Electroporetic transfection of pepper protoplasts with plant potyviruses

Journal of Virological Methods 179 (2012) 154–160

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Electroporetic transfection of pepper protoplasts with plant potyviruses Nubia Velasquez a , John F. Murphy a,∗ , Sang-Jin Suh b a b

Department of Entomology and Plant Pathology, 209 Life Sciences Building, Auburn University, AL 36849, United States Department of Biological Sciences, 101 Life Sciences Building, Auburn University, AL 36949, United States

a b s t r a c t Article history: Received 15 June 2011 Received in revised form 19 October 2011 Accepted 27 October 2011 Available online 9 November 2011 Keywords: Tobacco etch virus Capsisum Electroporation Gene Pulser Xcell Electroporation System

Potyviruses are a persistent threat to bell pepper (Capsicum annuum L.) production worldwide. Much effort has been expended to study the resistance response of pepper cultivars at whole plant levels but with only limited effort at the cellular level using protoplasts. A pepper protoplast isolation procedure is available but an inoculation procedure is needed that provides consistent and highly efficient infection. An electroporation-based procedure for inoculation of potyviruses was developed using a base procedure developed for Cucumber mosaic virus (CMV). The final parameters identified for efficient potyvirus infection of pepper protoplasts involves two 25 ms pulses, 200 V each pulse with a 10 s interval between pulses. Depending on the method of detection, e.g., ELISA versus RT-PCR, potyvirus RNA inoculum ranged from 10 to 40 ␮g with infection detection occurring with samples of 50,000–100,000 protoplasts. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Viruses in the genus Potyvirus infect a broad range of plants in most climatic regions, causing severe economic losses in many important crop species (Lopez-Moya and Garcia, 1999). The potyvirus virion consists of a single flexuous, filamentous particle containing a single strand of single-stranded messenger sense RNA of approximately 9500 nucleotides (Dougherty and Carrington, 1988). The potyviral RNA has a 5 genome-linked protein (Murphy et al., 1991; Riechmann et al., 1989; Siaw et al., 1985) and a 3 poly A tail (Hari et al., 1979) and encodes a single polyprotein that is cleaved autocatalytically into at least ten proteins (UrcuquiInchima et al., 2001). Studies on virus–host interactions have, more recently, added significant information on mechanisms by which plants resist virus infection (Boevink and Oparka, 2005; Culver and Padmanabhan, 2007; Nelson and Citovsky, 2005; Truniger and Aranda, 2009). Host resistance genes have been identified (Fraile and García-Arenal, 2010; Kang et al., 2005; Maule et al., 2007; Palukaitis and Carr, 2008) and their function determined in relation to the virus infection cycle (Diaz-Pendon et al., 2004; Lin et al., 2007; Soosaar et al., 2005). As these virus–host interactions are dissected in greater detail, there is a need for studies at the cellular level using plant protoplasts. The availability of a protoplast system to study virus infection provides greater quantitative accuracy for evaluations by having a synchronous infection process and collection and

∗ Corresponding author. Tel.: +1 1 334 844 1954; fax: +1 1 334 844 1947. E-mail address: [email protected] (J.F. Murphy). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.10.015

testing of known cell numbers. Protoplast studies allow determination of whether a plant’s resistance mechanism is directed at replication or movement. For example, if virus titer in an inoculated leaf of a resistant host is low relative to a known susceptible host, the low titer could be due to limited replication and accumulation within individual cells or limited movement of virus within the tissue being evaluated. In the latter case, the virus may have accumulated in individual cells to a similar level in both resistant and susceptible hosts; however, in the resistant host movement is limited and, therefore, fewer cells become infected leading to lower virus titers for those leaves. If the virus accumulates to relatively similar levels in protoplasts isolated from both susceptible and resistant hosts, this implies the resistance is not directed at replication and accumulation at the cellular level but a limitation on virus movement. For this type of study to be effective, a protoplast inoculation procedure is needed that is efficient and highly consistent. The original viral RNA inoculation procedure developed for pepper protoplasts involved electroporation using the Hoefer Scientific’s ProGenetorTM 1 (Murphy and Kyle, 1994). When this electroporation apparatus was no longer available, a polyethylene glycol (PEG) procedure (Loesch-Fries and Hall, 1980) was adapted for pepper protoplasts and used successfully in numerous virus-pepper studies (Deom et al., 1997; Guerini and Murphy, 1999; Turina et al., 2003). The PEG inoculation procedure, however, had several drawbacks. Of primary concern, among others, was a lack of consistency within and between experiments due to complications with the PEG solution and the inoculation procedure (J.F. Murphy, unpublished data). In an effort to obtain a high level of consistency among inoculations, an electroporation system

