Mechanical wounding of potato tubers induces replication of potato virus S

Physiological and Molecular Plant Pathology (1996) 49, 33–47

Mechanical wounding of potato tubers induces replication of potato virus S J. K. M and M. E. V* Department of Biochemistry, Microbiology and Molecular Biology, UniŠersity of Maine, Orono, ME 04469-5735, U.S.A. (Accepted for publication February 1996)

The replication of potato virus S in potato tuber tissue was induced by wounding. An acidic 32 kDa polypeptide accumulated in the polysome fraction of tubers upon wounding, concomitant with a transient increase in translational activity which is an integral component of the tuber wound response. We isolated and identified this polypeptide as the potato virus S coat protein. Immunoblot analyses indicated that the coat protein increased 2- to 20-fold in tubers within 4 h of wounding. Northern blot analyses showed a parallel increase in potato virus S genomic RNA during the same time frame. Both potato virus S RNA and coat protein were barely detectable in tubers before wounding. By contrast, the significant amounts of coat protein detected in leaf and stem did not increase further upon wounding or treatment with abscisic acid or methyl jasmonate. The 32 kDa polypeptide was not detected in seed tubers certified as virus-free. The observation that potato virus X and potato virus Y were also induced 5- to 10-fold within 4 h of wounding led us to conclude that the induction of potato virus S in tubers resulted from the transient release from dormancy and general stimulation of tuber metabolism that occurs upon wounding. Wound-induction of potato virus S, potato virus X and potato virus Y may have implications on potato field practices or provide greater sensitivity in immunological screening of seed tubers for viruses. # 1996 Academic Press Limited

INTRODUCTION

Potato tubers present a complex, biphasic response to mechanical wounding that includes a general stimulation in macromolecular activity in this quiescent organ. Within 30 min to 4 h after wounding, mRNAs and proteins accumulate that are involved in phenylpropanoid metabolism [5, 7, 11, 13, 22, 53, 56 ], isoprenoid metabolism [8, 10, 33, 44, 60 ] and suberization [26, 37 ], including phenylalanine ammonialyase, DAHP synthase, HMG CoA reductase and polyphenol oxidase. During this initial period, the abundant mRNAs encoding patatin and proteinase inhibitor II are degraded, indicating operation of a mechanism for selective translation of woundresponse mRNAs [7, 11 ]. A secondary response is evident 12–24 h after wounding that leads to formation of a wound periderm and includes DNA synthesis [7, 12 ], cell *Author to whom correspondence should be addressed : Dr. Michael E. Vayda, Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, 5735 Hitchner Hall, Orono, ME 04469-5735, U.S.A. Abbreviations used in text : ELISA, enzyme-linked immunosorbent assay ; ISMV, iris severe mosaic potyvirus ; NEpHGE, non-equilibrium pH gel electrophoresis ; PVS, potato virus S ; PVX, potato virus X ; PVY, potato virus Y ; SDS-PAGE, SDS-polyacrylamide gel electrophoresis ; TCA, trichloroacetic acid. 0885–5765}96}070033­15 $18.00}0

