Changes in Serological Reactivity of Papaya leaf distortion mosaic

J. Gen. Plant Pathol. 68 : 89-93 (2002)

Short Communication

Changes in Serological Reactivity of Papaya leaf distortion mosaic virus Caused by Papain in Carica papaya L. and Its Detection Using Antipain or Papain Tetsuo MAOKA'*, Shinya TSUDA', Tomio USUGV, Chiyoichi NODA3 and Mabito IWASAKll ABSTRACT Losses in serological reactivity of Papaya leaf distortion mosaic virus (PLDMV) were demonstrated. An antibody, IgG-papaya, raised against PLDMV purified from papaya (Carica papaya L.) did not react with virus particles in Cucumis metuliferus leaf extracts in ELISA or SSEM-PAG (serologically specific electron microscopy using protein A-gold). In addition, IgG-papaya and IgG-Cucumis raised against PLDMV purified from C. metuliferus did not react with virus particles in papaya leaf extracts after western blotting. From results of electrophoresis, the coat protein (CP) of PLDMV purified from papaya had degraded and migrated in two bands. Similar degradation was also observed when virus purified from C. metuliferus was treated with papain. These results indicated that the CP of PLDMV purified from papaya was degraded during the purification process by papain in the host plant. IgG-papaya was reactive to papaindegraded CP, while IgG-Cucumis was reactive to both intact and degraded CP. Modified serological methods using antipain (a protease inhibitor) or papain were established to detect PLDMV.

(Received February 16, 2001 ; Accepted September 26, 2001) Key words: Papaya leaf distortion mosaic virus, coat protein, proteolytic cleavage, serology, papain, antipain, papaya.

Papaya (Carica papaya L.) is one of the important fruit trees in tropical and subtropical areas. Papaya leaf distortion mosaic virus (PLDMV), the most destructive factor in papaya production in Japanlo) and Papaya ringspot virus type P (PRSV-P) have been reported to be the viruses causing papaya disease in JapanS,'O).The viruses, both belonging to the genus Potyvirus, have some similar characteristics. However, PLDMV has been shown to differ from PRSV-P in terms of serology and genomic The 3-terminal regions of the genomic RNAs of these viruses have been sequenced, and the putative amino acid sequences of the coat protein (CP) determined"J2). In preliminary experiments using some of the serological methods with antibodies we made against PLDMV purified from papaya and Cucumis metuliferus, we could not detect PLDMV. The objectives of this study, therefore, were to clarify the cause of the discrepancy in our results obtained using different serological methods and

to establish reliable serological methods for detecting PLDMV in leaf extracts. The leaf distortion mosaic (LDM) isolate of PLDMV (J56P, formerly 56P'O))) which has been maintained in papaya, was propagated in papaya and C. metuliferus. PLDMV in papaya was purified as follows. Infected papaya leaves were homogenized with a Waring blender in four times their weight of 0.1 M borate buffer (BB), pH 8.5, containing 10 mM sodium N,N-diethyldithiocarbamate trihydrate (DIECA). After being squeezed through a cotton cloth, the extract was clarified by adding carbon tetrachloride to 15%, stirring for 1min at room temperature, and then centrifuging at low speed (6000 X g for 15min). Polyethylene glycol 6000 (PEG), NaCl and Triton X-100 were added to the resultant supernatant to final concentrations of 7% (w/v), 0.1 M and 2% (w/v), respectively. The mixture was stirred for 30 min at 4°C and centrifuged at low speed. The pellet was resuspended in BB with a volume one-tenth of that of the clarified

National Agricultural Research Center for Hokkaido Region, Hitsujigaoka, Toyohira, Sapporo 062-8555, Japan National Agriculture Research Center, Tsukuba 305-8666, Japan Okinawa Subtropical Station of the Japan International Research Center for Agricultural Sciences, Maezato, Ishigaki 907-0002, Japan * Corresponding author (E-mail : maokat @ affrc.go.jp)

