Plan t Cell Repor t s (1994) 1 3 : 3 9 7 - 4 0 0 Plant Cell Reports (�9 Springer-Verlag 1994

Isolation and viral infection of Capsicum leaf protoplasts

John E Murphy and Molly M. Kyle

D e p a r t m e n t o f Plant Breeding and Biometry , Cornel l Univers i ty , I thaca, N.Y. 14853, U S A

Received 23 A u g u s t 1993/Revised vers ion received 25 J a n u a r y 1994 - C o m m u n i c a t e d by R. L. Gi lber t son

Summary. A protocol for protoplast isolation was developed and tested with five Capsicum genotypes representing two cultivated species, C. annuum and C. chinense. Key variables included growth conditions for source plants and the concentration of mannitol used as osmoticum. Protoplasts isolated from each of the genotypes became infected when inoculated via electroporation with viral RNA from either pepper mottle potyvirus, tobacco etch potyvirus or cucumber mosaic cucumovirus.

Key words: Protoplasts-Potyviruses-Cucumoviruses Capsicum-Pepper

Introduction

The ability to isolate plant protoplasts and infect them with viruses has played an essential role in the study of plant-virus interactions at the cellular and molecular levels. This approach has contributed significantly to studies focused on virus replication and on the elucidation of mechanisms by which plants resist virus infection (Ponz and Bruening 1986, Zaitlin and Hull 1987, Dawson and Hilf 1992).

While significant advances have occurred in understanding the molecular aspects of plant viral gene structure and expression, similar progress is not apparent in defining how viral gene products function to cause disease in susceptible interactions or the mechanisms by which selected genotypes interrupt various aspects of the infection process. We have identified several resistant Capsicum genotypes that do not accumulate detectable amounts of virus in inoculated leaves and in which systemic infection is not established (Murphy et al. 1994). The ability to isolate and infect protoplasts from these resistant plants could provide more information about the nature of the resistance observed at the whole plant level, e.g., whether resistance is a consequence of inhibition of viral cell-to-cell movement or interference with viral replication.

Correspondence to: M. M. Kyle

Protocols have been described for the isolation of Capsicum protoplasts (Saxena et al. 1981, Diaz et al, 1988, Jacobs, 1991). However, only Jacobs (1991) reported a procedure to infect protoplasts and this was with pepper mild mottle tobamovirus (PMMV). In this report we describe procedures for the isolation of protoplasts from five C a p s i c u m genotypes representing two species, C. annuum and C. chinense, as well as a general protocol for inoculation using viral RNA from either pepper mottle potyvirus (PeMV), tobacco etch potyvirus (TEV) or cucumber mosaic cucumovirus (CMV). These three viruses are generally considered to be among the most destructive Capsicum viruses.

Materials and methods

Plant growth conditions and protoplast isolation. Capsicum genotypes that were tested as sources for protoplast isolation included C. annuum 'NuMex RNaky ' , 'Early Calwonder' and 'Avelar ' and two C. chinense accessions, PI 152225 and PI 159236. Seeds were surface sterilized by stilting in a solution of 10% sodium hypochlorite, 0.1% Tween 20 for 8-10 rain followed by three rinses in double-distilled water (200 ml each rinse).

The genotype C. annuum NuMex RNaky, was selected to identify the appropriate growth conditions for plants that would subsequently be used for protoplast isolation. One environment tested was a greenhouse where plants were grown in a modified Comell mix with supplemental metal halide lighting at 23-28 ~ C. In a second environment, plants were grown in a modified ComeU mix in a growth chanlber maintained at 26 ~ C during the day and 22 ~ C at night with a 16 h photoperiod supplied with incandescent and fluorescent lights (120 ~tE m-2s-1). The third environment tested was a Percival incubator maintained at 26" C with a 16 h photoperiod (80 [xE m'2s "1) with plants grown on either Murashige and Skoog medium (MS medium, Gibco BRL, Grand Island, N.Y.), pH 5.7-5.8, solidified by addition of baetoagar to 0.8% or in modified Comell mix.

Mannitol was used as osmoticum and concentrations of 0.40 M to 0.70 M were examined with each of the Capsicum genotypes used in this study. Pectolytie and cellulytic enzymes included macerase, cellulysin (Calbiochem, San Diego, CA) and pectolyase (Sigma Chemical Co., St. Louis, MO).

Protoplast source plants were allowed to grow until the first pair of true leaves were 1-2 cm in length (not including the petiole). Only true leaves were used for protoplast isolation.

