Effect of two digestive enzymes and pH on the dsRNA of endornaviruses of bell pepper and melon under in vitro conditions
- 131 Downloads
The objective of this investigation was to determine the in vitro effect of two common digestive enzymes, amylase and pepsin, and pH on the integrity of the RI dsRNA of bell pepper endornavirus (BPEV) and Cucumis melo endornavirus (CmEV) evaluated by gel electrophoresis and reverse-transcription PCR (RT-PCR).
We conducted experiments on the in vitro effect of two common digestive enzymes, amylase and pepsin, and pH on the structural integrity of the replicative intermediate (RI) dsRNA of bell pepper endornavirus (BPEV) and Cucumis melo endornavirus (CmEV), evaluated by gel electrophoresis and reverse-transcription polymerase chain reaction.
The effect of the amylase, pepsin, and pH treatments on the dsRNA of both viruses was similar. Amylase did not appear to affect the structural integrity of the dsRNA. In contrast, gel electrophoresis analysis of pepsin-treated dsRNA samples showed an abnormal electrophoretic migration and evidence of partial dsRNA degradation. DsRNAs from both fruits were partially degraded when exposed to a pH value of 2.0 and completely degraded at a pH value of 1.0.
The results of this investigation suggest that when exposed to pepsin and pH values lower than 2.0, the RI of BPEV and CmEV lose their structural integrity. Therefore, when consuming endornavirus-infected bell pepper or melon, our digestive organs are exposed to both fragmented and full RI dsRNA of these two viruses.
KeywordsAmylase Capsicum annuum Cucumis melo Plant virus Pepsin Pepper mild mottle virus Replicative intermediate dsRNA
Plant viruses have been shown to be present in fruits, roots, leaves, seeds, and some processed plant products commonly available in grocery stores (Pavan et al. 2008; Willenborg et al. 2009; Colson et al. 2010; Okada et al. 2011; Sabanadzovic et al. 2016; Szostek et al. 2017). Some of these viruses, such as cowpea mosaic virus (CPMV) and pepper mild mottle virus (PMMoV), have been shown to be stable and infectious under simulated gastric conditions (Zhang et al. 2006; Berardi et al. 2018). Several studies have shown that plant viruses are common in human feces and, in some cases, their infectivity has been confirmed and their genome assembled (Zhang et al. 2006; Colson et al. 2010; da Costa et al. 2019). The presence of these viruses is likely the result of consumption of virus-infected plant products (Colson et al. 2010; da Costa et al. 2019).
In plant virology, the terms acute and persistent have been used to describe symptomatic and asymptomatic reactions of plants to viruses (Roossinck 2010). Most acute plant viruses can render the host unfit for certain environments. In contrast, persistent plant viruses do not appear to affect the phenotype of the host (Boccardo et al. 1987; Roossinck 2010; Fukuhara 2019). The virus family Endornaviridae includes viruses that infect fungi, oomycetes, and plants (Fukuhara 2019; Valverde et al. 2019). In plants, endornaviruses are persistent viruses which have not been shown to cause diseases (Fukuhara 2019). The genome of plant endornaviruses consist of linear ssRNA ranging in size from approximately 13–18 kb, lacking capsid protein and cell-to-cell movement, and indirect evidence suggests that they are present in all tissues of the infected plant (Fukuhara 2019; Valverde et al. 2019). Endornaviruses have been reported to infect economically important agronomic crops such as barley, common bean, and rice (Wakarchuk and Hamilton 1985; Zabalgogeazcoa and Gildow 1992; Fukuhara et al. 1993). Pepper (Capsicum annuum), an important vegetable crop, has been shown to be infected with bell pepper endornavirus (BPEV), a virus with a genome of about 14.7 kb in length (Okada et al. 2011). In the USA, all tested commercial cultivars of bell pepper have been found to be infected with BPEV (Okada et al. 2011). Melon (Cucumis melo), another food crop cultivated for fresh fruit consumption and nutritional value, has been reported infected with Cucumis melo endornavirus (CmEV) which has a genome of about 15 kb (Sabanadzovic et al. 2016). As in the case of bell pepper, all melon commercial cultivars tested in the USA were infected with CmEV (Sabanadzovic et al. 2016). The replicative intermediate (RI) double-stranded RNAs (dsRNAs) of these endornaviruses can be readily and consistently extracted from infected plants (Okada et al. 2011; Sabanadzovic et al. 2016; Khankhum et al. 2017).
The objective of this investigation was to determine the in vitro effect of two common digestive enzymes, amylase and pepsin, and pH on the integrity of the RI dsRNA of BPEV and CmEV evaluated by gel electrophoresis and reverse-transcription PCR (RT-PCR).