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was developed using a Gene Pulser Xcell Electroporation Apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.). This report describes the electroporation parameters evaluated to obtain infection of pepper protoplasts by potyviral RNA. 2. Materials and methods 2.1. Virus isolates and their purification Viruses used in this study included three Potyvirus species, Tobacco etch virus strain HAT (TEV), Pepper mottle virus strain Florida (PepMoV) and Potato virus Y strain NN (PVY). Cucumber mosaic virus strain Fast New York (CMV) was used as an infection control because successful infection of pepper protoplasts occurs with a broad range of electroporation conditions (Velasquez and Murphy, unpublished data). Each virus was maintained by mechanical passage in Nicotiana tabacum L. cv. Kentucky 14 in an insect-free, temperature controlled greenhouse (24 ± 4.5 ◦ C day/20 ± 3.5 ◦ C night) at Auburn University, AL, U.S.A. Each of the potyviruses, PepMoV, PVY and TEV, was purified from systemically infected Kentucky 14 tissue as described previously (Guerini and Murphy, 1999; Murphy et al., 1990). CMV was purified according to Roossinck and White (1998) with minor modifications. Viral RNA was isolated from each potyvirus preparation by treatment with Proteinase K and phenol, chloroform extraction as described by Guerini and Murphy (1999). CMV RNAs were isolated by several cycles of phenol, chloroform extraction according to Palukaitis and Zaitlin (1984). A sample of each viral RNA preparation was analyzed by electrophoresis through a 1% agarose gel, stained with ethidium bromide and visualized by ultraviolet light. Viral RNA concentration was measured using a Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, U.S.A.) and stored at −65 ◦ C. 2.2. Protoplast isolation Capsicum annuum L. cv. Calwonder seeds (Harris® seeds, Rochester, NY, U.S.A.) were surface sterilized by treatment with 1% sodium hypochloride (Murphy and Kyle, 1994). Plants were grown in magenta boxes (77 mm wide × 77 mm long × 97 mm tall; Sigma–Aldrich, Inc., St. Louis, MO, U.S.A.) in a temperaturecontrolled chamber (Percival Scientific, Inc. Perry, IA, U.S.A.) at 25 ◦ C under 12,000 lx illumination for 16 h and darkness for 8 h at 22 ◦ C. Calwonder leaf protoplasts were isolated as described by Murphy and Kyle (1994) and modified by Guerini and Murphy (1999). Protoplasts generated from these plants were washed and concentrated by three cycles of centrifugation at 294 × g at room temperature, and maintained in 0.42 M mannitol as osmoticum. Protoplast numbers were determined with a hemacytometer by light microscopy at 20× magnification. Protoplast viability was determined as a measure of phenotypic quality. A protoplast of good phenotypic quality showed little, if any, swelling with an even distribution of chloroplasts. During the incubation process, following electroporation, protoplasts often showed some swelling and polarization of chloroplasts. The basis for protoplast phenotypic quality was from initial experiments that involved treatment with fluorescein diacetate and visualization at 40× magnification using an inverted microscope (Axiovert 200 M; Carl Zeiss, Oberkochen, Germany) equipped with a FITC filter (488 nm excitation wavelength and 520 nm emission wavelength) (Huang et al., 1986). The percentage of viable protoplasts was determined from the number of protoplasts of high phenotypic quality at 24 h post-inoculation divided by the number of protoplasts of high phenotypic quality initially inoculated.