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Leaves were sliced from the midrib to the leaf margin parallel with secondary veins; slices were approximately 1 mm apart. Sliced leaves were plasmolyzed, added to enzyme solution, and incubated at 25 ~ C for the appropriate amount of time (samples were shaken during incubation using a Lab Line Orbit Environ Shaker, Lab Line Instruments Inc., Melrose, IL). After f'dtration through nylon mesh, protoplasts were collected by eentrifugation at 100 x g for 2.5 rain and were resuspended in a solution of the appropriate mannitol concentration for that particular Capsicum genotype. This process of washing the protoplasts by centrifugation and resuspension in mannitol was repeated three times. Protoplasts were counted using a hemocytumeter and viability was assessed by staining with fluorescein diacetate (Huang et aL 1986). Protoplast quality as stated in this report was determined phenotypically, i.e., the degree of protoplast swelling and chloroplast distribution within the protoplast over time. Protoplasts of good phenotypic quality showed no or only slight swelling and chloroplasts were evenly distributed, although some swelling and polarization of chloroplasts were typical during the course of incubation. This measure o f protoplast quality was based on initial studies that identified viable protoplasts relative to their phenotypic appearance after fluorescein diacetate staining.

Protoplast inoculation. The inoculation of protoplasts with viral RNA was done by electroporation using a single ring electrode connected to a ProGenetor I apparatus (Hoeffer Scientific, Inc., San Francisco, CA), and applying two 5 msee pulses of 300 volts (Cart and Zaiflin 1991). Protoplasts resuspended in the appropriate mannitol solution at concentrations of 0.5-1.0 x 10 ~ protoplasts/ml were inoculated in 2 ml aliquots. Inoeuium typically consisted of 1-5 lag of viral RNA delivered in a volume of less than I0 lal; mock-inoculum was similar but did not include viral RNA. The incubation medium used after electroporation of protoplasts was a modification of that described by Add and Takebe (1969): a solution of the appropriate mannitol concentration containing 0.2 mM KILaPO4. 1.0 mM KNO~, 0.1 mM MgSO4, O. 1 mM CaCL z , 1.0 gM KI and 0.0t I.tM CuSO 4. The antibiotics carbenicillin, cephaloridine and nystatin (Sigma Chemical Co.) were added at concentrations of 100, 100 and 4 lag/ml, respectively.

Samples analyzed for virus infection consisted of 50,000 protoplasts, collected at specific times post-inoculation. Each sample was concentrated by centrifugation, lysed using a protein dissociation buffer (Laemmli 1970), frozen by submersion in liquid nitrogen and stored at -80 ~ C. Viral coat protein (CP) accumulation in protoplast samples was determined by western blot analysis following SDS-polyacrylamide gel electrophoresis (12.5% resolving gel) (Laemmli 1970). Proteins were electrobloued (Towbin et al. t979) to nitrocellulose, probed with immunoglobulin made to PeMV, TEV-F or CMV-Fny and detected using an alkaline phosphatase detection system (Leafy et al. 1983). Antiserum to PeMV was obtained from Dr. T.A. Zitter (Coruell University), to CMV from Dr. D. Gonsalves (Comdl University) and to TEV from the American Type Culture Collection (Rockville, ME)).

PeMV (strain V-1182 from T.A. Zitter) and TEV-F (HAT isolate from Dr. T.P. Pirone, University of Kentucky) were propagated for vires purification in Nicotiana tabacum 'Kentucky 14' and purified according to Murphy et al. (1990). Viral RNA was isolated by SDS-sucrose density gradient centrifugation (Murphy et al. 1990) or by treatment of virus particles with SDS (final amount 0.5%) and Proteinase K (100 lag/m1) for 15 min at 37 ~ C. This solution was then subjected to several cycles of phenol:chloroform extraction and the RNA was precipitated with sodium acetate, pH 6.0, (final concentration 0.1 M) and 2.5 volumes of absolute ethanol at -20 ~ C. CMV-Fny (from Dr. R. Provvidenti, Comell University) was propagated in N. tabacum Kentucky 14 and virus was purified according to Palukaitis and Zaitlin (1984). CMV-Fny RNAs were isolated using the SDS- Proteinase K/phenol extraction procedure described above.

Results

We have developed an optimized protocol that has been applied to five genotypes representing two species of Capsicum and have identified key variables that appear to be genotype-specific with respect to protoplast isolation.

