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INFECTION OF PROTOPLASTS DERIVED FROM LIQUID SUSPENSION CULTURE OF SOYBEAN WITH COWPEA MOSAIC AND SOUTHERN BEAN MOSAIC VIRUSES: A NEW SYSTEM FOR STUDYING VIRUS/HOST INTERACTIONS By Nancy Priscilla Jarvis A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1978 ABSTRACT INFECTION OF PROTOPLASTS DERIVED FROM LIQUID SUSPENSION CULTURE OF SOYBEAN WITH COWPEA MOSAIC AND SOUTHERN BEAN MOSAIC VIRUSES: A NEW SYSTEM FOR STUDYING VIRUS/HOST INTERACTIONS By Nancy Priscilla Jarvis A system has been developed for studying synchronous plant virus infection and replication that allows for axenic conditions to be maintained and yields a high degree of reproducibility between experiments. This was achieved by isolating and inoculating proto- plasts from liquid suspension culture. Soybean cultures were initiated on agar and maintained in liquid medium. Protoplasts were enzymat- ically prepared from the culture tissue, washed in sorbitol and resuspended in a virus inoculation medium. After 20 minutes, proto- plasts were washed with sorbitol plus 10 mM CaCl and resuspended 2 in culture medium. Local lesion assays on Pinto bean leaves and fluorescent anti- body assays showed infection of the inoculated soybean protoplasts by cowpea mosaic virus (CPMV) and southern bean mosaic virus (SBMV). Nancy P. Jarvis The contents of the inoculation medium were critical to obtaining a high percent of infected protoplasts. With CPMV, 70-80% of the protoplasts were infected with 0.5 ug CPMV/ml, 1.5 ug poly-L- ornithine/ml, 10 mM K phosphate buffer, pH 6.3, and 0.5 mM CaCl2 at 23 C. Poly-L-ornithine was essential for infection. Phosphate buffer stimulated infection only in the range of pH 5.8 to 7.0, with infection peaking sharply at pH 6.3. The stimulatory effect of calcium on protoplast infection was dramatic. Only 10-12% infection occurred when the inoculation medium was not amended with calcium, while 70-80% infection was obtained when 0.5 mM CaCl2 was added to the inoculum. This seven-fold stimulation by Ca++ was considerably decreased by washing the proto- plasts in sorbitol plus 10 mM CaCl2 prior to inoculation. Very little protoplast infection was obtained at an inoculation temperature of 0 C, but nearly maximal infection could be obtained at 12-15 C. After this, increasing the temperature of inoculation increased the percent of infection only slightly. Infection of soybean protoplasts by southern bean mosaic virus was greatest at 2-2.5 ug SBMV/ml, 2 ug poly-L-ornithine/ml, 10 mM Tris-H01 buffer, pH 8.0, and 1 mM MgSOu plus 0.5 mM CaCl A maximum 2. of only 30-35% of the protoplasts were infected under these conditions. The infection was dependent on poly-L-ornithine. Infection in the presence of Tris-H01 buffer was nearly pH-independent over the range of pH 7.2 to 8.6. Several other relatively high pK buffers were also suitable for infection studies. The presence of a combination of calcium and magnesium salts increased the percent of infection 10-fold. Nancy P. Jarvis Soybean tissue culture can provide a reproducible, homogeneous source for isolation of viable protoplasts. These protoplasts can be infected with outstanding reproducibility. This system, the first of its kind to be described, is suitable for use in plant virus/host interaction studies, especially those where synchrony of infection, axenic culture, defined growth conditions and reproducibility are of utmost importance. This thesis is dedicated to my mother and father whose continuing love, support and enthusiasm have given me the confidence and courage to pursue my goals. ii ACKNOWLEDGEMENTS I would sincerely like to thank Dr. Harry Murakishi for continual support and encouragement and patient counseling during my graduate studies, and also for providing a laboratory environment conducive to pursuing research objectives. I am grateful to Dr. Norman Good and Dr. Peter Carlson whose broad knowledge has contributed in many ways toward this research and many other areas of science and philos- ophy. I would especially like to thank Mark Lesney for many hours of invaluable discussion and many suggestions that contributed greatly to the progress of this research and the preparation of this thesis. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . viii LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . ix INTRODUCTION AND LITERATURE REVIEW . . . . . . . . . . . . . 1 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . 9 Propagation and purification of CPMV . . . . . . . . . . 9 Propagation and purification of SBMV . . . . . . . . . . 10 Culture initiation . . . . . . . . . . . . . . . . . 11 Protoplast isolation and inoculation . . . . . . . . . . 11 Protoplast viability . . . . . . . . . . . . . . . . . . 15 Infectivity assay . . . . . . . . . . . . . . . . . 15 Mounting and staining of protoplasts . . . . . . . . . . 16 Photography . . . . . . . . . . . . . . . . . . 17 Antibody preparation and conjugation . . . . . . . . . . 17 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Tissue culture . . . . . . . . . . . . . . . . . . . . . 20 Protoplast inoculations . . . . . . . . . . . . . . . . 21 Observations of fluorescent protoplasts . . . . . . 2“ Development of infectivity over time . . . . . . . 2h Conditions affecting infection . . . . . . . . . . . . . 32 Buffer . . . . . . . . . . . . . . . . . . . . 32 Poly-L-ornithine . . . . . . . . . . . . . . . . . 37 Salt environment . . . . . . . . . . . . . . . . . “0 Virus concentration . . . . . . . . . . . . . . . . “5 Additional calcium effects . . . . . . . . . . . . ”5 Temperature of inoculation . . . . . . . . . . . . 51 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . 55 Culture growth . . . . . . . . . . . . . . . . . . . . . 56 Growth rate . . . . . . . . . . . . . . . . . . . . 57 Clump size . . . . . . . . . . . . . . . . . . . . 57 Culture age . . . . . . . . . . . . . . . . . . . . 57 Inoculation conditions . . . . . . . . . . . . . . . . . 57 iv SUMMARY Buffer used Presence of polycation . Virus concentration Salt environment . APPENDIX A . APPENDIX B . APPENDIX C . LITERATURE CITED . Page 58 60 62 63 68 69 70 78 79 Table 1. Table 2. Table 3. Table A. Table 5. Table 6. Table 7. Table A1. Table A2. Table A3. Table AM. LIST OF TABLES Composition of R3 Medium Composition of Protoplast Culture Medium Effect of the Addition of Various Buffers to the Inoculation Medium on Percent of Protoplasts Infected with CPMV and with SBMV Effect of Various Concentrations of Poly-L- 0rnithine on Percent of Protoplasts Infected with SBMV . . . . . . . . . . . . . . The Effect of the Addition of Various Salts to the Inoculation Medium on Percent of CPMV-Infected Protoplasts . The Effect of the Addition of Various Salts to the Inoculation Medium on Percent of SBMV-Infected Protoplasts . Effect of CaCl on Infection of Protoplasts by CPMV When Present in the Pre-Inoculation Wash, Inoculation Medium or Post-Inoculation Wash Growth of Soybean Callus on Agar Medium Supplemented with Various Hormones . CPMV Infection of Protoplasts With or Without Poly-L-ornithine in the Presence of Various Inoculation Buffers SBMV Infection of Protoplasts under Various Inoculation Conditions in the Presence of HEPES Buffer . . . . . SBMV Infection of Protoplasts in the Presence of HEPES at Various pH's . vi Page 12 1A 36 no 42 NH 51 69 73 7A 75 Table A5. Table A6. Table A7. SBMV Infection of Protoplasts in the Presence of HEPES Buffer Using Various Virus Concentrations . . . . . . . . CPMV Infection of Protoplasts in the Presence of HEPES Buffer Using Various Virus Concentrations . . . . . Effect of Mg(N03) on Infection of Protoplasts When Present in t e Inoculation Medium at Various Concentrations . . . vii Page 76 77 78 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 1. 2a & b LIST OF FIGURES Growth curve of liquid suspension culture of soybean in R3 medium . . . . . . . SBMV-infected protoplasts stained with fluorescent antibody . . CPMV-infected protoplasts stained with fluorescent antibody Time course of SBMV synthesis in protoplasts Time course of CPMV synthesis in protoplasts Effect of pH on percent of SBMV-infected protoplasts . . . . . . . . Effect of pH on percent of CPMV-infected protoplasts . . . . . . . . . . . Effect of various concentrations of poly-L- ornithine on percent of protoplasts infected with CPMV The Effect of CPMV concentration on percentage of protoplasts infected in the presence of various salts The effect of SBMV concentration on percentage of protoplasts infected in the presence of various salts Effect of temperature of inoculation on percent of protoplasts infected with CPMV viii Page 23 26 27 29 31 3A 3A 39 H7 ”9 5A BES bistrispropane CMV CPMV 2'N'D DIPSO EDTA FITC HEPES HEPPS HEPPSO 2ip LS salts MES MOPS MOPSO PLO SBMV TAPS TAPSO TMV LIST OF ABBREVIATIONS N,N-bis(2—Hydroxyethyl)-2-aminoethanesulfonic acid bis(2-Hydroxyethyl)imino-tris(hydroxymethyl)propane cucumber mosaic virus cowpea mosaic virus (Z'A'-dichlorophenoxy)acetic acid 3- N-(bis-Hydroxyethyl) -amino -2-hydroxypropane- sulfonic acid ethylenediamine tetraacetic acid fluorescein isothiocyanate N-2—Hydroxyethylpiperazine-N'-2-ethanesulfonic acid N-Hydroxyethylpiperazine-N'-propanesulfonic acid N-Hydroxyethylpiperazine-N'-2—hydroxypropanesul- fonic acid 6( -Dimethylallylamino)-purine