# 1996 Academic Press Limited

34

J. K. Morelli and M. E. Vayda

division [2 ] and induction of gene products associated with these processes such as the hydroxyproline-rich cell wall protein extensin, the glycine-rich cell wall protein and histones [7, 11, 12, 38 ]. Notable increases in translational activity accompany both phases of the wound response as is evident by greater incorporation of $&S-methionine into TCA-precipitable polypeptides by wounded tubers than non-wounded tubers [31, 55, 56 ]. Our present data indicate that the general stimulation of tuber macromolecular metabolism upon wounding leads to enhanced replication of contaminating viruses, specifically potato virus S (PVS). PVS is a common, aphid-transmissible carlavirus which infects members of the Solanaceae and Cheonopodiaceae. PVS infection alone is asymptomatic, even in high titres, however, PVS synergistically exacerbates the severity of infection by other viruses, such as potato virus X (PVX) and potato virus Y (PVY). Accordingly, reductions in potato yield of 10–20 % have been attributed to PVS infection when in combination with other viral agents [19 ]. PVS virions are filamentous particles with dimensions of 650 nm¬12 nm [24, 27 ]. The genome is comprised of a single-stranded, positive-sense, 7±5 kb RNA molecule with a relative mobility of 2±39¬10' Da [29 ]. The 32±5 kDa coat protein is encoded within an open reading frame located near the 3« end of the genomic RNA [27 ]. Two low abundance, subgenomic RNAs of 2±5 kb and 1±3 kb have been observed which map to the extreme 3« terminus of the viral RNA genome, but it is the 1±3 kb subgenomic RNA which is the apparent template for translation of the 32±5 kDa coat protein, at least in Šitro [17, 30, 50, 51 ]. The 5« terminal segment of the 1±3 kb subgenomic RNA is thought to act as a translational enhancer for expression of the coat protein [50, 51 ]. Addition of this 101 nucleotide segment immediately upstream of the coat protein sequence to a reporter construct greatly enhances translation of that message both in Šitro [50 ] and in ŠiŠo [51 ]. Crosby and Vayda [11 ] noted that a 32 kDa protein accumulated in the polysome fraction of tubers within 4 h of wounding. The appearance of this polypeptide coincided with the dramatic increase in protein synthetic activity in tubers. Polyribosomes isolated from wounded tubers exhibit greater activity in run-off translation assays than do polysomes from non-wounded tubers [11 ] indicating that the increased protein synthesis is due, at least in part, to enhancement of the translational machinery. The accumulation of the 32 kDa polypeptide in the polysome fraction of wounded tubers led us to surmise that this protein might play a role in the woundinduced activation of protein synthesis or selective translation of wound-response mRNAs. To the contrary, we have identified the 32 kDa polypeptide as the PVS coat protein. In this paper we provide evidence that viral replication occurs during woundinduced stimulation of tuber macromolecular metabolism. MATERIALS AND METHODS

Plant material Mature potato tubers (Solanum tuberosum cv. Russet Burbank) were obtained from the Maine Agricultural and Forest Experiment Station Aroostook Farm, purchased in a local market, or harvested from greenhouse-grown plants at the University of Maine. Certified virus-free seed tubers, and PVS, PVX and PVY infected plants were provided by Dr. Feridoon Mehdizadegan and the Maine Seed Potato Board. After harvest,

Induced replication of potato virus S

35

tubers were stored in the dark for 2 weeks at room temperature, then stored at 4 °C until needed. Tubers were rinsed briefly with warm soapy water and surface sterilized with 5 % hypochlorite (v}v), dried and allowed to equilibrate at room temperature for 16 to 24 h prior to experimentation. A 3 g, cross-sectional sample of a tuber was removed using either a 1 cm diameter cork borer or a scalpel, and immediately frozen in liquid nitrogen. The remainder of the tuber was wounded by insertion of a P-200 micropipette tip to a depth of 3 cm. Multiple wounds were inflicted throughout the tuber, spaced approximately 0±3 cm apart. Three gram samples of the tissue surrounding and including the wound sites were removed at the times indicated in the Figures. Nonwounded and wounded samples of each experiment were taken from the same tuber. Although the absolute amount of 32 kDa protein present varied among tubers, the relative increase observed upon wounding was consistent in repeated trials. Tuber phosphoproteins were labelled by incorporation of $#P-orthophosphate as described by Crosby and Vayda [11 ]. Polysome isolation Polysomes were isolated from tubers using a modification of the procedures described by Crosby and Vayda [11 ], Strommer et al. [45 ] and Vayda [54 ]. A 3 g core of tuber tissue was removed as described above, frozen in liquid nitrogen and ground in a Miracle Mill (Markson Scientific). Tissue was thawed in 10 ml of polysome buffer comprised of 200 m Tris-HCl pH 9±0, 400 m KCl, 60 m magnesium acetate, 50 m EGTA, 250 m sucrose, 0±01 % Triton X-100 and 15 m β-mercaptoethanol, then clarified by centrifugation at 15 000 g. The supernatant was centrifuged through a 1±5  sucrose cushion with the same buffer in a Beckman 70.1 Ti rotor at 45 000 g for 4±5 h at 4 °C. The resulting pellet was resuspended in 200 µl of diethylpyrocarbonatetreated dH O. Insoluble material was removed by centrifugation in an Eppendorf # microcentrifuge at 12 000 g for 5 min at 4 °C. Pilot samples were further fractionated by centrifugation through a 5 % to 20 % linear sucrose gradient [11 ] to confirm the presence of oligomeric ribosomes. Large scale preparation of polysomes for bulk isolation of the 32 kDa polypeptide was accomplished by parallel extraction of twenty 3 g cores from 4 h wounded tubers. Two-dimensional gel electrophoresis Two dimensional resolution of polypeptides was accomplished using the methods of O’Farrell et al. [34 ], Dunbar [14 ] and Sanders et al. [41 ] with the following modifications. Non-equilibrium pH gel electrophoresis (NEpHGE) gels (9  urea, 5 % 3–10 carrier ampholytes (Serva, Pharmacia), 3±5 % : 0±2 % acrylamide : bis-acrylamide (US Biochemicals), 0±05 % ammonium persulfate, 0±05 % TEMED), were prepared in glass tubes with 2 mm internal diameter and polymerized overnight at room temperature before use. A 200 µl sample of each polysome sample was lyophilized using a Speed-Vac (Beckman) and resuspended in 65 µl of sample buffer containing 300 m NaCl, 1 m EDTA, 1 m EGTA, 2 % Triton X-100, 5 m ascorbic acid, 100 m dithiothreitol, 1±6 mg l−" protamine sulfate, 20 µg ml−" leupeptin and 4 % 3–10 carrier ampholytes (Serva, Pharmacia), and incubated at room temperature for 30 min. Thirty-five micrograms of urea were dissolved in each sample in a subsequent 30 min incubation at room temperature and samples were clarified at 40 000 g for 30 min at