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2.3. Electroporation conditions Initial efforts developed an electroporation procedure for inoculation (and infection) of pepper protoplasts with CMV. This procedure did not work, however, for potyviruses but was used as a basis for evaluation of electroporation parameters. A Gene Pulser Xcell Electroporation System (Bio-Rad Laboratories, Inc.) was used for all experiments. The standard procedure (developed for CMV) included inoculation of 500,000 protoplasts suspended in 0.42 M mannitol (molarity was appropriate for Calwonder protoplasts) and 3 mM CaCl2 at a final volume of 800 ␮l in an electroporation cuvette that had a 0.4 mm gap (USA Scientific, Inc., Ocala, FL, U.S.A.). Viral RNA (inoculum) was added to the protoplast solution immediately before electroporation which involved two 5 ms pulses of 150 V each pulse with a 0.1 s interval between pulses. The inoculated protoplasts were placed on ice for 15 min, centrifuged at ∼218 × g for 2 min at room temperature, and resuspended in incubation medium containing 0.2 mM KH2 PO4 , 1.0 mM KNO3 , 0.1 mM MgSO4 , 0.1 mM CaCl2 , 1.0 ␮M KI, and 0.01 ␮M CuSO4 (Aoki and Takebe, 1969; Murphy and Kyle, 1994). An antibiotic mix containing carbenicillin, cephaloridine, and nystatin (Sigma Chemical, St. Louis, MO, U.S.A.) was added at final concentrations of 100, 100, and 4 ␮g/ml, respectively. A square wave electroporation method was used for all experiments. The protoplast suspension was placed in 60 mm × 15 mm Petri dishes and kept in a Percival Growth Chamber with light of 12,000 lx for 16 h at 26 ◦ C and darkness for 8 h at 22 ◦ C. The following electroporation parameters were evaluated for potyvirus infection of pepper protoplasts: pulse length, voltage, number of pulses and time between pulses and amount of viral RNA used as inoculum. The pulse length was tested using PVY RNA as inoculum and the remaining parameters were tested using TEV RNA as inoculum. We chose to use TEV for subsequent experiments due to availability of TEV RNA and the use of TEV in related projects currently underway. The final procedure was evaluated by testing PepMoV, PVY and TEV. Each experiment was performed three times and included two control treatments, CMV RNA inoculum as a positive infection control and a negative (mock) inoculation control that consisted of water.

2.4. Determination of infection by enzyme-linked immunosorbent assay (ELISA) Protoplasts were counted at 24 h post-inoculation (hpi) using a hemacytometer, samples consisting of 100,000 cells were pelleted by two pulse runs at 12,000 × g (Sorvall MC-12V table-top microfuge; Du Pont Co., Newton, CT, U.S.A.), resuspended in 100 ␮l of ELISA general extraction buffer (as described in the ELISA instructions, Agdia, Inc., Elkhart, IN, U.S.A.) and lysed by repeated pipetting with beveled pipette tips. Each sample was examined by light microscopy to determine efficient lysis of the protoplasts. For detection by ELISA, a commercial ELISA kit (Agdia, Inc.) was used specific to each virus, and performed according to the manufacturer’s instructions. The coating antibody and protoplast (virus) sample steps were each kept in a moist chamber at 4 ◦ C for at least 12 h. The alkaline phosphatase conjugated antibody step was incubated in a moist chamber at 37 ◦ C for 3 h. Substrate (1 mg/ml para-nitrophenylphosphate in 10% diethanolamine, pH 9.8) reactions were allowed to develop at room temperature for 1 h and then recorded using a Sunrise microtiter plate reader (Phenix Research Products, Hayward, CA, U.S.A.). A sample was considered positive for the presence of virus if the ELISA absorbance value at 405 nm was greater than the healthy control threshold. The healthy control threshold was determined from the average ELISA absorbance

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value plus three standard deviations of at least two samples of mock inoculated protoplasts.