Linsmaier and Skoog salts 2-(N-morpholino)ethanesulfonic acid 3-(N-morpholino)propanesulfonic acid 3-(N-morpholino)-2-hydroxypropanesulfonic acid poly-L-ornithine southern bean mosaic virus 3- N-(tris-Hydroxymethyl)-methylamino -propane- sulfonic acid 3- N-(tris-Hydroxymethyl)-methylamino -2-hydroxy- propanesulfonic acid tobacco mosaic virus ix INTRODUCTION AND LITERATURE REVIEW Basic mechanisms of virus infection and replication processes have been the focus for many molecular and microbiologists over the past 20 years. The literature on animal and bacterial viruses is voluminous - knowledge of replication processes is highly refined and virus-host interactions have been well characterized in many systems. Plant virus studies, however, have lagged dismally far behind. Much of this has undoubtably been due to the lack of a suitable experimental system for the study of plant virus replication. Intact plants, though appropriate for host range and transmission studies, were found to be unsuitable for replication studies for three major reasons. First, the initial inoculation of whole plants results in the infection of an extremely small percentage of cells. This means that the first synchronously produced intermediates and products of replication are diluted greatly by the presence of healthy cells. Secondly, the subsequent spread of virus results in asynchronous virus replication, making it impossible to study sequential events in replication. Thirdly, uptake studies are extremely difficult and inefficient in whole plants. Separated cells, enzymatically digested from infected leaves were an improvement in that they were able to take up labeled pre- cursors, and sustained virus synthesis was successfully studied gE'3 (Jackson, gt El- 1972). However, as in intact plants, replication was asynchronous and a relatively small population of cells was infected. Also cells degenerated rapidly. The development of a system for cold-temperature synchronized replication in leaves (Dawson and Schlegel, 1973) and in tissue culture (White gt El. 1977) improved percent of cells infected some- what and synchronized the final stages in virus synthesis, but early infection of cells was not actually synchronized. Therefore, it was a major breakthrough when techniques were developed by Cocking (1966) for the isolation and infection of proto- plasts from leaf mesophyll tissue, and were subsequently refined by Otsuki and Takebe (1969), allowing an efficient and synchronous infection of a high precentage of cells. Since these pioneering studies, over 25 protoplast-virus systems have been introduced and used successfully for studying virus replica- tion as well as various other aspects of plant-virus interactions. These are well discussed in several recent review articles (Takebe, 1975; Takebe, 1978; Zaitlin and Beachy, 1974a; Zaitlin and Beachy, 197ub). Protoplasts for such studies have been isolated almost exclusive- ly from mesophyll cells, and although often over 90% of the cells can be synchronously infected (Takebe, 1978), the system has disad- vantages. One problem has been microbial contamination. Although leaf sections can be surface sterilized and media autoclaved, anti- biotics must still be added to mesophyll protoplast culture medium to avoid bacterial and fungal growth during short-term experiments. A second problem has been in growing suitable plants for proto- plast isolation. Plant age, lighting conditions, soil type, tempera- ture, etc. are critical to the isolation of viable and uniformly infectible protoplasts (Kubo gt al., 1975), and these parameters are often difficult to regulate due to seasonal variation throughout the year, and have been difficult to duplicate between laboratories. These two problems have been circumvented in the case of tobacco (Murakishi, §£.§l°: 1971) and more recently with soybean (Wu and Murakishi, 1978) through the inoculation of agar- or liquid-grown cells. Although this system avoids problems of microbial and large scale cell variation and labeled precursor uptake studies pose less problems, the infection is 100 to 1000 times less efficient due to the presence of cell walls and cell clumps than in tobacco protoplast studies (Takebe and Otsuki, 1969). Also, only a relatively small percentage of cells are initially infected. An examination of the advantages and disadvantages of systems using callus cultures and those using protoplasts from mesophyll leads to the obvious conclusion that a procedure using protoplasts derived from tissue culture would avoid some of the problems and would incorporate the primary advantages of each system. Although Takebe has indicated the feasibility of this type of study using Kings tissue culture (Takebe, unpublished results), no workable system has hitherto been developed for the inoculation of protoplasts from tissue culture. It was in an attempt to fulfill this need for a usable, efficient system that the present study was undertaken. Soybean provides an excellent host for this type of study for several reasons. Cultures of soybean are readily initiated and main- tained (Gamborg 22 al., 1968; Wu and Murakishi, 1978) and protoplasts have been isolated from liquid suspension by Kao gt al., (1970) and others (Chu and Lark, 1976). Moreover, protoplasts have not as yet been successfully isolated from soybean meSOphyll tissue, evidencing a need for the use of an alternate system. Another distinct advantage to using soybean is its potential for adaptation to the study of many different viruses. Soybean has been reported to be susceptible to over 50 viruses (Sinclair and Dhingra, 1975), including such well characterized viruses as alfalfa mosaic, cowpea chlorotic mottle, cowpea mosaic, southern bean mosaic, bean pod mottle, soybean mosaic and cucumber mosaic viruses. COWpea mosaic virus (CPMV), a multicomponent icosahedral virus (Breuning and Agrawal, 1967) was chosen for this study for several reasons. It is easily propagated and purified in large quantities and is stable in vitgg (Van Kammen, 1967). Also, a precedent exists for protoplast infection by CPMV in that both cowpea (Hibi gt 31., 1975; Beier and Breuning, 1975) and tobacco mesophyll protOplasts (Huber gt al., 1977) have been efficiently infected with the virus. Southern bean mosaic virus (SBMV) was also used in this study. It is an icosahedral single component virus of 6.6 x 106 daltons (Miller and Price, 19A6), and as with CPMV, it is easily purified and quite stable. Although there has been no precedent for the study of this virus in protoplasts, soybean tissue culture has been suc- cessfully inoculated with SBMV (Wu and Murakishi, 1978). This in- creased the probability that it would prove infectious in protoplasts from the same tissue culture. Preliminary work in this lab (Jarvis and Murakishi, 1978) indeed indicated the feasibility of using the soybean protoplast-SBMV system for virus studies. Between A0 and 50% infection of tissue culture- derived protoplasts was obtained. This thesis describes the depth of that preliminary work and recounts the continued studies of the infection of soybean proto- plasts by SBMV. In addition it includes the investigation of con- ditions necessary to obtain efficient infection by CPMV. It has been shown for other protoplast systems that several factors are critical in obtaining efficient infection of protoplasts. These factors must be optimized in each protoplast-virus system to yield maximal infection. 1) Viability of cells: Viability and infectability of proto- plasts is critically dependent on the growth conditions of source tissue and on the protoplast isolation procedures (Kubo gt_§l., 1975). Unless these parameters have been optimized, protoplasts degenerate rapidly and little or no virus synthesis occurs. 2) Presence of a polycation: The infection in many protoplast- virus systems has been shown to be dependent on or stimu- lated by the presence of a polycation (most commonly poly- L-ornithine) in the inoculum (Takebe, 1978). Optimal poly- L-ornithine concentrations generally are between 0.5 ug/ml and 1.5 ug/ml. It has been hypothesized that the role of the polycation is to decrease electrostatic repulsion between the virus particle and the cell membrane, both 3) A) of which often bear a net negative charge. Buffering conditions: All protoplast systems studied thus far have required a buffered inoculum for virus in- fection to occur. The most common buffer employed is K citrate at pH 5.0 to 5.5 (Takebe, 1975); phosphate buffer, pH 6.5 to 6.7 (Kubo gt al., 197“) and Tris KCl buffer at pH 8.0 (Motoyoshi and Oshima, 1976) have been more recently introduced and have been found to be superior to K citrate in some systems. Othergparameters: The effect of the presence of various salts and the temperature of inoculation on percent of cells infected have been investigated in only a few systems. A calcium preinoculation wash of protoplasts has been found by Motoyoshi gt El. (197“) to decrease infection of tobacco protoplasts by cowpea mosaic chlorotic mottle virus. Cal- cium or magnesium salts, when included in the inoculum at 10 mM, completely inhibited infection of several kinds of protoplasts by brome mosaic virus (Furusawa and Okuno, 1978). Infection of tobacco protoplasts by cowpea chlorotic mottle virus has been shown to occur independently