Plant growth conditions

We were able to isolate viable Capsicum leaf proto_plasts only from plants grown in magenta boxes on MS medium in a Percival incubator. Upon seed germination, magenta boxes were sealed with 3M filter tape; at least one day prior to protoplast isolation the 3M filter tape was replaced by magenta box covers. Plants were allowed to grow to the two to four true leaf stage at which time the true leaves were used as protoplast sources. Generally, half-strength MS medium resulted in a higher percent seed germination and faster plant growth than full-strength MS medium. This was especially true for the C. chinense PI 152225 and PI 159236 plants. We were not successful at the isolation of intact viable protoplasts from plants grown in a greenhouse, growth chamber, or in the modified Cornell mix in a Percival incubator.

Protoplast isolation

The optimized procedure was as follows: true leaves used for protoplast isolation were sliced (See Materials and methods), plasmolyzed by submersion in a mannitol solution of the appropriate concentration (see below) and shaken at 170 rpm for 10 rain at 25 ~ C. The mannitol solution was decanted and the plasmolyzed leaves were submerged in enzyme solution which consisted of 1% (w/v) macerase, 0.25% (w/v)pectolyase and 1% (w/v) cellulysin dissolved in mannitol, pH 5.47-5.50. The leaves in enzyme solution were subjected to vacuum inf'lltration for 1-2 min and then shaken at 70 rpm at 25 ~ C for approximately 4 h. This procedure consistently provided yields of 650,000 protoplasts per 0.5 g of Capsicum leaf tissue (a typical leaf weighed 0.04 g).

Protoplast quality was not affected when pectolyase was omitted from the enzyme solution (e.g., 1% macerase and 1% cellulysin), however, the presence of pectolyase significantly reduced the amount of time necessary for cell release.

Preliminary experiments indicated that a single osmoticum was not appropriate for all of the genotypes used in this study. Therefore, the effect of mannitol concentration on protoplast yield and phenotypic quality was examined for each of the Capsicum genotypes. A given mannitol concentration was used for every step in the protoplast isolation procedure, i.e., a solution consisting of the same mannitol concentration was used for plasmolysis, the enzyme solution, washing and isolation, the inoculation medium and in the incubation medium.

C. annuum NuMex RNaky and Early Calwonder protoplast quality was better in solutions that consisted of 0.60 M mannitol while isolation of C. annuum Avelar protoplasts required a solution of 0A2 M mannitol. The isolation of C. chinense PI 152225 and PI 159236 protoplasts required solutions that consisted of 0.43-0.45 M mannitol. Viable protoplasts were not isolated from C. annuum Avelar and the C. chinense accessions when mannitol concentrations were above 0.45 M. C. annuum NuMex RNaky and Early Calwonder protoplasts tolerated mannitol concentrations that ranged from 0.50 M to 0.70 M. However, solutions of 0.60 M mannitol provided protoplasts of better quality, particularly after incubation for 24 h or more, i.e., protoplasts did not swell as extensively and chloroplasts remained distributed thoughout the cell for a longer peroid of time before becoming polarized.

Inoculation of protoplasts

The host-virus combination selected to develop a standard inoculation protocol was C. annuum NuMex RNaky and PeMV. Infection of leaf protoplasts by viral RNA (1-5 lxg) occurred when using the ProGenetor 1 electroporation apparatus with the parameters described by Carr and Zaitlin (1991) with 0.60 M mannitol as the electroporation medium. The other Capsicum genotypes used in this study also became infected using this procedure and in each case a solution that consisted of the appropriate mannitol concentration for that particular genotype was used as the inoculation medium.

Using the isolation and inoculation procedure described in this report, protoplasts from C. annuum NuMex RNaky, Avelar and Early Calwonder were

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successfully infected by PeMV, TEV-F and CMV- Fny. Protoplasts from C. chinense PI 152225 and PI 159236 plants were infected by CMV-Fny but resisted infection by PeMV and TEV-F (Murphy et al. 1994). Successful infection was determined by the accumulation of viral CP detected by western blot analysis of protoplast samples collected over time. As illustrated in Figure 1, PeMV, TEV-F and CMV-Fny CP accumulation was detected from samples of inoculated C. annuum NuMex RNaky protoplasts collected up to 48 h post-inoculation (hpi). In each ease, virus CP was not detected from samples collected at 1 hpi, but the respective coat proteins were detected from samples collected at 24 and 48 hpi. Virus CP was never detected from samples of mock-inoculated protoplasts.

We examined the effect of using different voltage during electroporation on PeMV infection of C. annuum NuMex RNaky protoplasts as measured by PeMV CP accumulation. As voltage was increased from 200 to 400, so did the accumulation of PeMV CP. Coincident with increased voltage, however, was an increase in both the size of protoplast aggregates and the duration of aggregation. At 400 volts, protoplasts remained aggregated throughout the course the experiment, while samples subjected to 200 and 300 volts tended to become dispersed within 24 hpi. We selected 300 volts for general use throughout the course of the experiments.