36

J. K. Morelli and M. E. Vayda

23 °C. The supernatant was loaded to a NEpHGE gel in a Protean II apparatus (BioRad). Electrophoresis of proteins was from anode to cathode at 300 V constant voltage for 4 h, using 0±01 N phosphoric acid as the anode buffer, and 0±02 N NaOH as the cathode buffer. The run was monitored by observing the progression of 10 µg cytochrome c through a reference gel. Tube gels were removed and layered on top of a 15 % SDS-acrylamide gel with 1 % melted agarose in SDS-PAGE running buffer. Following SDS-PAGE, gels were either stained with Coomassie blue, silver stained or dried for autoradiography. Purification of the 32 kDa protein Polysomes were isolated from 4 h wounded tuber tissue as described above, and polypeptides were resolved by NEpHGE and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The 32 kDa region was transferred electrophoretically to an ImmobilonP membrane (Millipore) and stained with Ponceau S to identify the polypeptide induced upon wounding. The spot was excised, rinsed in dH O to remove stain, and # the polypeptide was eluted with 4  guanidine-HCl, 0±1 % Triton X-100 (Sigma) as described by Petterson et al. [35 ]. The eluted protein was dialyzed against 5 m ammonium carbonate and lyophilized. Samples of the purified polypeptide were used for polyclonal antiserum production in rabbits or linked to an agarose matrix (Hydrazine AvidGel, Bioprobe International) for affinity purification of rabbit antisera [21 ]. Protein gel blot analysis Immunoblots were prepared essentially as described by Towbin [48 ] and Harlow and Lane [21 ]. Briefly, polypeptides resolved by SDS-PAGE were transferred to a HybondC nitrocellulose membrane (Amersham) at 85 V for 45 min in 25 m Tris base, 192 m glycine and 15 % methanol with a mini-Trans-blot cell (Bio-Rad). Membranes were blocked in 25 m Tris-HCl pH 7±4, 500 m NaCl, 0±1 % Tween-20 containing 0±5 % BSA, and incubated for 1 h in blocking buffer containing a 1 : 10 000 dilution of affinity-purified anti-32 kDa antisera. Blots were incubated subsequently with goat anti-rabbit IgG conjugated to horseradish peroxidase (Sigma). Detection of bound second antibody was achieved using the ECL chemiluminescent kit (Amersham). Amino acid sequence analysis Polypeptides in the polysome fraction of a 4 h wounded tuber were resolved by NEpHGE}SDS-PAGE and transferred to an Immobilon-CD membrane (Millipore) using a semi-dry blotting apparatus (Millipore SDE). The spot corresponding to the induced 32 kDa polypeptide was identified by the Millipore reverse staining kit, excised and submitted to the Harvard Microchemistry Facility (Cambridge, MA, U.S.A.) for further analysis. The polypeptide was digested with trypsin and the two largest tryptic peptides were sequenced by automated Edman degradation. RNA gel blot hybridization Total RNA was isolated from 3 g cores of potato tuber tissue frozen in liquid nitrogen, ground to a find powder using a Miracle Mill and immediately extracted using the phenol method described previously [9, 40, 43 ]. Total RNA was resolved by

Induced replication of potato virus S

37

formaldehyde agarose gel electrophoresis, transferred to Hybond-N (Amersham) and probed by gel blot hybridization using methods described previously [9, 11, 40 ]. The PVS genomic cDNA generously provided by Dr. G. A. De Zoeten is described by Monis et al. [29 ]. A 1±6 kb fragment containing the coat protein gene sequence was obtained by digestion with EcoRI. The insert encoding a segment of potato 28S rDNA [9, 12 ] was isolated by digestion of the construct pR101 with EcoRI. Restriction fragments were isolated from agarose gels using the Gene Clean II system (Bio 101, Inc). Probes were labeled using α$#P-dCTP by the random primer method (Boehringer Mannheim).