of inoculation temperature over the range of A C to 23 C (Motoyoshi gt al., 197A). However, Alblas and Bol (1977) have shown that virus yield from infected protoplasts is much higher when inoculation is carried out at A C rather than at the standard 23 C. Clearly, then, optimal infection is obtained under different inoculation conditions for each protoplast-virus system. We have found that infection of soybean protoplast by CPMV and SBMV is greatly influenced by all of these parameters. Therefore, this present study was conducted to examine each of these parameters and find the com— bination of inoculation conditions necessary to achieve a high per- centage of infection by CPMV and SBMV. As a result of the investiga- tion of these various parameters, several interesting phenomena surfaced, giving possible leads for future virus-membrane interaction research. Hopefully this system has reached the level of sufficient control and reproducibility to be easily adopted by other researchers for continuation of these studies. MATERIALS AND METHODS Propagation and purification of CPMV. The Sb strain of cowpea mosaic virus (CPMV), generously donated by Dr. G.B. Bruening, Depart- ment of Biochemistry and BiOphysics, University of California, Davis, was propagated in Vigna sinensis cv. Blackeye by inoculating newly expanded primary leaves with virus diluted in 0.1 M K phosphate buffer, pH 7.0. Infected leaves were harvested 1“ days after inoculation and stored at -25 C until use. Virus was isolated using a modifica- tion of the polyethylene glycol-NaCl method described by van Kammen (1967). Frozen leaves were triturated in 0.1 M K phosphate buffer, pH 7.0, 1.5 ml per gram of tissue, in a Waring blender at N C. The homogenate was expressed through A layers of cheesecloth and chloro- form-butanol (1:1) was added to the filtrate to 8% of the final volume (Soong and Milbrath, 1975). The mixture was stirred for 60 minutes at A C and coagulated plant debris was removed by centrifuga- tion for 10 minutes at 10,000 rpm (Sorvall type 88-34). NaCl was added with stirring to the supernatant to 0.2 M and A% (w/v) poly- ethylene glycol (m.w. 5,700-6,700) (General Biochemicals, Chagrin Fall, OH) was dissolved into the mixture (Hebert, 1963). Precipitate was allowed to form at room temperature for 1 hour and was collected by centrifugation at 10,000 rpm for 15 minutes. This pellet was resuspended in 0.1 M K phosphate buffer, pH 7.0 at 1/5 the original volume, and the suspension was subjected to 2 rounds of differential centrifugation at 10,000 rpm for 15 minutes and 34,000 rpm for 90 minutes (Rotor no. 50, Spinco model L centrifuge) (van Kammen, 1967). ‘1. 10 The final pellet was resuspended in 0.1 M K phosphate buffer, pH 7.0 and was freed of microorganisms by passage through a sterile ultrafine Corning sintered glass filter. The preparations had 260/280 nm absorbancy ratios of between 1.58 and 1.70. An extinction coeffi- cient of 8 cmZ/mg at 260 nm was used to estimate virus concentration (van Kammen, 1967). Propagation and purification of SBMV. The bean strain of southern bean mosaic virus (SBMV), was propagated in leaves of Phaseolus vulgaris cv. Bountiful. Infected leaves were harvested 14-18 days after inoculation and stored at -25 C until use. SBMV purification was accomplished according to the procedure used for CPMV with the following modifications. Leaf tissue was triturated in 0.2 M K phos- phate buffer, pH 7.5. Virus was precipitated using 0.2 M NaCl and 6% w/v polyethylene glycol. The virus-PEG precipitate was collected by centrifugation at 10,000 rpm for 15 minutes and the pellet was resuspended in 0.02 M K phosphate buffer, pH 7.2 at 1/10 the original sap volume. The suspension was subjected to 2 rounds of differential centrifugation (10,000 rpm for 15 minutes and 22,500 rpm for 2.5 hours) and the final pellet was resuspended in 0.02 M K phosphate buffer, pH 7.2 (Shepherd and Fulton, 1962). The preparation was cleared of brown pigment by passage through a 2 cm X 7 cm column of DEAE cellulose and freed of microorganisms as was CPMV. These preparations had 260/280 nm absorbancy ratios of 1.60-1.65. Virus concentration was estimated by using an extinction coefficient of 5.8 (Sheperd, 1971). 11 Culture Initiation. Callus culture of soybean (Glycine max cv. Harosoy 63) were initiated from hypocotyl sections on R3 agar medium (Table 1) as described by Wu and Murakishi (1978), and main- tained by transfer to fresh R3 every four weeks. To initiate liquid suspension culture, a section of callus was transferred to 30 or 50 ml of medium in 125 or 250 ml Erlenmeyer flasks and grown under continuous light of 861 lux (80 ft-c) from Gro-Lux fluorescent lamps at 23 C :_2 C on a rotary shaker at 80 rpm (model 61A0, Eberbach Corporation, Ann Arbor, MI). In these studies, R3A medium was used for liquid culture, a modified R3 medium containing 2X the concentra- tion of 2'uv dichlorophenoxy acetic acid present in R3, and no indole- 3-acetic acid. The liquid suspension culture was maintained on R3A for one year, transferring 1 ml culture per 5 m1 fresh medium every A days, prior to use as a protoplast source. Protoplast Isolation and Inoculation. All manipulations of culture cells and protoplasts, excluding assays, were performed under sterile conditions in a laminar flow hood (Contamination Control, Inc., Kulpsville, PA). Protoplast isolation was accomplished using a modification of the procedure outlined by Constabel (1975). Liquid suspension cultures of soybean, 36 to A8 hours from subculture, were transferred to 50 ml graduate conical tubes. Cells were washed once with R3A and allowed to resettle for 10 minutes. The supernatant fluid was carefully removed and digestion medium was added in an amount equal to the settled cell volume. Driselase at 2% (Kyowa Hakko Kogyo Co. , N.Y., NY), 1% Macerase and 2% Cellulysin (both Cal-biochem) in 0.4 M sorbitol, pH 6.0 were used in the digestion 12 Table 1 Composition of R3 Medium1 Linsmaier and Skoog minerals2 NHuNO KNO 3 CaCl '2H 0 2 2 MgSOu°7H20 KH2P04 H3B03 MnSOu'HZO ZnSOu°7H20 KI Na2MoOu'2H20 CuSOu°5H20 CoCL2'6H20 3 NaZEDTA FeSOu Thiamine-HCl Pyridoxine-HCl Nicotinic Acid Inositol Sucrose (2'4'-dichlorophenoxy)acetic acid Indole-3-acetic acid Kinetin amount/l .4 —A—-\ CDOOCDOCJ\CJ‘\Ozmm .nw cam mm ostfim 26 n N otzmflm mm onsmflm 27 Figure 3. CPMV-infected protoplasts stained with fluorescent anti- body. I, infected protoplasts; N, non-infected protoplasts. 28 .mummHQOBOLQ wouooucflu>zmm scam A1..1..I.v osflu cufiz mcoflmwa HMQOH no Lopes: on» can A oIIIIIIIIoV mafia Qua: mpwmaqouOLQ uncomonozam unmetom .m5LH> on» no zufi>wuoomcfi ocafimmuou on smog cpcflm no mm>mo~ ham: :0 vmzmmwm mama to .COHpoohcw owmucooamq oCHsLoumc on muonflpcm uncommgozam spa: vocflmpm one: use :OHumHSOOCH memm mosflu monLm> um vopmo>hmc one: muwmaaouoam .mpawm mooxm cam Lofimsmcflq momentum mfiuos mHmn new o.m mm .Lphhsn Homumflgh 28 o— .Hs\oqm w: N no mocomopq on» CH HS\>zmm w: m cows woundsoocfl egos mummHQouonm .mpmmaqouonq :fl mflmosuc>m >zmm no mmgzoo mafia .: oLDMHm 29 o ----- a uaawnu 1101331 m , 2 ‘32 .0. ? . E u ‘s 1p . ‘ ------- _____ -o._ ,2» ¢:.-._ - O O O 0 v (0 N '- O——"" SlSV'ldOlOlld 1113033401111 % 60 20 30 40 50 TIME OF INCUBATION , hours 10 Figure 4 30 .mumeQOponq vmuoomcfl1>2mo scum A9111111ov mew» spas mCOHmoH Hmooa no tones: on» new “nu 10v mafia Spas mummHQOBOLQ accomogozam pcoogom .msgfl> on» no mpfi>wpommcfl mafienmpov o» ammo oucwm mo mm>mma mam: co pozmmmm who: no .c0flpommcfl ommpcmopma mafiagopwc on uponflucm vasomotozam npfiz uocflmpm one: cam coapmHSOOCfi Loumm mmer msoflgm> pm popmm>gms who: mummHQOpOLm .mpamm wooxm cam Lofimemcflq spmcmnum waves mam: cam m.m ma .memsn opmnamocd M 28 or .Hs\oqm wz.m mo oocommga on» CH Ha\>2mo wn.m spas oopmasoocfl one: mummHQOpozm .mpmmHQOBOLQ :H mwmozpczm >zmo mo omuzoo mafia .m mgsmflm 31 o------o 113mm: "(“831 N 1- O P l -10 O O O 8 8 <1 co a 0—' SlSV'IdOlOUd 111308380111! % 1O 20 3O 4O 50 60 TIME OF INOIJBATION ,llours Figure 5 32 Conditions Affecting Infection (1) Buffer. Buffering of the soybean protoplast preparation during inoculation was found to be necessary to obtain infection with both CPMV and SBMV. Table 3 shows the effect of four of the buffers tested in CPMV infection. Citrate, the buffer most widely used in virus-protoplast studies (Takebe, 1978), allowed ~1% infection at pH 5.5 and was found to be somewhat toxic to the protoplasts. A similar low infection was observed with Tris-HCl buffer, pH 8.0, although the viability was greatly improved at this pH. With 10 mM phosphate buffer at pH 6.3, however, a large percentage of the cells were infected and viability was high. The pH optimum for phos- phate buffer infection is shown in Figure 7. Peak infection of 60% occurs at pH 6.3; infection drops off rapidly at higher and lower pH's. In the above case the pH study was done using inoculum medium containing Linsmaier and Skoog (LS) salts (Linsmaier and Skoog, 1965) which were found to greatly increase infection in a wide range of conditions (an effect which will be discussed later in this section). Interestingly, MES at pH 6.3 did not allow infection (Table 2). Other buffers were investigated for their use in protoplast infection. These are discussed in Appendix B. Although it allowed very little infection with CPMV, 10 mM Tris- HCl at pH 8.0 was found most effective in stimulating infection of soybean protoplasts by SBMV (Table 3). Less than 5% infection was obtained using K phosphate buffer at pH 6.3 or citrate buffer at pH 5.5, while 36% occurred with Tris-H01, pH 8.0. Table 3 also shows that higher pK buffers in general were the most effective of those 33 Figure 6. Effect of pH on percent of SBMV-infected protoplasts. Protoplasts were inoculated with 5 pg SBMV/ml in the presence of 2 pg PLO/ml, 10 mM Tris-HCl buffer at various pHs, plus 0.5 mM CaClZ. Protoplasts were harvested at 48 hours and stained with fluorescent antibody to determine percent infection. Figure 7. Effect of pH on percent of CPMV—infected protoplasts. Protoplasts were inoculated with 5 ug CPMV/ml in the presence of 2 pg PLO/ml, 10 mM K phosphate buffer at various pHs and with (r---*) or without (~—-—-—-—*) half strength Linsmaier and Skoog salts. Protoplasts were harvested at 48 hours and stained with fluorescent antibody to determine percent infection. % numscm mmuns 34 Figure 6 an .