Total RNA was extracted from 100,000 electroporated protoplasts using a Plant RNeasy kit (Qiagen Inc., Valencia, CA, U.S.A.). Each sample was tested for TEV RNA and the pepper 18S ribosomal RNA gene (18S rRNA), with the 18S rRNA used as an internal control to assure that a similar number of protoplasts was evaluated among samples. Following cDNA synthesis for the respective RNA species, two sets of forward and reverse primers were used for PCR amplification, one specific for TEV detection and the other for 18S rRNA. All primers were generated through Integrated DNA Technologies, Inc. (Coralville, IA, U.S.A.). The TEV primers used were: TEV-Fw 5 -GCAGCCAACACTGAGGCAAA-3 and TEV-Rv 5 AGCGGAAAGCAAAGACACGC-3 . These primers amplify a 375 bp (nucleotides 8962–9210) portion of the coat protein coding region. The 18S primers were: 18S-Fw 5 -TTAGACTGCTCAAAGCAAGC3 and 18S-Rv 5 -CCAAGAATTTCACCTCTGAC-3 , which amplify a 75 bp region of the pepper 18S rRNA gene (GeneBank accession AA840641). CMV RNA was amplified, from inoculated protoplast samples, using primers: CMV-Fw 5 -TCCCACGGCGATAAAGGACT3 and CMV-Rv 5 -GGGGAACAGCAGTGTACGTT-3 . These primers amplify a 353 bp fragment (CMV RNA1, nucleotides 136–488). cDNA synthesis was performed with 1 ␮l of total RNA, 0.1 ␮M of reverse primers, 400 ␮M dNTPs and SuperScript III Reverse Transcriptase following the instructions of the manufacturer (Invitrogen, Carlsbad, CA, U.S.A.). Two PCR reactions were made for every sample, 3 ␮l of the cDNA was added to a Taq PCR amplification cocktail containing 1× PCR buffer (50 mM KCl and 10 mM Tris–HCl, pH 8.3), 1.5 mM MgCl2 , 200 ␮M dNTPs, 0.4 ␮M of each TEV or CMV primer, and 1 U of Taq polymerase (Invitrogen). For the second reaction, the cocktail was prepared in the same way but included 2 mM MgCl2 and 0.4 ␮M of each 18S primer. PCR reactions were performed in a Multigene Gradient thermal cycler (Labnet, Woodbridge, NJ, U.S.A.), with an initial denaturation step at 94 ◦ C for 1 min followed by 35 cycles (94 ◦ C for 1 min, 53 ◦ C for 30 s, and 72 ◦ C for 1 min). A final extension step was at 72 ◦ C for 10 min. The amplification with 18S primers was performed in the same way, but the annealing temperature was 55 ◦ C. The amplification products were analyzed by electrophoresis in a 2% agarose gel and the amplicons visualized by ethidium bromide staining under ultraviolet light. Total RNA extracted from 100,000 nonelectroporated protoplasts was used as template for the negative control, and purified virus RNA was used as positive control. 3. Results The procedure developed for successful infection of pepper protoplasts by CMV using the Gene Pulser Xcell Electroporation System did not lead to detectable infection of pepper protoplasts using potyviral RNA. Therefore, we evaluated each inoculation parameter in an effort to obtain potyvirus infection of pepper protoplasts. 3.1. Electroporetic pulse length The duration of time that the protoplasts are exposed to an electric pulse is an important parameter for successful virus infection (Saunders et al., 1989a). The effect of pulse duration on PVY and CMV infection of pepper protoplasts was tested using two 5 ms pulses, two 25 ms pulses or one pulse of 50 ms (Fig. 1). For each pulse treatment, 150 V was used and a time interval between pulses of 0.1 s. PVY RNA inoculum was 40 ␮g and CMV RNA inoculum was 10 ␮g. PVY was detected, based on a positive ELISA value,

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Fig. 1. Effect of electroporetic pulse length on infection of Potato virus Y (PVY) and Cucumber mosaic virus (CMV) in pepper protoplasts. Virus was detected by enzymelinked immunosorbent assay (ELISA) from a sample of 100,000 protoplasts collected at 24 h post-inoculation. Each bar represents the mean ELISA value from three separate experiments for the respective treatment. The horizontal bar represents the healthy control (mock inoculation) treatment threshold determined from the average ELISA value plus three standard deviations. ELISA values above the horizontal threshold bar are positive for detection of virus.