Discussion

We have developed a procedure for the isolation of Capsicum leaf protoplasts that is extremely consistent and provides yields of 650,000 protoplasts per 0.5 g of

Anti-PeMV Ig A n t i - T E V - F Ig A n t i - C M V - F n y Ig

Fig. 1. Western blot analysis of virus- and mock-inoculated C. annuum NuMex RNaky protoplasts. Each lane represents a sample of 50,000 protophsts collected at 1, 24 or 48 hpi. Samples were probed with immtmoglobulin fig) to PeMV, TEV-F or CMV-Fny. A 50 ng sample of purified virus was included as coat protein marker for each of the viruses, PeMV (P), TEV-F (T) and CMV-Fny (C) and their locations are indicated by the arrow.

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leaf tissue. This procedure is applicable, with appropriate variations in osmoticum, for protoplast isolation from several diverse C. annuum genotypes and C. chinense PI 152225 and PI 159236. In addition, we have been able to infect protoplasts from each of these genotypes via electroporation with viral RNA (infection of C. annuum NuMex RNaky protoplasts by each of the three viruses is illustrated in Figure 1). Jacobs (1991) attempted electroporation of Capsicum protoplasts with PMMV virions but reported that results were inconsistent.

There have been several reports on the isolation of Capsicum protoplasts (Saxena et al. 1981, Diaz et al. 1988, Jacobs 1991), and in each case, source plants were grown on MS-based media under controlled conditions. We were not able to isolate viable Capsicum protoplasts unless source plants were grown under conditions similar to those required for regeneration which conlrasts with growth requirements for other plant species, e.g., tobacco (Takebe et al. 1968) and tomato (Motoyoshi and Oshima 1975).

The general procedure for Capsicum protoplast isolation differed in each report (Saxena et al. 1981, Diaz et al. 1988, Jacobs 1991), however, the most important difference was the choice of osmoticum used for protoplast isolation. We found that C. annuum NuMex RNaky and Early Calwonder protoplasts could be isolated using 0.50 M to 0.70 M mannitol, while narrower ranges of mannitol concentrations were required for C. annuum Avelar and C. chinense PI 152225 and PI 159236 protoplasts. Saxena et al. (1981) used 0.50 M mannitol to isolate C. annuum California Wonder protoplasts while Diaz et al. (1988) used 0.70 M mannitol for several C. annuum lines and a C. chinense accession. Jacobs (1991) used 0.40 M sorbitol as osmoticum to isolate protoplasts from a C. annuum accession and a 0.70 M mannitol solution as inoculation medium. This variation in osmoticum, as described by Saxena et al. (1981), Diaz et al. (1988) and Jacobs (1991), agreed with our data that showed different osmoticum requirements between and within Capsicum species. Furthermore, this variation may not only be genotype-specific but may also reflect different requirements with respect to the type of tissue from which protoplasts were isolated. For instance, Saxena et al. (1981) and our procedure used true leaves, while Jacobs (1991) used cotyledons, and Diaz et al. (1988) apparently used various cell types from Capsicum shoots.

We varied the voltage parameter in an attempt to optimize the inoculation of Capsicum protoplasts, although 300 volts was routinely used in our

experiments as a compromise between a greater level of infection (more PeMV CP accumulated in protoplasts subjected to 300 volts than 200 volts) and protoplast aggregation (more aggregation occurred with 400 volts than 300 volts). We have not determined whether the increase in accumulation of viral CP observed with increased voltage was due to more PeMV accumulation per protoplast and/or represented a greater number of protoplasts that became infected. Although more PeMV CP accumulated at 400 volts, the high degree of protoplast aggregation that resulted made it difficult to accurately count and thus collect specific numbers of cells following inoculation.

We are now applying these procedures to Capsicum genotypes that display resistance at the whole plant level in an attempt to unravel the mechanisms by which these plants resist virus infection.

Acknowledgements. We wish to thank Dr. John Carr for his assistance with the ProGenetor 1 electroporation unit and for critical review of this manuscript and Janette Lynn Jacobs for providing portions of her M.S. Thesis. This work was supported in part from a grant from the Cornell Center for Advanced Technology in Biotechnology which is sponsored by the New York State Science and Technology Foundation, a consortium of industries, and the National Science Foundation, and USDA NRI/CGP Award No. 91-373001-6564. MMK is a Burroughs Welle~ne Fund Fellow of the Life Sciences Research Foundation.

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