Sandwich ELISA assay The abundance of PVX, PVY and PVS antigen in wounded tubers was assessed using PathoScreen Sandwich Immunoscreening Kits (Agdia Inc., Elkhart, IN, U.S.A.) specific for each of these viruses. A 0±5 g cross-sectional sample of tuber tissue was removed prior to wounding, or 4 h after wounding, and immediately frozen in liquid nitrogen. The samples were ground to a fine powder using a mortar and pestle, and thawed in 1 ml of extraction buffer containing phosphate buffered saline pH 7±4, 2 m PMSF, 10 µg ml−" leupeptin, 50 m EGTA and 10 m β-mercaptoethanol. Insoluble material was removed by centrifugation at 12 000 g for 5 min at 4 °C. Ten micrograms of soluble protein was made up to 100 µl in phosphate buffered saline containing 0±1 % Triton X-100 provided with the kit, and incubated overnight in microtitre plate wells coated with PVX-, PVY- or PVS-specific antibody. After sample removal, wells were rinsed five times with phosphate buffered saline containing Triton X-100. 100 µl of the second antibody, diluted 1 : 1000, was incubated in each well for 2 h at 23 °C. This second antibody consisted of either PVX-, PVY- or PVS-specific antibody conjugated to alkaline phosphatase. After removal of the second antibody, wells were washed five times with phosphate buffered saline containing Triton X-100, then incubated with 100 µl of 1 mg ml−" p-nitrophenyl phosphate substrate for 1 h at 23 °C. Colour development was terminated by the addition of 50 µl 3  sodium hydroxide, and its absorbance measured at 405 nm using a Titrex Multiskan PLUS plate reader (Flow Laboratories, McLean, VA, U.S.A.).

RESULTS

An acidic 32 kDa polypeptide is induced by wounding tubers The results shown in Fig. 1 confirmed the observation of Crosby and Vayda [11 ] that a 32 kDa polypeptide accumulated in the polyribosome fraction of wounded tubers. The polysome fraction contained ribonucleoprotein complexes that were isolated from tissue homogenates based upon their sedimentation through sucrose in the presence of 200 m KCl, their ability to serve as templates for run-off translation in Šitro [3, 4, 11, 54 ], and their sensitivity to RNase A. To identify the induced species, polypeptides in the polysome fraction of non-wounded (Fig. 1A) and 4 h wounded (Fig. 1B) tubers were resolved by two-dimensional NEpHGE}SDS-PAGE analysis. Relative to reference ribosomal proteins (L2 and S6 in Fig. 1A and B), only one

38

J. K. Morelli and M. E. Vayda 4

6

pI

9 12

(A)

35

*

L2

kDa

S6

27

18

(B) 35

*

L2 S6

kDa 27

18

(C)

35 kDa 27

F. 1. Two-dimensional resolution of polysome-associated proteins. Polypeptides present in the polysome fraction of non-wounded tubers (Panel A) or the same tuber 4 h after wounding (Panel B) were resolved by NEpHGE and SDS-PAGE and stained with Coomassie blue. (Panel C) Polypeptides in the polysome fraction of a 4 h wounded tuber were resolved by NEpHGE}SDSPAGE, electrophoretically transferred to Hybond-C (Millipore), and probed using affinitypurified anti-32 kDa rabbit antiserum. Presence of bound antibody was detected using a horseradish peroxidase conjugated second antibody and Western Blue substrate (Promega). The pH of the first-dimension NEpHGE gels and mobility of SDS-PAGE molecular markers (BioRad) is indicated.