— 3 SBMV E 30 - a. 5 2° ’ W E g 10 r * I l l l l l l l 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 III of TRIS IIOI BUFFER 70#:1111111 60" 50.... 40-- 30-- 2o-- ............... ............ 4"” N """' ------- 10r- 5:6 58 era 62 6:4 6:6 638 1'0 712 7. p11 of 111031111111: BUFFER Figure 7 35 Table 3. Effect of the Addition of Various Buffers to the Inoculation Medium on Percent of Protoplast Infected with CPMV and with SBMV. Protoplasts were inoculated in the presence of 2 ug PLO/ml, a buffer as indicated, and either 5 ug CPMV/ml plus 0.5 mM CaCl2 in experiment 1 or 5 ug SBMV/ml Linsmaier and Skoog salts, half media strength in experiment 2. Protoplasts were harvested 48 hours after inoculation and stained with fluorescent antibody to determine percent of CPMV- or SBMV-infected protoplasts. Table 3 Experiment 1. CPMV 36 Buffer Percent Fluorescent Protoplasts 10 mM K citrate, pH 5.5 1% 10 mM Tris-HCl, pH 8.0 4% 10 mM K phosphate, pH 6.3 66% 10 mM MES, pH 6.3 2% Experiment 2. SBMV Buffer Percent Fluorescent Protoplasts 10 mM K phosphate pH 6. 0 10 mM Tricine, pH 8. 21% 10 mM Bistrispropane, pH 7. 32% 10 mM Tris-HCl, pH 8. 36% 10mM TAPS, pH 8. 29% 10 mM TAPSO, pH 7. 20% 10 mM BES, pH 7. 15% 10 mM DIPSO, pH 7. 31% 10 mM HEPES, pH 5. 8% 37 tested; tricine, pH 8.0, Bistrispropane at pH 7.5, BES at pH 8.0 and others were able to allow some infection, although the percentage was not as high and the viability was lower than with Tris-HCl. It is apparent from Figure 6 that the Tris-stimulated infection is nearly independent of pH over the range of 7.1 to 8.6. (2) PolyeL-ornithine. Poly-L-ornithine (PLO) has been found to have a profound effect on infection of protOplasts by CPMV and SBMV. Figure 8 shows that under these conditions, no infection occurred in the absence of PLO. As increasing concentrations of PLO were added to the inoculum, an increasing percentage of infected cells resulted. At a PLO concentration of 1.5 ug/ml, maximal infec- tion of 74% occurred. Higher PLO concentration proved to be supra- optimal — the percent of infected cells dropped slightly at 2 ug/ml and 2.5 ug/ml. Infection of soybean protoplasts by CPMV was possible in the absence of PLO only when a sulfonic acid buffer was used in the inoculum at low pH. This is discussed in Appendix B. SBMV infection also is dependent on the presence of PLO. Table 4 shows that no infection occurred without PLO, 19% of the proto- plasts were infected at 1 ug PLO/ml, and when the PLO concentration was raised to 2 ug/ml, a substantial increase to 30% infection occurred. PLO levels above 2 ug/ml proved to be toxic to protoplasts under these conditions. 38 Figure 8. Effect of various concentrations of poly—L—ornithine on percent of protoplasts infected with CPMV. Protoplasts were inoculated with 5 pg CPMV/ml in the presence of 10 mM K phosphate buffer,pH 6.3, 0.5 mM CaClZ, and various concentrations of Poly-L- ornithine (PLO). Protoplasts were harvested 48 hours after inoculation and stained with fluroescent antibody to determine percent of CPMV—infected cells. 39 O O O O 0 O O O 8 7 6 5 4 3 2 1 ”51:95.... 588331.: 1.5 2.0 2.5 Flo , pg/ml 1.0 0.5 Figure 8 40 Table 4. Effect of Various Concentrations of Poly-L-ornithine on Percent of Protoplasts Infection with SBMV. Poly-L-ornithine Percent Fluorescent concentration, pg/ml Protoplasts 2 30% 1 19% o 0% Protoplasts were inoculated with 5 pg SBMV/ml in the presence of 10 mM Tris-HCl buffer, pH 8.0 and Linsmaier and Skoog salts at half media strength plus various concentrations of poly-L-ornithine. Protoplasts were harvested 48 hours after inoculation and stained with fluorescent antibody to determine percent of SBMV-infected protoplasts. (3) Salt Environment. Various salts were added to the inocula- tion medium in order to determine their effect on percentage of cells infected by CPMV and SBMV. Table 5 shows the results of the addition of several salts to CPMV-inoculation medium. Clearly experiment 1 shows that the cationic moiety of the salt largely determines the effect on infection; the type of anion appears to be of little or no consequence. In both experiment 1 and 2 all magnesium salts tested increased CPMV infection by about 40%. This increase may be obtained with as little as 1 mM MgSOn (see Appendix C). Addition of a magnesium chelator (EDTA) dramatically decreased infection, 41 Table 5. The Effect of the Addition of Various Salts to the Inoculation Medium on Percent of CPMV-Infected Protoplasts. Proto— plasts were inoculated with 5 pg CPMV/ml in the presence of 2 pg PLO/ml, 10 mM K phosphate buffer, pH 6.3 and ammendments as listed. Protoplasts were harvested 48 hours after inoculation and stained with fluorescent antibody to determine percent of CPMV—infected protoplasts. 42 Table 5 Experiment 1. Inoculum Percent Fluorescent Amendments Protoplasts none 18% 5 mM Mg(N03)2 24% 5 mM MgCl2 26% 5 mM MgSOu 22% 2.5 mM MgSOu + 2.5 mM MgEDTA 8% 5 mM MgEDTA 6% 5 mM NiCl2 0% 5 mM CaSOu 65% 5 mM CaHu(POu)2, H20 55% 5 mM NaNO2 17% 5 mM KNO3 5% 5 mM LiCl 5% Experiment 2. Inoculum Percent Fluorescent Amendments Protoplasts none 11% 1 mM MgSOLl 15% 0.5 mM CaCl2 62% 1 mM CaC12 57% 1 mM CaSOu 64% 0.25 mM Ca012 + 1 mM MgSOu 45% 0.5 mM CaCl2 + 1 mM MgSOu 64% 1 mM CaCl2 + 1 mM MgSOu 67% 1.5 mM CaCl2 + 1 mM MgSOu 66% LS salts, half strength media concentration 60% 43 2+ ions were added to the inoculum. A second even when extra Mg divalent cation-containing salt, NiC12, completely inhibited infec- tion by CPMV. All monovalent cations tested were also not effective at stimulating infection. Na+, K+ and Li+ ions either did not affect percentage of infection or were quite inhibitiory. Ca2+ ions, how- ever, caused a dramatic increase in infection. Both experiments showed that various concentrations of calcium salts stimulated between 57 and 65% infection. In the presence of 0.5 mM CaCl 62% infection 2: occurred, nearly a 6-fold increase over the non-amended control. To investigate the possibility that a combination of stimulatory salts might allow an even higher percent of infected cells, magnesium and calcium salts were mixed in various proportions in the inocula- tion medium. Experiment 2 in Table 5 shows the results. Infection stimulated by CaCl2 was not significantly further increased by the addition of 1 mM MgSOu. Also, the combination of salts present in soybean culture medium was tested for its stimulatory ability. Linsmaier and Skoog (LS) salts (Linsmaier and Skoog, 1965) at 0.5 media strength contains 10 mM NHuNO3, 9 mM KNO 1.5 mM CaClZ, 0.75 3, mM MgSOu and 0.6 mM KH2P04’ This mixture stimulated 60% infection, which was not an increase in percentage Over that obtained with CaC 2 alone. In fact, no combination of salts tested was significantly more effective at stimulating CPMV infection than 0.5 mM CaC12. However, a disadvantage of using only CaCL2 in the inoculum is the tendency for debris to accumulate with the protoplasts. When Ca2+ ions are added to the inoculum, cell membranes from broken protoplasts tend to clump and pellet with the protoplasts. Although 44 this phenomenon does not appear to affect viability of protoplasts or virus proliferation, this type of preparation may not be suitable for some studies unless a purification step is added. This problem may also be alleviated by inoculating in the presence of a combina- tion of salts, such as those in the LS medium. The membrane-aggrega- tion phenomenon was considerably lessened when several salts were combined. Infection by SBMV is also dramatically affected by the addition of divalent cations (Table 6). However, the stimulatory effects of magnesium and calcium appear to be additive, having 6% infection with MgSOLl alone, 19% with CaCl2 alone and 30% when they are in combination. These two salts appear to account for the stimulatory effect of LS salts on percent of infection. Table 6. The Effect of the Addition of Various Salts to the Inoculation Medium on Percent of SBMV-Infected Protoplasts.