in the treatment consisting of two 25 ms pulses only. The ELISA absorbance value for PVY treatments consisting of two 5 ms pulses or one pulse of 50 ms was not above the healthy control threshold. Successful infection of CMV occurred with each of the pulse treatments, although the ELISA absorbance value was significantly greater for the two 25 ms pulse treatment than the other two treatments. The two 5 ms pulse treatment resulted in a significantly greater CMV accumulation than the one pulse of 50 ms. 3.2. Number of electroporetic pulses The number of pulses was evaluated using parameters that included 150 V and 25 ms for each pulse with a 0.1 s interval between pulses. The TEV RNA inoculum amount was 40 ␮g and 500,000 pepper protoplasts were inoculated for each treatment. A single electroporetic pulse did not result in a positive ELISA absorbance value; however, a positive ELISA value occurred with two, three and four pulse treatments (Fig. 2A). The three pulse treatment had a significantly higher ELISA value than the two pulse treatment; however, there was nearly a 15% drop in protoplast viability from two to three pulses and a slightly greater decline between two and four pulses. We selected the two pulse treatment as a compromise for a strong ELISA reaction for virus accumulation and lesser negative effect on protoplast viability. 3.3. Voltage levels The amount of voltage was evaluated and, in each case, the treatment included two 25 ms pulses with a 0.1 s interval between pulses. Each of the treatments, 150, 200, 250 and 300 V, led to a positive ELISA absorbance value for detection of TEV from protoplast samples (Fig. 2B). The ELISA absorbance value was significantly greater for the 200 V treatment than the 150 and 300 V treatments and the 300 V treatment was significantly less than each of the other treatments. With each increase in voltage, protoplast viability decreased by approximately 10% from one treatment to the next with the largest decline between 200 and 250 V treatments. These voltage data provided sound evidence that for TEV RNA electroporation, 200 V resulted in better protoplast infection while maintaining decent protoplast viability.

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Fig. 2. Effect of electroporation conditions on Tobacco etch virus (TEV) infection (bar graphs) and pepper protoplast viability (line graphs). TEV infection was determined by enzyme-linked immunosorbent assay (ELISA) at 24 h post-inoculation. Each bar represents the mean ELISA value from three separate experiments for the respective treatment. The horizontal bar represents the healthy control (mock inoculation) treatment threshold determined from the average ELISA value plus three standard deviations. ELISA values above the horizontal threshold bar are positive for detection of virus. Tests were performed to determine effects on TEV accumulation and protoplast viability evaluating (A) the number of 25 ms pulses of 150 V, (B) the amount of voltage used for each electroporetic pulse of 25 ms, (C) the time interval between two 25 ms pulses of 200 V, and (D) the amount of TEV RNA inoculum and number of protoplasts tested for detection of virus infection.

3.4. Interval time between electroporetic pulses Experiments were performed to test the time interval between electroporetic pulses. These experiments used the base parameters of two 25 ms pulses of 200 V each pulse. Inoculum consisted of 40 ␮g of TEV RNA and inoculation of 500,000 pepper protoplasts. The time interval between pulses of 0.1 s, used in the original electroporation procedure developed for CMV, did not result in a positive average ELISA value for TEV inoculation, although one of the three experiments had an ELISA value slightly above the threshold (Fig. 2C). TEV was detected from 5 to 10 s interval treatments with the highest ELISA value occurring for the 10 s interval treatment, although the average ELISA value was not significantly greater than the 5 s interval treatment. TEV was not detected from protoplast samples from the 20 to 40 s interval treatments (Fig. 2C). Protoplast viability was lowest for the 0.1 s time interval treatment with at least a 10% increase in viability with longer time intervals. The 10 s time interval between electroporetic pulses was selected as a base parameter for subsequent experiments. 3.5. Viral RNA inoculum amount and number of protoplasts tested The parameters identified from the previous experiments were used to evaluate the amount of TEV RNA inoculum and protoplast sample number needed to detect TEV accumulation by ELISA (Fig. 2D). TEV was not detected from any of the 10 ␮g TEV RNA treatment samples that tested 50,000 protoplasts (Fig. 2D). The average ELISA value for detection of TEV for the 10 ␮g TEV RNA treatment