Seeds of BPEV-infected bell pepper cv. Marengo and seeds of CmEV-infected melon cv. PMR-45 were planted and the plants were grown to maturity in a greenhouse. Fruits were collected and tested for endornavirus by dsRNA extraction, gel electrophoresis, and RT-PCR as described below and infected tissues were used in all the experiments. Fruits from a BPEV-free line of bell pepper cv. Marengo generated in previous research were used as the source of BPEV-free fruit tissue (Okada et al. 2011). Approximately 2.0 g of fresh tissue from endornavirus-infected bell pepper and melon fruits was dissected with a scalpel and macerated into a paste with a mortar and pestle in 1.0 mL of tris buffer (0.1 M NaCl, 0.05 M tris, 0.001 M EDTA, pH 6.8). Two grams of tissue from BPEV-free bell pepper fruits were also macerated. Five hundred milligrams of the tissue macerate was placed in a 2.0 mL microcentrifuge tube, and the appropriate enzyme treatment was applied. To test the effect of pH, approximately 10 g of tissue was dissected from the fruits and macerated in 5.0 mL tris buffer. The macerated tissue was placed in a beaker, pH adjusted with 1.0 M HCl to the appropriate value, and 0.5 g of the macerate placed in 2.0-mL tubes. Negative controls consisted of tris buffer–treated tissue macerates. The amounts of amylase and pepsin, used to treat tissue macerates, were based on reports of common concentrations of these enzymes in humans (Kalipatnapu et al. 1983; Roberts et al. 2007; Mandel et al. 2010; Foltz et al. 2015; Liu et al. 2015). Similarly, the range of pH values included pH values commonly detected in human gastric media (Zhu et al. 2006; Lu et al. 2010). All enzyme and pH treatments were conducted in duplicates and experiments were repeated at least twice. To each 0.5 g of macerated tissue, α-amylase from human saliva type XIII-A (Sigma-Aldrich) 10 U/μL was added to adjust the amylase concentration in the macerate to 2.0, 3.0, and 4.0 mg/mL. The macerated tissue was vortexed at 1000 rpm for 30 s at room temperature, and dsRNA was extracted. Pepsin from porcine gastric mucosa (250 U/mg) (Sigma-Aldrich) was added to each 0.5 g of macerated tissue to final concentrations of 0.5, 0.7, and 1.0 mg/mL. After vortexing at 1000 rpm for 30 s, the mixture was incubated in an Orbit Environ Shaker (Lab Line Instruments Inc., Melrose Park, IL, USA) at 37 °C, 150 rpm for 2 h, and dsRNA was extracted. To determine if the 2-h incubation period had an effect on the dsRNA extraction, buffer-treated tissue macerates, subjected to the incubation conditions, were also extracted. Hydrochloric acid (1 M) was added to 0.5 g of macerated tissues to obtain the following pH values: 1.0, 2.0, 3.0, and 4.0. Samples were vortexed at 1000 rpm for 30 s and incubated in an Orbit Environ Shaker (Lab Line Instruments) at 37 °C, 150 rpm for 2 h, and dsRNA was extracted. To determine the effect of a combined treatment of amylase, pepsin, and pH to the endornavirus dsRNA, we conducted the treatments described above simultaneously on bell pepper and melon tissue macerates. Although, our primary objective was to test the dsRNA of two persistent viruses (BPEV and CmEV), we also conducted experiments with purified preparations of dsRNA from the acute virus PMMoV. Foliar tissue of an endornavirus-free line of bell pepper Marengo infected with a Louisiana isolate of PMMoV was used as the source of dsRNA (Escalante et al. 2018; Okada et al. 2011).
After the appropriate experimental treatment, dsRNA was extracted from the macerated fruit tissues as described by Khankhum et al. (2017). The extracted dsRNA was suspended in 35 μL of water and treated with 1 unit of RNase-free DNase I (Sigma-Aldrich). After staining with 5 μL of 100X GelRed® (Biotium, Hyward, CA, USA), 15–25 μL aliquots were loaded in 1.2% agarose gels prepared with 45 mM Tris-borate 1 mM EDTA buffer. A 1-kb Molecular Ruler (Bio-Rad, Hercules, CA, USA) was included as marker. Gels were run for 1.5–2.0 h, and results were recorded with a GelDoc-It2 Imager (UVP, Upland, CA).
We also evaluated the effect of pH, amylase, and pepsin on 40 μL of purified BPEV (300–400 ng) and CmEV (200–300 ng) dsRNA extracted from 0.5 g of tissue macerate. The same enzyme concentrations, pH, and incubation conditions used with the macerated tissue treatments were used to treat purified dsRNAs. After each treatment, the dsRNA was ethanol precipitated and resuspended in 35 μL of tris buffer and 15–25 μL aliquots electrophoresed as described above. Negative controls consisted of tris buffer–treated dsRNA.