* Inoculum Percent Fluorescent Amendments Protoplasts None 3% 1 mM MgSOu 6% 1 mM Mg(NO3)2 6% 1 mM CaCl2 19% 1 mM MgSOll + 0.5 mM CaC12 30% LS salts, one tenth media concentration 30% LS salts, half media concentration 32% 45 *Protoplasts were inoculated with 5 ug SBMV/ml in the presence of 2 ug PLO/ml, 10 mM Tris-HCl buffer, pH 8.0 and amendments, as listed. (4) Virus Concentration. A study was undertaken to determine the effect of virus concentration on the percent of protoplasts infected in the presence of various salts. Figure 9 shows curves of CPMV concentration vs. percent fluorescence without additional salts and in the presence of 0.75 mM MgSOu, 0.5 mM CaCl2 or LS salts, 0.5 media strength. Without salts, infection reached a maximum at 1 ug CPMV/ml and never rose above 15%. MgSOLI generally increased percent of infection, reaching a maximum of 40% around 1 ug CPMV/ml. Addition of 0.5 mM CaC12, however, increased infection to over 70%. At 0.1 ug CPMV/ml, infection was already 52% and maximum infection occurred at 0.5 ug CPMV/ml. The addition of LS salts at 0.5 media strength generally had the same effect as did CaCl2 alone. SBMV infection was much less efficient than CPMV infection (Figure 10). Percent of infection rose slowly and did not level off until 2 to 2.5 ug SBMV/ml. Infection was actually maximal at much higher concentrations, although no attempt was made to increase the virus concentration above 10 ug/ml. (5) Additional Calcium Effects. Calcium has been shown to be highly stimulatory to virus infection, however this effect is definitely dependent on when the protoplast and/or virus is exposed to the calcium ions. Table 7 shows the results of an experiment where CaCl2 was included in the preinoculation sorbitol wash, the inoculation medium, the post inoculation sorbitol wash, or a com- bination of the three. When the standard prewash sorbitol-post wash I 46 .cowuoomcfi >Zmo no pcoonma mcHSLopon on weaponwpcm acoomoLOSHm npfiz umcwmum cam cofiumasoocH Leona mason w: coumm>nmn one: mummHQODOLm .Naomo 28 m.o Avv pew Apxmp oomv muamm wooxm new LmHmEmCHA newcotpm waves «Hm: on “zomwz 28 m>.o ADV “mpamm Hmcoflpflucm 0: Amy usswnme coaumHSOOCH on» on venom one: mucosucmem wcwzoaaom one .>zmo mo mcoflumnpcmocoo macanm> new .m.o ma .memsn oumcawoca x 28 OF .Hs\oqm m: N uo mocomona on» Ca umpwHDOOCH one: mummHQOponm .mpamm macatm> mo oocommna map :H pmpommcfl mummHQOponq mo mwmpcoonmq co coflumzpcoocoo >2mo no pommmo one .m enamHm m wgzmwm a? sis—.528 is 47 .v 0 N main 2— .0 11011 as”: .__ 1011 3.8 3 .o Lil-h ||||||| .u IIIIIII *\ ‘l'nn-r SlSV'IdOlOHd 11130831101113 % 48 .mpwmaQOpona uopoomnfi1>zmm no owmunoonoa on» onflsnouon on zoonflnnm pcoomonosam nuflz nonflonm one noflpmazoocfl noumm mason w: uoumo>nmn onoz mummagononm .>zmm no mnowuonunoonoo mSOHnm> unm Apxou oomv mpamm mooxm use LonEman nnwnonpm «Hon .o.m ma .Loumsn Homumflne Es OF .He\onm ms N no oonomona on» 2H nonmasoonfi onoz mummHQononm .nonoomnfl mummfiaonona no omonnoonoa no coanmnnnoonoo >zmm mo noommo one .0? onswflm 49 SlSV1d0101ld 11130531101113 % 1O 31111111 couctmmou, ”/1111 Figure 10 50 sorbitol plus CaCl2 was used, familiar results were obtained; with 032+ in the inoculum, 63% infection was obtained, and 12% when no Ca2+ was included. However, when 10 mM CaCl2 was included in the re-inoculation wash medium, only 31% infection was obtained from the calcium-containing inoculum, representing only 1/2 of that ob- tained with no pre-wash. When no CaCl2 was in the inoculum, infec- tion actually increased slightly when cells were washed with CaCl2 prior to inoculation. The post-wash affected neither the percent of cells infected when calcium was present nor that when calcium was absent. However under these conditions, calcium was definitely affecting the proto- plasts. When CaCl2 was included in the wash medium, protoplasts clung tenaciously together in a slime-like mass, and became extremely hard to resuspend. No calcium pre-wash experiments were done with SBMV. 51 Table 7. Effect of CaCl2 on Infection of Protoplasts by CPMV when present in the Pre-Inoculation Wash, Inoculation Medium or Post- Inoculation Wash. Pre-wash Inoculum Post-wash Percent Fluorescent Amendment Amendment Amendment Protoplasts None None 10 mM CaCl2 12% None 0.5 mM CaCl2 10 mM CaCl2 63% 10 mM CaCl2 None 10 mM CaCl2 20% 10 mM CaCl2 0.5 mM CaCi2 10 mM CaCl2 31% None 0.5 mM CaCl2 None 65% None None None 10% Protoplasts were washed with either 0.3 M sorbitol or 0.3 M sorbitol plus 10 mM CaCl2 and inoculated with 5 ug CPMV/ml in the presence of 2 ug PLO/ml, 10 mM K phosphate buffer, pH 6.3. The inoculum medium was amended with 0.5 mM CaCl2 or not, as indicated. After inoculation, protoplasts were washed with 0.3 M sorbitol or 0.3 M sorbitol plus 10 mM CaClZ. Protoplasts were harvested 48 hours after inoculation and stained with fluorescent antibody to determine per- cent of CPMV-infected protoplasts. (6) Temperature of Inoculation. A study was made to determine percent of infected protoplasts vs. temperature of inoculation. Tubes of inoculation medium were equilibrated in water baths to temperatures between 0 C and 35 C after the 10 minute PLO—virus incubation period. Protoplast pellets were briefly chilled or warmed 52 in the water baths prior to inoculation. Results are given in Figure 11. Percent infection was lowest at 0 C and rose sharply to about 12-13 C. After this temperature, increase in percent infection occurred only slowly with further increase in temperature. Two distinct lines may be drawn intersecting at 12-13 C. Between 0 C and 12—13 C, percent of infected protoplasts increased 4.4 per degree; after 12-13 C, an increase of only 0.45 percent infection per degree was observed. 53 Figure 11. Effect of temperature of inoculation on percent of proto- plasts infected with CPMV. Protoplasts were inoculated with 5 pg CPMV/ml in the presence of 1.5 pg poly-L-ornithine/ml, 10 mM K phosphate buffer and 0.5 mM CaCl2 for 20 minutes at various temperatures. Protoplasts were harvested 48 hours after inoculation and percent fluorescence was determined by staining with CPMV-specific antibody. 54 00 0 00 0 O 98 7 65 4 3 32:32... 5335:: .K. 20' 10' 20° 30° 40° TEMPERATURE ,°C 10° Figure 11 DISCUSSION Protoplasts have been used in plant virus research since the mid 60's. A procedure for the efficient inoculation of tobacco mesophyll protoplasts with tobacco mosaic virus was introduced by Takebe in 1969, and since then many other protoplast-virus systems have been developed. In these studies, protoplasts have been iso- lated almost exclusively from mesophyll tissue. Although development of this system was a great advance over the use of whole plants for virus study, difficulties in achieving sterility, uniformity and reproducibility still plague plant virologists. In an attempt to overcome these problems, we have developed the first workable system, to our knowledge, for the efficient inocu- lation of protoplasts from a new source — tissue culture. This system has many advantages over those using mesophyll protoplasts. First, tissue culture and tissue culture protoplasts may be manipulated under sterile conditions, obviating the necessity for antibiotics which are universally used in mesophyll protoplast culture. This absolute freedom from microbial growth and potentially inhibitory chemicals available through the use of tissue culture-derived proto— plasts is of critical importance to any biochemical study. Secondly, callus protoplasts often regenerate cell walls more readily than protoplasts from leaf (Constabel, 1975). This property, combined with the ability to maintain sterile cultures, allows long-term experiments to be undertaken. 55 56 Perhaps the most important advantages of using tissue culture protOplasts, however, are the ease of maintenance and, particularly, uniformity of tissue. Laboratories using mesophyll protoplasts must cope with many problems inherent in using whole plants as source material. Seasonal, even diurnal fluctuations, variation between plants and parts of plants, variations of exact conditions between labs, etc. can cause considerable non—uniformity of material, and therefore some degree of irreproducibility. Although growth and isolation conditions for the tissue culture protoplast system are not less critical than those for the mesophyll protoplasts, they are less of a source for irreproducibility because growth conditions for callus - light, temperature, growth media, etc. - may be more easily defined and regulated. With regular subculturing, a source of homogeneous cells is continuously available for protoplast iso- lation. ‘We hope that these defined conditions will allow this system to be easily adapted by other laboratories for use in specific virus- protoplast studies. Those conditions which are critical to the success of the soy- bean protoplast-SBMV and -CPMV systems have been examined in this thesis and can be divided into two categories - culture growth and inoculation conditions. Culture Growth. Clearly optimal culture growth is important in the isolation of viable and infectible protoplasts. Three factors are of utmost importance in this regard. 