Induced replication of potato virus S

39

polypeptide (*) was more abundant in 4 h wounded tuber polysomes than those from non-wounded tubers. The induced polypeptide exhibited an apparent pI of 5±5–6±0, and thus, was one of the few proteins in the polyribosome fraction that displayed an acidic pl. The acidic 32 kDa polypeptide was isolated from preparative NEpHGE}SDSPAGE electroblots for production of antisera in rabbits. Figure 1C demonstrates that the affinity-purified polyclonal antiserum reacted exclusively with the acidic 32 kDa species. Although not readily detected in Coomassie blue stained SDS-PAGE gels (Fig. 2A), the immunoblot shown in Fig. 2B clearly demonstrates that minute amounts of the 32 kDa polypeptide were present in the polysome fraction of non-wounded tubers. The abundance of the acidic 32 kDa protein increased an average of 6-fold within 4 h after wounding ; this induction varied 2- to 20-fold in various experimental trials. Immunoblots showed that sprouting tubers consistently contained higher amounts of the 32 kDa polypeptide than tubers at harvest, or stored post-harvest for 1–5 months at 4 °C (data not shown). The induction of this polypeptide, in parallel with observed increases in translational activity [31, 55 ] led us to question whether this protein was a component of the translational apparatus or a regulator of translational activity. Acidic ribosome-associated phosphoproteins have been proposed as regulators of eukaryotic protein synthesis [28, 39, 49, 59 ]. N

W

(A)

35 kDa 27

18

(B) 35 kDa 27 F. 2. Accumulation of the 32 kDa polypeptide after wounding. (Panel A) Polypeptides in the polysome fraction of a non-wounded tuber (Lane N) and that same tuber 4 h after wounding (Lane W) were resolved by SDS-PAGE and stained with Coomassie blue. (Panel B) A duplicate gel of the same samples was transferred to Hybond-C (Amersham), incubated with affinitypurified anti-32 kDa antiserum, and visualized using the ECL system (Amersham). The mobility of known polypeptide molecular weight markers is indicated.

40

J. K. Morelli and M. E. Vayda

Crosby and Vayda [11 ] reported that the induced 32 kDa species is phosphorylated. In contrast the dimensional analysis revealed that the vast majority of $#Porthophosphate incorporated into polysomal proteins of wounded tubers was present in a single 31 kDa species with a pI of 9±0 (Fig. 3). This phosphoprotein was identified as ribosomal protein S6 by virtue of its basic isoelectric point and mobility relative to other peptides in the characteristic two-dimensional pattern of ribosomal proteins [14, 42 ]. This result was consistent with observations of S6 phosphorylation in other plant species [1, 15, 42 ]. Ribosomal protein S6 becomes phosphorylated at multiple sites in a wide variety of organisms when translational activity is stimulated [32, 46, 47, 57 ] although neither the role of, nor requirement for, S6 phosphorylation in induction of protein synthesis has been established. pI 4 35

6

9 S6

kDa 27

F. 3. Phosphoproteins in the polysome fraction of a 4 h wounded tuber. 0±5 mCi $#Porthophosphate was presented to a tuber 3 h after wounding and incubated for 1 h at 20 °C. Polypeptides present in the polysome fraction were resolved by NEpHGE}SDS-PAGE and stained by Coomassie blue (not shown). Autoradiography of dried gels was performed with Kodak X}AR5 film. pH and molecular weights indicated are relative to known markers.

Several observations suggested that the acidic 32 kDa polypeptide was not involved in translational regulation. Fig. 4 demonstrates that considerable amounts of this polypeptide were detectable by immunobloting in the polysome fraction of potato leaves and stems. However, no increase in the abundance of the 32 kDa protein was observed after wounding potato shoots, stems or mature leaves (data not shown). Further, application of the wound-related hormones abscisic acid and methyl jasmonate did not increase the steady-state level of the 32 kDa protein in either shoot or tuber tissues (data not shown). The highest steady-state level of the 32 kDa polypeptide was found in older leaves (Fig. 4, lane 2) ; young leaves, shoots and flowers, which exhibit higher levels of protein synthesis, contained significantly less of the acidic 32 kDa polypeptide (Fig. 4, lanes 3–6). Translational competence was not affected by addition of affinity-purified anti-32 kDa antisera to wounded tuber polysomes assayed by run-off translation in Šitro (Table 1). These results indicated that neither the presence nor abundance of the 32 kDa protein correlated with enhanced translational activity. Thus, we concluded that increased abundance of the acidic 32 kDa in wounded tubers was the result of increased translational activity, rather than a cause.

