2018, simplifies the framework for novel foods (European Parliament and the Council of the European Union, 2015). The regulation creates a centralized authorization system for novel foods and establishes the EFSA at the center of the risk assessment. It applies to cloned foods until more specific legislation is enacted.
Foods derived from the descendants of cloned animals fall outside the scope of the definition of a novel food under the 2015 Novel Foods Regulation. This means that products of descendants of cloned animals are not subject to the pre-market authorization, labeling, or traceability requirements of these regulations. Since the descendants of clones will provide food products, consumers will not know whether their food was derived from a cloned animal unless new requirements are imposed.
Dissatisfaction with the existing regulations on cloned food led the EU Commission to submit two separate proposals dealing with animal-cloning and derived foods in 2013. The first proposal was for a Directive on Animal Cloning for Farming Purposes that would provisionally ban such practices (European Commission, 2013a). The second complementary proposal was for a Directive on Cloned Foods that would temporarily prohibit placing products on the market from clones as well as their importation (European Commission, 2013b).

Affect and Availability Heuristics
Decisions about risk are influenced by affect and availability heuristics, the mental shortcuts people take when making judgments and choices. An affect heuristic involves a person's response to a risk based on experiences (Pachur et al., 2012). The availability heuristic looks at the information available to people making decisions (Mase et al., 2015).
Affect and availability heuristics may influence decisions made by the three groups of people who determine safeguards that will govern animal meat products. The first group of decisionmakers is composed of the scientists who determine what to examine, how the examination will proceed, and how to interpret the findings. While scientists are highly trained and seek to be objective in verifying scientific accounts of nature, science is messy (Lather, 2010). Scientists do not have a set of standardized questions or rules to follow; rather, they decide what questions should be asked (Haack, 2003). Scientists decide what experiments to conduct, so they make choices based on their knowledge and past experiences.
The second group of decisionmakers is comprised of legislators who enact provisions governing the use of technologies. They have choices in determining which scientific findings are the most credible, whether oversight is needed, and what limitations to establish. Opinions of constituents and advocacy groups, which may not be based on science, are often important (Miller, 2009). Once legislative pronouncements have been made, regulators are in charge of developing regulations to implement the legislation. They may evaluate the same science as the scientists but may make choices influenced by legislative directives, staffing, available expertise, and timeframes (US FDA, 2010).
When scientists, legislators, and regulators assess the risks of new technologies, such as beta agonists and cloned food products, their past experiences with related technologies may influence their evaluations of risk (Miller, 2009). The availability of information on related technologies and the regulation of these technologies may also impact their evaluations (EFSA, 2014). Given the uncertainties that accompany the assessment of risks, heuristics may lead individual decisionmakers to weigh the risks of new technologies dissimilarly and to ascribe divergent levels of precaution to accompanying risks (Stoutenborough et al., 2016). Due to their unique backgrounds and locations, individuals weigh risks of the same technology differently.
Cultural differences between the US and EU on food safety may have also influenced evaluations of risk. Europeans may have greater aversion to dread and unknown risk (Finucane et al., 2005). They also have been more supportive of actions to improve the welfare of animals used for food (Matheny & Leahy, 2007).