57 (1) Growth rate. A rapid growth rate is desirable for the following reasons. Fast growing cultures have a high proportion of recently divided "young" cells which have more easily hydrolyzed walls than do older cells. This allows protoplasts to be released more rapidly, avoiding prolonged exposure to enzymes which may have deleterious effects on cells, and possibly on virus-receptor sites. (2) Clump Size. Obtaining fine cultures with no more than 20 cell per clump is necessary for the isolation of a high yield of viable cells. Intuitively, smaller clumps with all cells exposed to the enzymes digest faster than do large clumps. Transferring from the top layers of the culture and frequent transfer help to select for fine cultures. (3) Culture Age. The optimal age for isolating protoplasts from culture is 36-48 hours from last subculture, or approximately at the first doubling time when the probability is high that a large population of the cells have recently divided. Culture of more than 48 hours tend to yield less viable protoplasts, and of those that are viable, fewer become infected with virus. This may correspond to the tendency toward increasing resistance to virus infection with age in many whole plants (Matthews, 1970). Inoculation Conditions. Once suitable protoplasts can be iso- lated, other factors become critical to obtaining a high percentage of infection. Four important factors to regulate are, buffer used, presence of a polycation, virus concentration, and salt environment. These factors are all intimately involved in defining the inoculation environment during which infection takes place. 58 (1) Buffer Used. The kind of buffer used, its concentration and pH have been shown, in the case of other protoplaSt systems, to be of critical importance for optimal infection. The most effec- tive buffering conditions depend on the virus and the type of proto- plasts used. Most of these same observations held true for obtaining a high level of infection of soybean protoplasts by CPMV and SBMV. Optimal buffering conditions for these two viruses are very different and perhaps reveal some interesting aspects of infection. CPMV infection: Out of the non-sulfonic acid buffers tried, only phospate was successful at stimulating infection of soybean protoplasts by CPMV. Interestingly enough, citrate was ineffective at stimulating any infection, although CPMV infection of tobacco (Huber, et al., 1977) and cowpea (Hibi 23 gl., 1975) meosphyll proto- plasts reached 75% and 95% respectively when K citrate buffer was used; yet cowpea mesophyll protoplasts were also infectible with CPMV using 0.1 M K phosphate buffer, pH 6.5, buffering conditions similar to those used successfully for soybean-CPMV infection with the soybean system, optimal CPMV infection occurred at pH 6.3 for phosphate buffer. However, MES buffer at pH 6.3 has no stimulatory effect on infection even though this is well within its optimal buffering range. Clearly then, stimulation is not simply a buffering 2r pH phenomenon. It is possible that K phosphate itself is causing a stimulation of infection as well as functioning as a buffer, or that MES is simultaneously buffering yet inhibiting infection. This phenomenon could be investigated through several approaches: ex- amining the effects of MES-phosphate combination buffers; adding 59 potassium ions to MES buffer; using sodium instead of potassium phos- phate buffer, or testing other non-sulfonic acid buffers with pKés around 6.3-6.7. SBMV infection: All buffers tested having high pK values suc- cessfully stimulated SBMV infection of soybean protoplasts at rela- tively high pH values. Buffers used at lower pH values (phosphate, pH 6.5 or citrate pH 5.5) were unsuccessful. Tris-HCl gave the best infection among those high pK buffers tested, and its effectiveness was relatively independent of pH over the range of 7.3 to 8.6. No data was obtained with phosphate at pH 7.2 to 8.6, although it would be interesting to note whether SBMV's inability to infect soybean protoplasts with phosphate buffer is due to the compound or simply the low pH. A higher concentration of Tris-HCl (50 mM) has been used successfully in other systems, (Motoyoshi and Oshima, 1976) and should be tested here. It can be seen, therefore, that infection of soybean protoplasts by CPMV and SBMV differ greatly in buffering requirements between the two viruses. SBMV infection appears to be stimulated at rela- tively high pH values; no infection is obtained with CPMV above pH 7 using Tris or phosphate buffers. CPMV's infection is stimulated by K phosphate buffer and is quite pH critical, while SBMV does not infect soybean protoplasts in the presence of K phosphate at pH 6.3 and its infection in the presence of Tris-HCl buffer is pH independent at the higher pH values tested. These differences may be paritally due to differences in electrophoretic mobility and ion and charge requirements of the two viruses, which will be discussed in the next sections. 60 (2) Presence of Polycation. Long chain polycations, when pre- sent in inoculation medium, have generally been stimulatory or essen- tial to virus infection of protoplasts (Takebe, 1978). It has been suggested that addition of poly-L-ornithine (PLO) to inoculation medium enhances binding of the virus to the protoplasts (Takebe, 1978) by forming a complex with the virus. This complex is then much more likely to become associated with the membrane, possibly at specific sites, and result in infection. Clearly the effective- ness of this interaction and the necessity for the cationic liaison depends on the net negative charge of the virus and on the charge of the membrane, or of specific sites on the membrane. It has been found that cowpea mesophyll protoplasts have been able to be infected with CPMV (Hibi et al., 1975) and cucumber mosiac virus (CMV) (Koike et 31., 1977) to a very high percent without PLO; also some tobacco mosaic virus (TMV) infection occurs in the absence of PLO in cowpea protoplasts (Koike et_§l., 1976). However, CPMV (Huber et al., 1977), CMV (Otsuki and Takebe, 1973) and TMV (Otsuki et al., 1972) infec- tivity in tobacco cells is completely dependent on PLO. Perhaps cowpea mesophyll protoplast membranes, or appropriate portions of the membranes are less negatively charged than corresponding areas of tobacco membranes. Virus charge also apparently plays a role in the interactions. Brome mosaic virus, strain V5 and pea enation mosaic virus have relatively high isoelectric points (Okuno 32 al., 1977; Motoyoshi and Hull, 1974) and their infection of tobacco mesophyll protoplasts (Motoyyoshi et al., 1974d; Motoyoshi and Hull, 1974) does not require 61 PLO. Thus these relatively positively charged particles can overcome the relative negative charge of the membrane or the membrane sites of tobacco protoplasts. With soybean tissue culture protoplasts, infection by both CPMV and SBMV was absolutely dependent on the presence of poly-L-ornithine under the conditions used in this study. CPMV infection: The requirement for PLO in the CPMV-soybean system perhaps could be considered as analogous to the situation found for the tobacco mesophyll-CPMV system. According to the PLO- charge neutralization theory, it could be that both tobacco and soybean protoplasts have membranes or membrane sites that are negative- ly charged, and CPMV, whose isoelectric point is between 3.7 and 4.5, would tend to be discouraged from binding to the highly negative sites unless PLO was present. A less negatively charged membrane, such as is hypothesized with cowpea mesophyll protoplasts, could allow infection in the absence of PLO. It would certainly be inter- esting to determine whether a virus (such as pea enation mosaic virus) with a relatively high isolecetric point would require PLO for infec- tion of soybean protoplasts. In the soybean-CPMV system the levels of 1.5 to 2 ug PLO/ml found optimal for infection are somewhat higher than normal. CPMV infection of tobacco and cowpea mesophyll protoplasts was optimal at only 0.5 ug PLO/ml. SBMV infection: Since SBMV infection of soybean absolutely requires PLO, we may predict, according to the aforementioned theory, that the isoelectric point of SBMV bean strain is relatively low. 62 Electrophoretic data has been presented for several other stains of SBMV and isoelectric points range from 4.5-6.0 (Magdoff-Fairchild, 1967). However, data regarding the bean strain used in this study is not yet available, although we probably may guess that the iso- electric point does not fall much above 6.0. Appropriate isoelectric potential data could help understand this system and play a role in supporting or revising the present theory. (3) Virus Concentration. For virus-protoplast interactions requiring PLO, percent of infected protoplasts generally increases in proportion to the log of the inoculum concentration over a certain range (Otsuki and Takebe, 1973; Otsuki et al., 1974). After the concentration giving the maximum percentage of infection is reached (usually 0.5 to 5.0 ug virus/ml), increasing the amount of virus in the inoculum does not generally increase percent of infection, and usually some protoplasts will not become infected even if excess virus is used. This type of curve, characteristic for mesophyll protoplasts infection, is also observed in CPMV and SBMV infection of soybean tissue culture protoplasts. CPMV infection: Percent of CPMV-infected protoplasts was de- pendent on virus concentration within the range of 0 to 0.5 ug CPMV/ml. Under optimal conditions, maximum infection occurred at 0.5 ug CPMV/ml and higher levels of infection could not be obtained by raising the virus concentration. Based on the average molecular weight for an infectious CPMV particle of 5.5 X 106 daltons, this represents approx- imately 1011 virus particles/ml or 5 X 105 