Induced replication of potato virus S

41 1

2

3

4

5

6

(A) 35 kDa 27 18

(B)

35 kDa 27

F. 4. Presence of the 32 kDa polypeptide in various potato organs. Polysomal proteins of 2 h wounded tuber (lane 1), fully expanded (third) leaves (lane 2), immature (seventh) leaves (lane 3), lower internodal stem segment (lane 4), upper internodal stem segment (lane 5) and flowers (lane 6) were resolved by SDS-PAGE and stained with Coomassie blue (Panel A) or transferred electrophoretically to Hybond-C (Amersham) and probed using affinity-purified anti-32 kDa antiserum (Panel B). The mobility of molecular weight markers is shown. T 1 Effect of affinity-purified anti-32 kDa antibody on incorporation of $&S-methionine by polysomes in run-off translation assay Template

RNA

Antibody

Incorporation*

Control RNA 4 h wounded tuber polysomes 4 h wounded tuber polysomes 4 h wounded tuber polysomes

2 µg 2 µg 2 µg 2 µg

— — 2 µg 20 µg

189 628³7998 62 258³4296 67 664³1340 67 420³1618

* Incorporation expressed as TCA-precipitable ct min−" recovered after at 30 min reaction at 20 °C [8 ]. Incorporation is the mean³SE for three independent trials.

The acidic 32 kDa polypeptide is the PVS coat protein Partial peptide sequences of the purified acidic 32 kDa polypeptide conclusively identified this species as the coat protein of PVS (Fig. 5). The induced acidic 32 kDa species and a reference 32 kDa ribosomal protein were isolated from two dimensional NEpHGE}SDS-PAGE blots for sequence determination. Direct sequencing of the isolated 32 kDa protein was not possible due to modification of the N-terminus, presumably by acetylation. Therefore, the acidic 32 kDa polypeptide was subjected to partial digestion with trypsin. The sequences of the two largest tryptic fragments obtained by automated Edman degradation are shown in Fig. 5A. These sequences exhibit 100 % identify to adjacent segments of the 32±5 kDa PVS coat protein amino acid sequence, deduced from the cDNA sequence of the Andean strain of PVS [18, 27 ]. The N-terminal sequence of the basic 32 kDa reference polypeptide confirmed this species as ribosomal protein L2, with 98 % identity to the tomato [16 ] ribosomal protein (Fig. 5B). Identification of the wound-induced 32 kDa polysomal protein as the PVS coat protein explained the abundance of this species in older shoot tissues, and the inability of anti-32 kDa antisera to affect protein synthetic activity in Šitro.

42

J. K. Morelli and M. E. Vayda FRAGMENT # 1

A

FRAGMENT # 2

32 kDa protein PVS coat protein B Potato RPL2 Tomato RPL2 F. 5. Amino acid sequence of the 32 kDa polypeptide. Acidic and basic 32 kDa polypeptides were isolated from the polysome fraction of wounded tubers by NEpHGE}SDS-PAGE. (Panel A) The amino acid sequences of the two largest tryptic fragments of the acidic 32 kDa polypeptide are shown aligned with the deduced polypeptide sequence of the PVS coat protein [18 ], GenBank Accession uA48549. (Panel B) The N-terminal sequence of the basic 32 kDa polypeptide aligned with tomato ribosomal protein L2 [16 ], GenBank Accession uP29766. Sequences were identified by BLAST search of the GenBank SwissPro database.

1 (A)

2

3

4

5

6

68 49 kDa 35 27 18

(B)

35 kDa 27

F. 6. Absence of the 32 kDa protein from certified virus-free seed tubers. Soluble protein extracts were made of tubers from PVS-infected plants (lanes 1, 2 and 3) or certified virus-free tubers (lane 4, 5 and 6), either prior to wounding (lanes 1 and 4), 4 h after wounding (lanes 2 and 5) or 12 h after wounding (lanes 3 and 6). Proteins were resolved by SDS-PAGE and stained with Coomassie blue (Panel A) or transferred electrophoretically to Hybond-C (Amersham) and probed with affinity-purified anti-32 kDa antiserum (Panel B). The mobility of molecular weight markers is shown.

Verification that the PVS coat protein was induced upon wounding is presented in Fig. 6. The 32 kDa polypeptide was not present in extracts of seed tubers certified as virus-free by the Maine Seed Potato Board (Fig. 6). Further, mechanical wounding did not induce the appearance of the 32 kDa polypeptide in these tubers (Fig. 6). By contrast, our immunoblot assay demonstrated that the 32 kDa polypeptide was present, and was induced by wounding of tubers from plants which tested positive for PVS in routine ELISA screening of nuclear material (Fig. 6). The observation that the 32 kDa polypeptide was present only in PVS-infected material confirmed that it was the PVS coat protein that we detected in the polysome fraction of wounded tubers.