from 100,000 protoplasts was below the healthy control threshold; however, one of the three samples used to generate the treatment mean was slightly above the threshold indicating a positive detection of virus (TEV treatment average was 0.135, TEV positive sample ELISA was 0.155, healthy control sample average was 0.144). TEV was detected from both 50,000 and 100,000 protoplast samples when 20 ␮g and 40 ␮g of TEV RNA inoculum was used, although the 20 ␮g TEV RNA treatment mean ELISA value for the 50,000 sample was close to the healthy control threshold. The 40 ␮g TEV RNA treatment had significantly greater ELISA absorbance values for both 50,000 and 100,000 protoplast samples compared with the 20 ␮g TEV RNA treatment. Furthermore, significantly more TEV was detected for the 40 ␮g TEV RNA 100,000 protoplast sample than the 50,000 sample (Fig. 2D). 3.6. Validation of the electroporation procedure for infection of potyviruses The electroporation parameters developed for TEV inoculation of pepper protoplasts (40 ␮g virus RNA used to inoculate 500,000 protoplasts using two 25-ms pulses of 200 V each with a 10-s time interval between pulses) were evaluated for the potyviruses, PepMoV and PVY (Fig. 3). Each virus accumulated to a detectable level as average ELISA values were above the respective healthy control thresholds. The amount of virus detected appeared to differ among virus treatments; it was not determined whether these differences were actual differences in the amount of virus that accumulated or reflected different detection sensitivities of the respective ELISA kits.

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Fig. 3. Electroporation of pepper protoplasts by Pepper mottle virus (PepMoV), Potato virus Y (PVY) and Tobacco etch virus (TEV) using a procedure developed for potyviruses. The inoculation procedure was two 25 ms pulses, 200 V each pulse with a 10 s interval between pulses. Viral RNA (40 ␮g) was used to inoculate 500,000 pepper protoplasts and a sample of 100,000 protoplasts was collected at 24 h post-inoculation with virus infection determined by enzyme-linked immunosorbent assay (ELISA). Each bar represents the mean ELISA value from three separate experiments for the respective treatment. The horizontal bar represents the healthy control (mock inoculation) treatment threshold determined from the average ELISA value plus three standard deviations. ELISA values above the horizontal threshold bar are positive for detection of virus.

3.7. Detection of infection of protoplasts by reverse transcription polymerase chain reaction (RT-PCR) Using the procedure developed in this report for inoculation of pepper protoplasts with potyviral RNA, the amount of viral RNA needed for inoculum to obtain detectable infection was re-evaluated comparing ELISA with RT-PCR as the method of detection. TEV was detected by ELISA from protoplast samples (100,000 protoplasts per sample) when 10, 20 and 40 ␮g of viral RNA was used as inoculum (Fig. 4). The amount of TEV detected for the 10 ␮g RNA inoculum treatment was only slightly above the healthy control threshold with increased amounts of TEV detected with each increase on viral RNA inoculum. A 375 bp amplicon was observed for each of the TEV RNA inoculum treatments with no corresponding amplicon from the negative

Fig. 4. Comparison of enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) for detection of Tobacco etch virus (TEV) infection of pepper protoplasts. TEV RNA inoculum amounts of 10, 20 and 40 ␮g [and a healthy (mock inoculated) control] was used to inoculate 500,000 protoplasts with samples of 100,000 protoplasts tested at 24 h post-inoculation by ELISA or RT-PCR for infection. The horizontal bar across the ELISA data represents the healthy control threshold. ELISA values above the horizontal threshold bar are positive for detection of virus.