5 particles/protoplast when 2.5—3 X 10 protoplasts/ml were inoculated. This optimum is sub- stantially lower than that found for CPMV-infection of mesophyll 63 protoplasts. In cowpea and tobacco mesophyll protoplasts, 3-4 ug CPMV/ml was needed to obtain maximum infection (Hibi, et 31., 1975) and Huber et al., 1977). SMBV infection: Infection of soybean tissue culture protoplasts was somewhat less efficient with SBMV than with CPMV, and maximum infection required a high concentration of SBMV. At 2-2.5 ug SBMV/ml, maximum percent of infection occurred. Although this percentage was relatively low, the shape of the curve is comparable to infection of viruses in many mesophyll protoplast systems. It is likely, however, that when conditions are optimized that it will be possible to obtain 100% infection of viable cells. (4) Salt Environment. Aside from the information that can be garnered from buffer studies, extremely little is known about the effect of the ionic environment on viral infection on plant proto- plasts. However, studies in several other systems make this aspect of protoplast-virus interactions potentially interesting. In several animal virus studies, ionic strength is of critical importance in early cell-virus interactions (Lonberg-Holm and Philipson, 1974). Monovalent cations in the medium promote maximum attachment of several viruses (Holland and McLaren, 1959; Neurath gt al., 1970). 2+ 2+ have also been shown to increase Divalent cations Mg and Ca attachment rate of Coxsackie A9 and human rhinovirus type 2 to HeLa cells. This stimulation can be inhibited by EDTA (Lonberg-Holm and Karant, 1972)- In whole plant studies there have been some indications that the presence of certain metallic ions can greatly affect the extent of infection. In 1956, Matthews and Proctor showed that when 64 Mg(NO3)2 at 10 mM was included in tobacco necrosis virus inoculum, number of lesions on Phaselus vulgaris cv. 'Black Prince', approxi- mately doubled over the no Mg(NO3)2 control. Ca(NO at 10 mM had 3’2 no effect on lesion numbers when applied alone, although it did partially annul the inhibitory (hypothesized to be chelating) effects of 0.01 N succinic acid. Potassium and ammonium nitrates had no effect on the production of local lesions. Matthews remorses that "With bacterial viruses it is possible to control the concentration of compounds in the medium in which virus establishment occurs. No such control is possible with plant leaves." Certainly the addi- tion of various metal ions in various ionic strengths is a simple matter in protoplast-virus studies. 2+ 2+ One group has reported having studied effects of Ca and Mg on protoplast infection. Furusawa and Okuno (1978) have observed that MgCl2 and CaCl2 at 10 mM inhibit infection of wheat, barley, maize and Japanese radish mesophyll protoplasts with brome mosaic virus. No data or methods were given. However, in the tissue culture system studied here, ionic strength and composition proved to be exceedingly important with SBMV and CPMV. Calcium salts in particular greatly stimulated infection. CPMV infection: Infection of soybean tissue culture protoplasts 2+ 2+ by CPMV was stimulated to different degrees by Mg and Ca . When 1 mM Mg(N03)2, MgSOu or MgCl2 were included in the inoculation medium, infection increased approximately 40% over the control where no divalent cation was added. This stimulation was strongly inhibited by the addition of ethylenediamine tetraacetic acid (EDTA), indicating chelation of the stimulatory cation(s). 65 A more marked, seven-fold increase was obtained when 0.5 mM CaCl2 was included in the inoculum. This is quite a dramatic increase in infection, particularly since the level of Ca2+ ions required is quite low. These salts could be having several effects on the system. (a) These divalent cations (especially Mg2+) could be having an effect by stabilizing the virion or the viral RNA, the net effect being an increased specific infectivity of the population. (b) The effect may be largely at the membrane. Calcium in particular is well known to have manifold effects on fluidity and permeability of the membrane (Hauser et_§l., 1976), on binding sites and a whole host of other phenomena. It is almost 2+ inconceivable that Ca would not have membrane effects in this system. It is highly possible that Ca2+ alters the membrane, perhaps at quite specific sites, allowing it to be more readily accessible to the virus. (0) A third ion effect may be involved here either alone or, likely, in combination with one or both of the above. This is that an effect on the virus-membrane interaction is occurring. It is possible that calcium ions can form a bridge between virus and cell. There is certainly precedence for Ca2+—induced binding between membranes (Kretsinger, 1976), and there is some indication that this type of interaction can occur between cells and macro- molecules (Lonberg-Holm and Philipson, 1974). 66 SBMV infection: Similar phenomena could well be occurring during the SBMV infection of protoplasts. However the observation that this infection appears to be stimulated by a combination of magnesium ions and calcium ions suggests that perhaps virus stabiliza- tion is a substantial factor (Magnesium effect) as well as calcium- mediated membrane or virus-membrane effects. The observation that a pre-inoculation CaCl wash greatly de— 2 creases the calcium-stimulated CPMV infection leads to further spec- ulation. In this case, calcium appears to be having a direct effect on the protoplasts. This could be occurring in several ways: 1) At 10 mM, CaCl2 could be altering the net charge or con- figuration of the hypothesized binding site, making conditions un- favorable for virus adsorption and/or uptake. 2) CaCl2 at 10 mM may be having a profound effect on the fluidity of the membrane. Its addition may have an inhibitory effect on the lateral or vertical movement in the membrane of any receptor, a function which might be necessary for binding (see next section on temperature). 3) A third hypothesis is that it might be essential that the membrane be exposed simultaneously to the virus and to calcium, and calcium pre-treatment inhibits the virus-calcium-membrane interaction necessary for infection. The decrease in infection at low inoculation temperatures is interesting for several reasons. First, it has not been reported for any other protoplast-virus system. Indeed, the reverse has been reported for one system (Alblas and B01, 1977) and no temperature 67 effect has been found for others (Motoyoshi et al., 1974b). Secondly, temperature-sensitivity provides a handle for the study of virus- membrane interactions. In these initial experiments, the transition appears to be rather distinct between the great increase in infection with temperature at low temperatures and a very small increase with temperatures above about 13-15 C. This observation, if it is substan- tiated by further experimental data, would be particularly valuable for understanding virus—membrane interaction. Speculations can be made regarding the basis for this temperature- sensitivity. It has been hypothesized in the case of temperature- sensitive animal-cell interactions (Holland and McLaren, 1959; Lonberg- Holm and Philipson, 1974) that a multi-protein receptor complex is necessary for virus adsorption and penetration. Thus the virus binds weakly to an initial receptor, and a second (or more) receptor must migrate to that binding site and engage in the interaction, forming a complex. This latter migration would of course be temperature- sensitive. Penetration of the virus into the membrane, if it is an active process, would also be inhibited at lower temperatures. Takebe (1978) has hypothesized that virus entry occurs by a pinocytotic mechanism which would be sensitive to low temperatures. SUMMARY A procedure has been described for the efficient infection of soybean protoplasts isolated from tissue culture with two plant viruses. Infection by southern bean mosaic virus requires the presence of poly—L-ornithine, and is stimulated by a combination of magnesium and calcium salts. The infection occurs in the presence of Tris- HCl buffer at pH 8.0 and several other buffers having relatively high pK values. Infection by cOWpea mosaic virus also requires poly-L-ornithine. This infection is stimulated seven-fold by the addition of 0.5 mM CaClZ. Of the buffers tested, only K phosphate was effective at stimulating infection. Infection occurred in a narrow pH range around pH 6.3. The infection could be inhibited by washing the protoplasts with CaCl2 prior to inoculation. Infection was also considerably decreased at temperatures below 10 C. This system is highly suitable for many plant virus-host studies for the following reasons: 1) Sterility may be maintained. 2) Protoplast source tissue is uniform and reproducible. 3) A constant tissue culture source is easily maintained. We hope that these advantages will make this new system valuable for the study of many aspects of plant virology. The calcium- and temperature-dependency of the interaction should provide a probe for further virus—membrane interaction studies. 