Induced replication of potato virus S

43

Viral replication in tubers is induced by wounding The northern blot shown in Fig. 7 illustrates that PVS genomic RNA increased rapidly upon wounding of tubers, in parallel to the observed increase in coat protein. The 7±5 kb genomic RNA increased greater than 10-fold within 4 h after wounding, with a further 2- to 4-fold increase by 12 h after wounding. In contrast, only a minor increase was observed in the steady-state level of the barely detectable 1±3 kb subgenomic RNA encoding the coat protein (Fig. 7, panel B). These results indicated that the increase in PVS coat protein was not due to preferential synthesis of the coat protein subgenomic RNA. Preferential translation of this message has been proposed by virtue of its 101 nucleotide non-translated leader which acts as a translational enhancer [50, 51 ]. Binding of this enhancer sequence by the PVS coat protein might explain the presence of the 32 kDa polypeptide in the polysome fraction. In contrast, the rapid and parallel increase in PVS genomic RNA and coat protein in this fraction indicated that viral replication in tubers was stimulated by mechanical wounding. The virion displays a sedimentation coefficient of 150 to 200 S [58 ] and so the presence of the PVS coat protein in the polyribosome fraction of tubers is explained by co-sedimentation of mature PVS virions with oligomeric polyribosome complexes. NW (A)

0.5

2

8

12

24 PVS 7.5 kb

(B) 2.5 kb PVS 1.3 kb (C) 28S rDNA

F. 7. Accumulation of PVS genomic RNA in wounded tubers. Total RNA [7, ,40, 42 ] was isolated from a potato tuber prior to wounding (NW) and fractions of that same tuber 0±5 h, 2 h, 8 h, 12 h and 24 h after wounding. The RNA gel blot was hybridized with a $#P-labeled 1±6 kb restriction fragment of PVS cDNA [29 ] and exposed to Kodak X}AR 5 film for 1 day (Panel A). Subgenomic PVS RNAs are detected by hybridization with the same probe in a 5 day exposure (Panel B). (Panel C) Blot reprobed with $#P-labeled potato 28 S rDNA [12 ] to demonstrate equal loading of total RNA.

44

J. K. Morelli and M. E. Vayda

Abundance of PVX and PVY is also induced by mechanical wounding of tubers Tubers which contained low titres of PVX or PVY exhibited an increase in the abundance of these viruses upon wounding. The full sandwich ELISA assay, used by certification programs for the routine screening of seed tubers, indicated that the abundance of PVX antigen increased greater than 10-fold within 4 h of wounding ; wounded tuber extracts exhibited an average A of 0±122 compared to an A of %!& %!& 0±010 for extracts of those same tubers taken prior to wounding. Similarly, PVY antigen increased 5-fold within 4 h after mechanical wounding ; wounded tuber extracts displayed an average A of 0±609 compared to A of 0±134 for extracts of the %!& %!& PVY-infected tubers taken prior to wounding. The ELISA assay indicated a similar increase in abundance of the PVS antigen as observed in previous immunoblot assays. The observation that three distinct potato viruses increase abundance upon wounding of tubers suggests that the induction is not specific for PVS, but rather is due to a general response of the tuber to an abiotic stress. DISCUSSION

Our results indicate that mechanical wounding of tubers induced replication of PVS genomic RNA, accumulation of PVS coat protein and assembly of PVS virions. These results identify the acidic 32 kDa polypeptide as the PVS coat protein and dispel the deduction by Crosby and Vayda [11 ] that the induced 32 kDa polypeptide may play a role in wound activation of protein synthesis. Induction of the acidic 32 kDa coat protein and replication of the virus appear to be associated with the general stimulation of tuber metabolism that occurs upon wounding. Mechanical wounding causes gross changes in tuber transcription [5, 12, 25 ], and increases in translation [11, 31, 55 ], cell division [2, 7 ] and respiration rates [23 ]. This induction is transient ; macromolecular synthesis rates return to basal levels approximately 72 h after wounding [25, 55 ]. Translational induction appears to be part of the general response to mechanical wounding which occurs both in the absence and presence of the opportunistic bacterial pathogen Erwinia carotoŠora [7, 31, 38 ]. This induction is distinct from the response to fungal infection and fungal elicitors which alters the expression of wound response genes involved in isoprenoid synthesis. For example, synthesis of lipoxygenase and members of the HMG CoA reductase family is affected dramatically by the presence of fungal pathogens, methyl jasmonate or arachidonic acid [6, 8, 9, 60 ]. The replication of PVS was greatest in the first 4 h after infliction of the wound, although virus abundance continued to increase modestly for 24 h after wounding. Thus, the transient induction of PVS replication is likely to be a result of the general activation of tuber metabolism during this period. Although PVS encodes its own replicase [27 ], viral replication may be dependent on host factors which may complex with the viral replicase [19 ]. Thus, increased abundance of translation factors [31 ], ribosomal components [20, 36, 55 ] or transcription factors could lead to the transient period of viral replication observed. A similar increase in ISMV genomic RNA and viral antigen was observed following mechanical wounding, or heat stress, of iris bulbs [52 ]. High titres of ISMV were found only in the vicinity of the wound site where enhanced metabolic activity was observed.