control sample (Fig. 4). In contrast to the increased accumulation of TEV detected by ELISA for each increased TEV RNA inoculum treatment, the intensity of the 375 bp amplicon was similar among TEV RNA inoculum treatments (Fig. 4). In an effort to confirm that the TEV RNA detected was replicated progeny rather than residual inoculum, an additional control was included that consisted of 40 ␮g of TEV RNA added to the protoplasts, as if performing electroporation, but with no electric pulse. These protoplasts were then treated similarly to those subjected to electroporation. For these samples, no 375 bp amplicon was observed indicating that the amplicon from the electroporated samples resulted from detection of progeny viral RNA. These results suggest that use of RT-PCR as the method of virus detection would allow less viral RNA inoculum needed for determination of a successful infection of the protoplasts. The internal control 18S (74 bp) amplicon had similar intensity among all treatments. 3.8. Final comparisons: CMV electroporation procedure versus the potyvirus virus electroporation procedure and ELISA versus RT-PCR detection TEV inoculation of pepper protoplasts using the procedure developed for potyviruses [Fig. 5, TEV(potyp)] had a strong positive ELISA absorbance value but TEV was not detected from protoplasts inoculated using the procedure originally developed for CMV [Fig. 5, TEV(CMp)]. These results were confirmed using RT-PCR whereby TEV was detected for the potyvirus electroporation procedure but not for the procedure developed for CMV (Fig. 5, upper panel). In contrast, CMV inoculation of pepper protoplasts using either the original electroporation procedure developed for CMV [Fig. 5, CMV(CMp)] or the newly developed procedure for potyviruses [Fig. 5, CMV(potyp)] resulted in positive ELISA absorbance values with corresponding detection using RT-PCR. No virus related amplicon was detected for TEV or CMV from the respective healthy sample. 4. Discussion Electroporation and PEG have been used to successfully infect pepper protoplasts with potyviral RNA (Deom et al., 1997; Guerini and Murphy, 1999; Murphy et al., 1998; Murphy and Kyle, 1994; Turina et al., 2003). If available, an electroporation procedure can be more reliable and reproducible than PEG transfection (Bates, 1999; Hibi, 1989; Saunders et al., 1989b). Initial efforts to obtain successful infection of pepper protoplasts with potyviral RNA, using the Gene Pulser Xcell Electroporation System, tested a procedure developed for CMV infection of protoplasts (Masiri et al., 2011). All attempts were unsuccessful. We then evaluated two modifications to the CMV electroporation protocol in an effort to obtain potyvirus infection: the amount of voltage and the amount of potyviral RNA used as inoculum. Neither parameter led to potyvirus infection of pepper protoplasts (data no shown). Electroporetic pulse length was shown to be an important parameter of the electroporation process. According to a proposed model, pores develop in the plasma membrane of the protoplast and these pores expand as a result of the electric pulse (Weaver and Chizmadzhev, 1996). Successful potyvirus infection occurred when pulse length was increased from that used in the CMV electroporation procedure. For the potyvirus–pepper protoplast system, an increase in electric pulse may lead to an increase in pore size, thereby allowing entrance of the potyviral RNA into the cell. The ability to obtain infection led to a systematic evaluation of numerous parameters in an effort to further develop and improve potyvirus transfection of pepper protoplasts. Each parameter revealed an improvement in the infection process, i.e., a greater

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Fig. 5. Comparison of the electroporation inoculation procedure developed for potyviruses (referred to as potyp) with the original electroporation inoculation procedure developed for Cucumber mosaic virus (CMV; procedure referred to as CMp). Tobacco etch virus (TEV) and CMV accumulation were detected by enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Viral RNA (10 and 40 ␮g for CMV and TEV, respectively) was inoculated using 500,000 protoplasts with samples of 100,000 (ELISA) or 50,000 (PCR) protoplasts tested at 24 h post-inoculation. The horizontal bar across the ELISA data represents the healthy control threshold. ELISA values above the horizontal threshold bar are positive for detection of virus.