Animal Production Technologies
Heuristics related to other animal production technologies would be expected to influence decisionmakers. An examination of the agricultural law literature over the past two decades discloses several technologies for which the US and EU proceeded differently with their regulatory oversight. Based on the contrasting decisions concerning authorization to use these technologies, six are selected for further evaluation and assessment of how they may have influenced decisionmakers who evaluated risks accompanying the use of beta agonists and cloning. While decisionmakers on both sides of the Atlantic would have had access to considerable information about the technologies, they would have been exposed to different governmental and public attitudes about the technologies.
In approaching their evaluations of beta agonists and cloning, decisionmakers would be expected to maintain consistency with established cultural institutions and public sentiments. Legislators and regulators in the EU had already adopted regulations forbidding the use of steroidal hormones, recombinant bovine somatotropin (rBST), and some non-therapeutic antibiotics (European Commission, 1981, 1988EU, 1996EU, , 1998b, whereas in the US, the use of these production inputs was considered important to the viability of the country's livestock industry. Decisionmakers in both the US and EU would have had access to information on their existing regulatory controls over concentrations of animals, tail docking, and the castration of pigs without anesthesia. The analysis of responses of US and EU regulators and producers to six animal production technologies shows a pattern (Table 2). Regulators in the US allowed the technologies to be adopted accompanied by measures to prevent harm. Considerable numbers of producers in the US embraced the technologies to maintain profitability and to enhance economic returns. However, EU regulators applied limitations and prohibitions. For those technologies allowed in the EU, producers were more reluctant to adopt them and expressed greater concern for animal welfare.
The use of steroidal hormones in the production of food animals is a technology that is regulated in the US and EU. The US Department of Agriculture commended the use of hormones in 1991 and, due to the economic advantages of adopting these agents, a significant number of US producers commenced using them. Steroidal hormones being used are estradiol benzoate, estradiol 17β, melengestrol acetate, progesterione, testosterone, trenbolone acetate, and zaranol (Schweihofer & Buskirk, 2016). In contrast, the European Community adopted a directive to preclude the administration of steroidal hormones in 1981 that was amplified through additional directives (European Commission, 1981, 1988EU, 1996). These provisions show a sustained antipathy to the use of steroidal hormones in food animal production in the EU.
The use of rBST in dairy cows is a second production technology. It is approved in the US but proscribed in the EU. In 1993, the US FDA approved the use of rBST in lactating dairy cows for the purpose of increasing the production of marketable milk (US FDA, 1993). Subsequently, this artificial hormone is available for use in the US in contrast with the EU where the use of rBST is banned (EU, 1999).
Another technology used for producing food animals on some farms involves the administration of non-therapeutic antibiotics. The US continues the use of these substances to enhance animal growth, although recent limitations should decrease usage (Centner, 2016;Sneeringer et al., 2015). This may be contrasted to the EU's 1998 regulation limiting the use of certain antibiotics in animal production (EU, 1998a).
Governmental limitations on the tail docking of pigs, including allowing the procedure without anesthesia, involve the use of a technology related to animal welfare. Tail docking is done to reduce biting by other pigs in confined areas (Larsen et al., 2015). Docking may have negative consequences for the welfare of the docked pig including acute pain and possibly long-term pain with the formation of neuromas in the tail stump (EFSA, 2007). Tail docking is prevalent in the US and is usually preformed a few days after birth without analgesia (Sutherland & Tucker, 2011). In the EU, the tail docking of pigs is only allowed where there is evidence that injuries to other pigs' tails have occurred (EU, 2001;EU, 2008;European Commission, 2001).  Rault et al., 2011;EU, 2008 Notes. + = greater use; -= less use.
The castration of male pigs without anesthesia involves another technology related to animal welfare (EU, 2008). In both the US and EU, it is generally performed without anesthesia or post-operative analgesia even though the vocalization of animals suggests it is painful (Rault et al., 2011). However, an EU council directive requires castration of pigs older than seven days be performed by a veterinarian using an anesthetic agent (EU, 2008).
A major difference between the US and EU concerning the production of livestock is that large cattle feedlots and integrated pig and poultry operations are prevalent in the US (US Government Accountability Office, 2008). The assemblage of large numbers of animals at production units known as concentrated animal feeding operations (CAFOs) creates economies of scale. In the EU, while farms have increased in size, there has not been the same embracement of CAFOs as occurred in the US. Under the EU's Common Agricultural Policy, the EU chose to focus on protecting small-scale farming viability (Barnes et al., 2016). With direct payments to farmers and an emphasis on farm diversification and environmental maintenance, producers in the EU retained their holdings.
A majority of these six technologies have been available for public scrutiny for more than a decade. Decisionmakers in the US and EU would have had information on the public attitudes, practices by agricultural producers, activities by business firms, and agendas of special interest groups about these technologies. The prevalence of use of these technologies in the US and their limited use or prohibition in the EU mean that American and European decisionmakers had different information, experiences, and beliefs when called upon to regulate beta agonists and cloning.
Given the prohibitions on using steroidal hormones, rBST, and some nontherapeutic antibiotics in the EU, it was consistent for decisionmakers to assess the risks and apply precaution in a manner that led to reaffirming the EU ban on beta agonists and the possible ban of the cloning of food animals. Animal health concerns in the EU, expressed in regulations on tail docking and castration, supported a finding that the cloning of food animals had adverse effects on surrogate dams. Affect and availability heuristics caused EU decisionmakers to be concerned about the potential for meat products to contain unhealthy residues and failing to maintain the well-being of food animals that had been expressed in earlier regulations governing other technologies. In the US, decisionmakers observed public acceptance of meat products from animals administered steroidal hormones and antibiotics as well as the stressful conditions for animals confined at CAFOs. With these observations, US decisionmakers concluded that the use of beta agonists and cloning were simply two additional technologies that could be made available to producers to reduce the costs of animal production.