Induced replication of potato virus S

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We have shown previously that wound induction of protein synthesis in potato tubers is limited to within 1 cm of the wound site [7, 11, 12, 31 ]. The observed increases in ISMV, PVS, PVX and PVY upon wounding or heat stress of storage organs suggest that virus replication is stimulated by abiotic stresses which transiently increase the metabolism of dormant plant organs. Wound-induced multiplication of viruses in plant storage organs may have important implications for field practices and seed screening. Many potato producers increase their seed stock by cutting mother tubers into halves or quarters prior to planting. This wounding event may inadvertently increase the amount of virus present as the shoot emerges, leading to mature plants with higher virus titres than plants emerging from non-wounded mother tubers. Whether wounding prior to planting has an effect on virus spread in the field is currently under investigation. Further, ISMV is often undetected by ELISA unless iris bulbs are first wounded, or stressed by incubation at 30 °C [52 ]. Our results demonstrate that the sensitivity of ELISA testing for certification of seed tubers may be increased significantly by wounding prior to analysis. This work was supported by grants to M.E.V. from the U.S. Department of Agriculture National Research Initiative Competitive Grants Program (award u9137100-6622) and the Maine Agricultural and Forest Experiment Station (uME38402). The authors wish to thank Dr. G. A. De Zoeten for providing PVS cDNA probe, Dr. William Belknap for production of initial anti-32 kDa antisera, the Harvard Microchemistry Facility for professional assistance in sequencing the 32 kDa polypeptide, Dr. Feridoon Mehdizadegan and the Maine Seed Potato Board for supplying certified seed, and Dr. Stellos Tavantzis for critical reading of this manuscript. This is publication u1995 of the MAFES. REFERENCES 1. Bailey-Serres J, Freeling M. 1990. Hypoxic stress-induced changes in ribosomes of maize seedling roots. Plant Physiology 94 : 1237–1243. 2. Barkhausen R. 1978. Ultrastructural changes in wounded plant storage tissue. In : Kahl G, ed. Biochemistry of Wounded Plant Tissues. Berlin : DeGruyter, 1–42. 3. Berry JO, Carr JP, Klessig DF. 1988. mRNAs encoding ribulose-1,5-bisphosphate carboxylase remain bound to polysomes but are not translated in amaranth seedlings transferred to darkness. Proceedings of the National Academy of Sciences USA 85 : 4190–4194. 4. Berry JO, Breiding DE, Klessig DF. 1990. Light-mediated control of translation initiation of ribulose-1,5-bisphosphate carboxylase in amaranth cotyledons. The Plant Cell 2 : 795–803. 5. Bevan M, Shufflebottom D, Edwards K, Jefferson R, Schuch W. 1989. Tissue- and cell-specific activity of a phenylalanine ammonia-lyase promoter in transgenic plants. EMBO Journal 8 : 1899–1906. 6. Bostock RM, Yamamoto H, Choi D, Ricker KE, Ward BL. 1992. Rapid stimulation of 5lipoxygenase activity in potato by the fungal elicitor arachidonic acid. Plant Physiology 100 : 1448–1456. 7. Butler W, Cook L, Vayda ME. 1990. Hypoxic stress inhibits multiple aspects of the potato tuber wound response. Plant Physiology 93 : 264–270. 8. Choi D, Bostock RM. 1994. Involvement of de noŠo protein synthesis, protein kinase extracellular Ca#+, and lipoxygenase in arachidonic acid induction of 3-hydroxy-3-methylglutaryl Coenzyme A reductase genes and isoprenoid accumulation in potato (Solanum tuberosum L.). Plant Physiology 104 : 1237–1244. 9. Choi D, Bostock RM, Avdiushko S, Hildebrand DF. 1994. Lipid-derived signals that discriminate wound- and pathogen-responsive isoprenoid pathways in plants : methyl jasmonate and the fungal

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