After the amylase, pepsin, and pH treatments of the fruit tissue samples, in addition to gel electrophoresis, the presence of BPEV and CmEV in the extracts was determined by RT-PCR. Similarly, RT-PCR was performed on purified dsRNA after the treatments. Two sets of primers were used for each virus. For the detection of CmEV, a pair of primers, Endor-F (5′AAGSGAGAATWATHGTRTGGCA 3′) and Endor-R (5′ CTAGWGCKGTBGTAGCTTGWCC 3′) were used as described by Valverde et al. (2011). For the detection of BPEV, we designed a pair of primers (BPE-3F: 5′- CCAGCCAACAAACCAAATGT -3′ and BPE-4R: 5′- CTGCCTAATGATGGCTGTTG -3′) which amplify 572 nT of the UDP-glycosyltransferase of BPEV. After initial denaturation at 94 °C for 1 min, the cDNAs were amplified by 40 cycles as follows: denaturation 1 min at 94 °C, annealing 1 min at 58 °C, extension for 2 min at 70 °C, and a final extension for 7 min at 70 °C. For RT-PCR detection of PMMoV, we used virus-specific primers which amplified a 387-bp fragment of the viral coat protein and followed the procedure described by Jarret et al. (2008). RT-PCR products were analyzed by gel electrophoresis as described above. A 100-bp PCR Molecular Ruler (Bio-Rad) was included as marker.
In this investigation, we defined dsRNA structural integrity as the intactness or state of degradation of the dsRNA evaluated by gel electrophoresis. We used RT-PCR to confirm the identity of the viruses and detect low concentrations of viral RNA, which may not be detectable by gel electrophoresis.
Our results show that under the simulated gastric conditions used in this investigation, the structural integrity of the RI dsRNA of BPEV and CmEV can be negatively affected. Electrophoresis and RT-PCR results of the pH treatments of endornavirus dsRNA support that depending upon the individual (human) pH value in their digestive system (which is affected by several factors including amount and type of food consumed), these dsRNAs are not degraded at pH 3 or above or may be partially degraded at pH 2. Pepsin treatments of dsRNA containing tissue showed limited dsRNA degradation. Therefore, when consuming these endornavirus-infected fruits, it is likely that our digestive organs are exposed to both fragmented and full-length RI dsRNA of BPEV and CmEV. The fate of these dsRNAs in the lower digestive system is not known. Nevertheless, it has been shown that infectious PMMoV can be recovered from human feces after consumption of virus-infected peppers (Colson et al. 2010; Zhang et al. 2006). It is possible that virus-infected tissues were not completely degraded during digestion and intact virions recovered form feces.
Oryza sativa endornavirus (OsEV) has been reported in some rice cultivars (Fukuhara et al. 1993). Kasumba et al. (2017) used the dsRNA of this virus to stimulate the immune response in mice and obtained a strong immune reaction. Moreover, the OsEV dsRNA treatment in mice suppressed replication of Influenza A virus at an early stages of infection. In another study using BPEV, Hajake et al. (2019) reported melanoma-suppressive effects in mice after injecting them with BPEV dsRNA. Furthermore, they found that BPEV dsRNA acted as an effective vaccine adjuvant. These reports illustrate potential practical applications of endornaviral dsRNAs.
It is not known if these dsRNAs have any effects in humans, although it has been shown that exogenous plant micro-RNAs acquired orally through food intake can regulate the expression of target genes in mammals (Zhang et al. 2012). The results of this investigation may be extrapolated to the replicative intermediate and genomic dsRNAs of viruses present in other plants and plant-derived food products.
The authors would like to thank C.S. Kousik, USDA Vegetable Laboratory, Charleston, SC, and M.J. Roossinck, Penn State University, State College, PA, for providing endornavirus-infected cucurbit and pepper seeds, respectively. We also wish to thank Andrea Hebert, Middleton Library, Louisiana State University, for proof reading the manuscript and the Episcopal High School, Baton Rouge ESTAAR program for sponsoring the internship of Adair McCanless and Allison Hultgren.
Partial support for this investigation was provided by the National Institute of Food and Agriculture, USDA.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.
- Colson P, Richet H, Desnues C, Balique F, Moal M, Grob J-J, Berbis P, Lecoq H, Harlé JR, Berland Y, Raoult D (2010) Pepper mild mottle virus, a plant virus associated with specific immune responses, fever, abdominal pains, and pruritus in humans. PLoS ONE 5(4):e10041. https://doi.org/10.1371/journal.pone.0010041 CrossRefPubMedPubMedCentralGoogle Scholar
- Del Vigna de Almeida P, Tridade Gregio AM, Naval Machado MA, Soares de Lima AA, Reis Azevedo L (2008) Saliva composition and functions: a comprehensive review. J Contemp Dent Pract 9:1–11Google Scholar
- Hajake T, Matsuno K, Kasumba DM, Oda H et al (2019) Broad and systemic immune-modulating capacity of plant-derived dsRNA. Jpn Soc Immunol. https://doi.org/10.1093/intimm/dxz054/5542381
- Jarret RL, Gillaspie AG, Barkley NA, Pinnow DL (2008) The occurrence and control of pepper mild mottle virus (PMMoV) in the USDA/ARS Capsicum germplasm collection. Seed Technol 30:26–36Google Scholar
- Kalipatnapu P, Kelly RH, Rao KN, Thiel DH (1983) Salivary composition: effects of age and sex. Acta Medica Port 4:327–330Google Scholar
- Pavan MA, Krause-Sakate R, Silva ND, Zerbini FM, Le Gall O (2008) Virus diseases of lettuce in Brazil. Plant Viruses 2:35–41Google Scholar
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.