Results
The use of beta agonists and cloning in food animal production show differences in opinion on what actions governments should take to address risks accompanying new technologies. Governments can choose not to act and allow new technologies to be unregulated, but legislative bodies in both the US and the EU have felt that beta agonists and cloning need to be regulated to protect animals, people, and the environment. However, the regulatory oversight in the US and EU has led to contrasting results. The evaluation of how scientists, legislators, and regulators assess risk and apply precaution helps explain the different conclusions on regulating these technologies. The decisionmakers in the US and EU viewed information differently given diverse regulatory institutions, market structure, interest groups, public attitudes, and politics. Concomitant with these various perspectives, decisionmakers would interpret data on beta agonists and cloning based on personal experiences, a perceived need to be consistent with regulations of other animal production technologies, and value judgments about the welfare of humans and animals used for food.

Assessing Risk
Risk plays a crucial role in the regulation of foods and is especially significant since scientific evidence is often inconclusive. Although scientific risk assessments may be perceived to be value-free, neutral, and unbiased (Haack, 2003), scientific inquiry incorporates notions of risk and conceptions of scientific evidence (Kang, 2013). Technologies include uncertainties, choices for perspectives, and value-loadings, so they are not comprised of stand-alone, factual answers (Saltelli & Funtowicz, 2004). Rather, the evaluation of risk involves defining risks, analyzing inconclusive scientific evidence, making assumptions, assigning probabilities, and discerning acceptable levels of potential damages.
While the evaluation of risk accompanying beta agonists and cloning involves scientific evidence, legislative and regulatory decisions to protect people, animals, and the environment involve the values of the decisionmakers. For beta agonists, the EU's prohibition is to protect the health of people. The EFSA Panel on Additives and Products or Substances used in Animal Feed concluded that there was no human study from which to derive an acceptable daily intake for beta agonist residues (Bories et al., 2009). This precluded the calculation of tolerances (maximum residue limits) for meat products so any product with a residue is unacceptable. For cloning, the EU's decision was not based on human health concerns but rather considered the evidence that the technology adversely affected the welfare of animals. It was felt that surrogate dams and animal clones had too many health problems (European Group on Ethics in Science and New Technologies, 2008).
In addressing beta agonists and cloning, the US FDA and the EFSA had nearly identical scientific data about the technologies, yet the agencies' assessments led to different policies. While the US decided to allow producers to use beta agonists and allows the cloning of food animals, the EU proscribed beta agonists and addresses cloning as a novel food. One explanation for the divergent assessment of risk is the heuristics affecting the decisionmakers. As a result of the decisionmakers' experiences with the six noted animal production technologies, affect and availability heuristics influenced their evaluations of risk and their decisions on how much precaution was needed to protect people, nonhuman animals, and the environment. These heuristics may account for the discrepancies in the US and EU regulations on beta agonists and cloning.

Welfare of Animals Used for Food
For cloning, the EU decisionmakers interpreted the data to conclude that the technology adversely affects surrogate dams and some offspring. By finding differences in the health of surrogate dams and cloned offspring, the decisionmakers in the EU acknowledged that animal welfare is important and could constitute a justification for not approving a new technology.
In 1998, the EU acknowledged concern for the welfare of animals produced for food with its adoption of a directive on the protection of animals kept for farming purposes (EU, 1998b). The 1998 directive provided the foundation for subsequent decisions to safeguard the welfare of animals used for food. In 1999, the Scientific Committee on Animal Health and Welfare concluded that rBST increases risks of clinical mastitis, the duration of the treatment of mastitis, and the incidence of foot and leg disorders; it also adversely affects reproduction and induces severe reactions at the injection site (EU, 1999). Based on these findings, the EU prohibited the use of rBST. The EU decisions on the tail docking and castration of pigs also were based on animal welfare concerns (EFSA, 2007;EU, 2001EU, , 2008. Given these precedents regarding the treatment of animals used for food and the assessments of dams and offspring, the decisionmakers consider it "prudent to provisionally prohibit the use of cloning in animal production for farm purposes of certain species" (European Commission, 2013a).
In the US, common law traditionally treats nonhuman animals as property. Individual US states have enacted animal cruelty laws to prevent abusive behavior, but these often do not address most issues affecting the welfare of animals used for food. Moreover, they are not always enforced (Friedrich & Wilson, 2015). In the absence of significant regulatory provisions protecting animal welfare in the US, decisionmakers declined to recognize any concerns about the health of surrogate dams used for cloning.