level of virus accumulation in protoplasts, or an improvement in inoculation conditions such as reduced amount of inoculum and better sensitivity of detection. The final parameters chosen, and shown to provide consistent infection by three potyvirus species, include two 25 ms pulses, 200 V each pulse with a 10 s interval between pulses. Depending on the method of detection, virus RNA inoculum can range from 10 to 40 ␮g and 50,000 to 100,000 protoplasts should be collected for evaluation. An interesting observation from this study was the broad range of parameters allowed for successful infection with CMV, which was in stark contrast to the potyviruses. The CMV genome consists of three RNA species with RNAs 1 and 2 able to replicate in the absence of RNA 3; however, all three RNA species are required for replication with coat protein accumulation (Palukaitis and GarcíaArenal, 2003). In contrast, the potyviral RNA is a single RNA species, although larger than each of the individual CMV RNA species (Adams et al., 2005; Rajamäki et al., 2004; Shukla et al., 1994). An explanation for the differences in electroporetic infection efficiency observed between CMV and potyvirus may reside in the overall charge of the viral RNAs. According to this model, the viral RNA becomes attached to the protoplast’s surface prior to the formation of electropores in the protoplast membrane with subsequent movement into the protoplast (Krassowska and Filev, 2007; Pliquett et al., 2007; Sukharev et al., 1992). The membrane binding step is required for entry into the protoplast and correlates strongly with the charge of a RNA molecule (Xie and Tsong, 1993). Although, no evidence to support this model for the CMV and potyvirus systems in pepper was obtained in this study, the model offers a plausible component of the inoculation process. Potyviral RNA has a genome-linked protein (VPg) covalently bound to the 5 -terminus of the RNA (Murphy et al., 1991; Riechmann et al., 1989) which was shown to be responsible for aggregation of RNA molecules (Guo et al., 2001; Luciano et al., 1991; Yambao et al., 2003). The aggregation of two or more potyviral RNA molecules could result in a complex too large for efficient inoculation of pepper protoplasts. This explanation corresponds with the greater level of each parameter needed for successful infection using potyvirus RNA as inoculum. The potyviral RNA used as inoculum in this study, however, was treated with Proteinase K during the extraction process. The Proteinase K treatment does not completely eliminate the VPg from the viral RNA 5 - terminus but leaves a small peptide still linked to the RNA (Murphy et al., 1991). It was not determined whether PVY or TEV RNAs aggregate with or without Proteinase K treatment and, therefore, this phenomenon

is unable to be addressed in relation to electroporation. Although, treatment of Tobacco vein mottling virus RNA with Proteinase K eliminated the VPg-induced aggregation of viral RNA molecules (Luciano et al., 1991). This suggests that the reduced efficiency of infection of potyvirus RNA in pepper protoplasts may not be due to aggregated RNA inoculum complexes since the inoculum was subjected to Proteinase K treatment. The complications encountered with efforts to obtain potyvirus infection in pepper protoplasts was consistent among each of the potyviruses tested, suggesting it is a potyvirus phenomenon. The ability to infect pepper protoplasts with CMV occurred over a broad range of inoculation parameters; this may be due to this virus’ greater ability to establish an infection. Similar observations were made with CMV infection of tobacco protoplasts (Saunders et al., 1989a). In all experiments performed involving infection of pepper protoplasts with CMV versus a potyvirus, regardless of whether the inoculation method was PEG or electroporation, CMV accumulated to detectable levels sooner and to a greater degree than the potyvirus (Guerini and Murphy, 1999; Murphy, unpublished data). This was also observed in studies evaluating infection of whole plants whereby CMV accumulated throughout tissues of the stem of infected plants at a much greater rate and level of accumulation than a potyvirus (Guerini and Murphy, 1999). CMV may serve as a good positive infection control for the sake of obtaining infection but, perhaps, it should be used with caution in comparative studies for potyvirus infection of pepper protoplasts. A reliable electroporation procedure for potyvirus infection of pepper protoplasts is described in this report. This procedure will be useful for studies to evaluate potyvirus replication and accumulation at the cellular level and understand the basis for resistance mechanisms. Acknowledgements We thank Aaron Rashotte for use of the Percival growth chamber. Funding for N. Velasquesz was provided by a grant from the Alabama Agriculture Experiment Station and the Department of Entomology and Plant Pathology, Auburn University. References Adams, M.J., Antoniw, J.F., Fauquet, C.M., 2005. Molecular criteria for genus and species discrimination within the family Potyviridae. Arch. Virol. 150, 459–479. Aoki, S., Takebe, I., 1969. Infection of tobacco mesophyll protoplasts by Tobacco Mosaic Virus ribonucleic acid. Virology 39, 439–448.

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