68 APPENDICES APPENDIX A Soybean callus growth medium study Table A1. Growth of Soybean Callus on Agar Medium Supplemented with Various Hormones Hormonal Increase in Composition (pg/ml) weight (g) 2'4'D Kinetin 2ip 1AA - — - - -5 - — 0.3 — -6 1 0 - - - 352 1.0 0.3 - — 1365 1.0 0.3 — 0.5 1135 1.0 - 0.3 - 1401 1.0 - 0.3 0.5 1355 0.5 0.3 - 5.0 1352 0.5 ~ 0.3 5.0 1277 Callus characteristics no growth no growth very friable, soft callus friable, dry callus same friable, moist callus same very compact, hard crumbly callus same *Sections of soybean hypocotyl were grown on R3 minerals, vitamins and sugars (Table 1) containing 0.8% agar, and supplemented with various combinations of hormones. After 2 months on agar medium, calluses were harvested and wet weights were recorded. 2'4'D alone stimulated some growth, but increased proliferation could be achieved by the addition of a cytokinin. While the addition of 1AA does not appear to further stimulate growth, its presence alters the callus characteristics. R3 medium, which contains 2'4'D, kinetin and 1AA, produced a very compact callus, having small and densely packed cells. R3A medium, containing twice as much 2'4'D and no 1AA, however, stimulated a friable and loosely-growing callus of large (highly vacuolated) cells. The compact callus grown on R3 proved to be most readily adapted to liquid suspension culture. 69 APPENDIX B Further inoculation buffer investigations Several sulfonic acid buffers were tested for their effectiveness in allowing or stimulating infection of soybean protoplasts by CPMV and SBMV. As shown in Table A3, CPMV infection was obtained with several of these compounds. These experiments demonstrate the following points: 1) Infection occurred in the presence of phosphate buffer only if poly-L-ornithine was included in the inoculum, 2) All the sulfonic acid buffers with the exception of TAPS allow some infection with or without poly-L-ornithine. 3) Tricine, a non—sulfonic acid buffer, does not cause CPMV infection under any conditions tested. 4) Percentage infection by phosphate is fairly constant between experiments, but there is considerable variation between percentage of infection in those experiments using sulfonic acid buffers. 5) The pK's of all the compounds used are much higher than 5, so that none of these compounds (with the possible exception of MES) are buffering substantially at the pH used in these experiments. There- fore their stimulatory effect is one other than buffering. Viability (data not shown) was far better for phosphate and tricine than any of the sulfonic acid buffers, especially MES. There- fore, due to buffer toxicity and variation in experimental results, under these conditions the sulfonic acid buffers are not suitable for use for protoplast inoculations. However, variation of pH, buffer concentration, etc. may alleviate these problems and may make their use more desirable. 70 SBMV infection could be stimulated by the sulfonic acid buffer HEPES. Results are shown in Tables A3-A5. Poly-L-ornithine was not essential for infection nor in most cases was significantly stimulatory (Tables A3 and A4). SBMV infection occurred equally well at inoculation temperatures of 4C and 230 (Table A3). From Table A4 it can be seen that lower HEPES pH favored a high percent of infection. Protoplast stability decreased greatly at a pH less than 5 (data not shown). In the presence of 10 mM HEPES, pH 5.0, percent of protoplasts infected increased as the concentration of SBMV in the inoculum was increased (Table A5). This was also found to be the case for HEPES- stimulated CPMV infection (Table A6). No poly-L-ornithine was in- cluded in the inoculation medium in these two experiments. Therefore, it can be concluded from these experiments that the characteristics of sulfonic acid-stimulated virus infection of soybean protoplasts differ dramatically from those observed for phosphate- or Tris—HCl—stimulated infections described in the body of this thesis. The major differences are (1) No poly-L-ornithine is required for sulfonic acid-stimulated infections while a polycation is absolutely essential in obtaining infection with other buffers. (2) Sulfonic acid-stimulated infection is equally effective at 4C and 23C; CPMV infection with PLO and phosphate is temperature sensitive (no data for SBMV). (3) Using phosphate and PLO or Tris—HCl and PLO, satura- tion is reached at a very low virus concentration, while with HEPES, even at 40—50 pg/ml, increasing virus concentration still causes an increase in percent of cells infected. (4) Viability is much 71 poorer with any of the sulfonic acid buffers at pH 5.0 than with phosphate or Tris-H01 at higher pH's. It appears that different mechanisms are involved in those infections occurring with sulfonic acid buffers and those obtained with non-sulfonic acid buffers. It is possible that the sulfonic acid buffers may be allowing infection simply by injuring the membrane and permitting entry of the virus particles, while the function of other less toxic buffers is to provide an environment which allows a more complex virus-cell interaction. Much more work must be done, however, to reveal the respective roles these buffers play in the infection process. 72 73 .ocwnnfinn0141zaoq mo oonomnm on» ca noflpoomnfl pnoonod mucomonqon mononpnonma :H nonssz wcfinaopm .mnson me no maaoo manumo>non kn vocwanopou mos cowpoomnw unoonom n .oonooooLOSHm pcoonoa Lou wnwcwsoxo new zuonflpno pcoooonosam owmwooqm1>zmo npfis .28 Or am mLGMMSD WSOHLN> .HO oocononq on» 2H Ha\ocwnpacLOIQI>Hoq m:.m psonpfiz no nufiz Hs\>zmo w:.mP nu“: nouoasoocfl onoz mumoHQOponmm Aomvmm 1 1 Apmvom Ammvom 1 1 AmFme 1 Amvmm AmFme 1 1 onm nose 1 Aesop ons_ Pas o_s Armvem Azmvmm nova. nova, as msofinm> no oonomonm A A n 1 APMVm: 1 AmmVPw 1 Ammvm mvw ANVQF ova? onem we we Apavos AFNvmz AmFVPm ono never 0* Amvmm ono me Aoovmm onmF 2* noncomnH mumoHQOponm oanmfl> unoonom Ammvmm Aomvms Amrvmo me Q Aomvzm 1 1 Aomvmm 1 Aosvoa Azovmw 1 Assam 1 ono 1 Amvs. Amvo 1 ono we P* ncosflnomxm o.m o.m o.m o.m o.m o.m o.m o.m o.m o.m o.m m.m mm wnommsm coflumazoonH on» Ca ocwanCLO141>Hom usonuflz Lo npfl3 mummaaouOLm mo 20auooQCH >Zmo ommHQ Ommmmm mmmmm mm: mum ommoz mmoz omm¢e mm2mm no“: mnwnflopm .mnson an no mumoaq nopona mcflpmo>non an conHSLouon mos noncomnfl onmoaaouona mo unoonom .Lommsn mmmmm no moonomonq on» :H Aonmv Ha\ocfinuHcL01numH0Q w: o.P psonpflz no npflz Hs\>zmm m: 0.09 no m.w new: oopoasoocw ono: mummaaouonmm Pm mm o._ c.09 am mm o o.o_ mm a o.P o.mF m: a o o.o_ mm . mm 0., m.m mm mm o m.m h: z 0.9 m.w om : 0 Wm noncomnfl mnmoflqouona nOHpoasoonH mo Aas\mzv coauonpnoonoo AHe\mnv cowpmnunoonoo oHnoH> unoonoa onsuonoasop onm >zmm «nommsm mmmmm.ao oonononm onn CH mnowuwcnoo coapoasoonH mSOHno> nouns ouooaaononm mo coauoomcH >2mm .m< oHan 75 Table A4. SBMV Infection of Protoplasts in the Presence of HEPES at Various pH's* Percent Viable Protoplasts Infected pi nun. m 4.8 36 44 5.2 21 32 5.8 10 39 6.0 3 0.5 6.4 0.5 0.5 7.2 0.5 0.5 7.6 0.5 0.5 *Protoplasts were inoculated with 10 pg SBMV/ml with (+) or without (—) 2 pg poly—L-ornithine (PLO)/ml in the presence of 10 mM HEPES buffer at various pH's. Percent of protoplasts infected was deter- mined by harvesting protoplasts 48 hours after inoculation, staining with SBMV—specific antibody and examining for percent fluorescence. 76 Table A5. SBMV Infection of Protoplasts in the Presence of HEPES Buffer Using Various Virus Concentrations’ SBMV percent viable concentration (pg/ml) protoplasts infected 0 0 1 5 2 13 5 21 1O 28 15 28 2O 45 30 52 40 56 50 61 *Protoplasts were inoculated at 4°C with various concentrations of SBMV in the presence of 10 mM HEPES buffer, pH 4.8. Percent infected protoplasts was determined by harvesting protoplasts 48 hours after inoculation, staining with SBMV-specific fluorescent antibody, and examining for percent fluorescence. 77 Table A6. CPMV Infection of Protoplasts in the Presence of HEPES Buffer using Various Virus Concentrations“ CPMV percent viable concentration (pg/ml) protoplasts infected 0 0 1 1 5 2 10 9 15 26 20 28 30 40 *Protoplasts were inoculated at 23°C with various concentrations of CPMV in the presence of 10 mM HEPES buffer, pH 5.0. Percent infected protoplasts was determined by harvesting protoplasts 48 hours after inoculation, staining with CPMV-specific antibody, and examining for percent fluorescence. APPENDIX C Magnesium concentration in the inoculum and its effect on CPMV infec- tion of protoplasts Table A7. Effect Of M8(N0 ) on Infection of Protoplasts when Present in the Inoculation Medium at Various concentrations'1 M8(NO3)2 % fluorescent concentration protoplasts 0 8 0.5 17 1.0 30 2.0 - 30 3.0 30 5.0 36 10.0 33 *Protoplasts were inoculated with 5 ug CPMV/ml in the presence of Z‘pg poly—L-ornithine/ml, 10 mM K phosphate buffer, pH 6.3 and various concentrations of Mg(NO )2. Protoplasts were harvested at 48 hours and percent fluorescenc was determined by staining with fluorescent antibody to CPMV. It can be seen from Table A7 that infection is maximally stimulated at 1 mM Mg(N03)2. Raising the concentration past this level does not further stimulate infection. 78 LITERATURE CITED LITERATURE CITED Alblas, F. and J.F. B01. 1977. Factors influencing the infection of cowpea mesophyll protoplasts by alfalfa mosaic virus. J. Gen. Virol. 36:175-85. Bachtold, J.G., H.C. Bubel, L.P. Gebhardt. 1957. The primary inter- action of poliomyelitis virus with host cells of tissue culture origin. 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