Discussion
Citizens of the US and EU have a lot in common. Both the US and the EU have approached the issue of food safety with great diligence and have adopted a wide array of provisions to protect people from harmful substances and products. Both America and Europe are known for their safe food products. Yet, they have not approached all issues in the same manner. The evaluation of beta agonists and cloning shows that the US FDA and the EFSA have approached the risks involving these technologies differently.
For beta agonists, the US FDA has set acceptable daily intake levels and tolerance levels, whereas the EU has decided that residues are unacceptable due to human health concerns. For animal cloning, the US FDA found no distinctions in products from cloned animals, so no further regulation was needed, whereas the EU found the technology adversely affected animal health.
In applying the precautionary principle, the EU grants regulators discretion in regulating risk that can lead to products being banned (Morag-Levine, 2014). However, the US also applies precaution in regulating risk. A comprehensive study of the application of precaution by the US and EU showed that political and institutional factors, legal systems, and the role of cost-benefit analyses did not account for the observed patterns of precaution (Weiner et al., 2014). Rather, the authors concluded that heuristic availability provided more important clues about the use of the precautionary principle and cross-cultural differences in risk perception (Weiner et al., 2014).
Heuristic availability is access to information that will affect a person's memory (Geurten et al., 2015). By drawing on personal experiences and individual senses, the availability heuristic affects people's perceptions of risk and regulatory oversight (Mase et al., 2015). For beta agonists and cloning, people who evaluated the risks, applied precaution, and authored the regulations had experiences and information that may have impacted their perceptions of the regulatory needs of these technologies. Since both of these technologies dealt with animals used for food, decisionmakers may have been influenced by events and existing regulatory controls over six agricultural technologies related to animal well-being and wholesome food (Table 2).
For decisionmakers in the EU, the affect heuristic may include public and governmental responses to the potential introduction of GE crops and the use of ruminant-derived protein in animal feed. Public concerns over the introduction of GE crops contributed to a strengthened precautionary principle including its application to food items (European Parliament and the Council of the European Union, 2002). In 1985, ruminant-derived protein was related to an outbreak of bovine spongiform encephalopathy (BSE), also known as mad cow disease, in the United Kingdom (Burka, 2011). Ten years later, BSE was related to humans dying from Creutzfeldt-Jakob disease. This development caused the public to become more skeptical of governmental regulations (Jasanoff, 2005). In the United Kingdom, 176 human deaths were connected to the disease, with deaths peaking in 2000 (Andrews, 2012).
Given the seriousness of the Creutzfeldt-Jakob disease outbreak in the UK, decisionmakers in the EU were likely to have had an enhanced concern about health dangers that might accompany the use of new technologies. With this background, the regulatory climate of the EU was less favorable to the use of steroidal hormones, rBST, non-therapeutic antibiotics, concentrations of animals, tail docking, and castration without anesthesia. When the decisionmakers analyzed the data concerning potential health effects related to beta agonist residues in meat products, they wanted to avoid another adverse public health problem and so concluded that the usage of beta agonists was not safe for humans.

Conclusion
The regulatory agencies of the US and EU applied precaution in reaching their decisions on the use of beta agonists and cloning but delineated contrasting responses. While the application of the precautionary principle by the EU is offered as explaining the differences, an evaluation of affect and availability heuristics offers an alternative explanation. The decisionmakers evaluated risks differently. American decisionmakers had information and experiences showing widespread acceptance by regulators of new animal production technologies, including steroidal hormones, rBST, non-therapeutic antibiotics, and other production practices. While there was concern about animal health and welfare, it was not at the level expressed in EU directives.
However, European decisionmakers had very different information and experiences. Decisionmakers would have known about the existing EU prohibitions on steroidal hormones, rBST, and non-therapeutic antibiotics. Turning to the issue of animal welfare, EU decisionmakers were constrained by the directive to ensure animals are not caused unnecessary suffering (EU, 1998b). In regulating cloning, decisionmakers would want to be consistent with the animal welfare standards embodied in directives on tail docking and castration. Furthermore, due to the disease outbreak of BSE, EU decisionmakers may have had negative emotional reactions to new animal technologies.
The analysis shows that decisionmakers in the US and EU adopted precaution in devising regulations over beta agonists and cloning. However, despite the internationalization of food safety standards, past experiences led decisionmakers in the US and EU to assess risk differently, caused the EU to have a greater concern about potential harm, and led the Americans to fail to find any adverse health concerns for animals receiving the technologies. While the EU's precautionary principle facilitates the rejection of technologies, the use of beta agonists and cloning was unacceptable because it would not be consistent with regulations governing other animal production technologies. Decisionmakers in the US and EU developed divergent regulatory approaches on the use of beta agonists and cloning technologies due to affect and availability heuristics.