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supernatant and centrifuged at low speed. The resultant supernatant was centrifuged at 125,000 X g for 90 min. The virus pellet was resuspended in BB. After low-speed centrifugation, the supernatant was layered on a 10-50% (w/v) linear sucrose gradient in BB and centrifuged at 112,000 X g for 150 min in an RPS-40T rotor (Hitachi, Tokyo, Japan). The zone containing the virus was collected with a Pasteur pipette, diluted with BB and centrifuged at 125,000 X g for 90 min. The pellet was resuspended in BB and centrifuged at low speed. The resultant supernatant was layered on a 49-73% linear density gradient of Omnipaque 350 (Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan)7)in BB and centrifuged at 175,000 X g for 15 hr in the RPS-40T rotor. The virus fraction was collected, resuspended as described, then centrifuged at 238,000 X g for 90 min. The virus pellet was suspended in a small volume of 10 mM BB, pH 8.5. From papaya, the best result was obtained using 0.1 M BB, pH 8.5, containing DIECA for the extraction and Omnipaque for the medium during equilibrium density gradient centrifugation. Omnipaque, a nonionic radiographic contrast medium, was used by Koganezawa and Yanase7)for purification of Apple stem pitting virus. The method used for purification of PLDMV from papaya was not used for purification of PLDMV from C. metuliferus because the virus yield was very low. PLDMV was therefore purified from C. metuliferus using a method previously described"), which involved maceration of infected leaves in 0.5M citrate buffer (CB), pH 7.0,

1 66.2k

7

45k 31k

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21.5k 14.4k

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3

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Fig. 1. SDS-polyacrylamidegel electrophoresis of coat protein subunits of PLDMV. Lane 1, Molecular weight markers with apparent molecular weights indicated (SDS-PAGE Molecular Weight Standards, Low Range, Bio-Rad Laboratories, Richmond, USA) ; lane 2, coat protein subunits of purified PLDMV from C. metuliferus (CP-C); lane 3, CP-C treated with 0.0001%papain ; lane 4, CP-C treated with 0.01% papain; lane 5, CP-C treated with papaya extract ; lane 6, coat protein subunits of purified PLDMV from papaya.

containing 0.1 M disodium hydrogen phosphate, clarification with carbon tetrachloride, and differential and cesium sulfate linear density gradient centrifugations. F&glectrophoresis, 40 ,ug of purified PLDMV from C. metuliferus in 50 ,ul of 50 mM CB, pH 7.0, was incubated with 0.0001% to & O l % of papain (w/v, Sigma, St. Louis, USA)') or 5 0 ~ of 1 healthy papaya leaf extract. After incubation for 30 min at room temperature, the solution was layered on a 40% sucrose cushion in 50 mM CB, pH 7.0 and centrifuged at 214,000 X g for 60 m h . The pellet was resuspended in 100 ,ul of 50 mM CB, pH 7.0. Electrophoresis of polypeptides was performed with 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Maoka et d 9 ) . Figure 1 shows the electrophoretic pattern of PLDMVCP in SDS-PAGE. The molecular weight of the major band for the CP of PLDMV purified from C. metuliferus (CP-C) was 37kDa (Fig. 1, l w e 2). The CF-C was degraded when it was treated with 0.01% papain (Fig. 1, lane 4) or with the papaya leaf extracts (Fig. 1, lane 5). Treatment with 0.0001% papain resulted in partial degradation of CP-C (Fig. 1, lane 3). The degradation products principally migrated in two bands with molecular weights of 30 and 28 kDa. CP preparation of PLDMV purified from papaya (CP-P) migrated principally in two bands as did the degraded CP-C (Fig. 1, lane 6). No band of CP-C or CP-P was observed when ultracentrifugation with 40% sucrose cushion after the papain treatment was omitted (data not shown). For analysis of the N-terminal amino acid sequences, CP-P was isolated by SDS-PAGE*),blotted onto a PVDF membrane (Japan Genetics, Tokyo, Japan), and then analyzed with an automated gas-phase protein sequencer (model 477A, ABI, Norwalk, USA) as described by Maoka et al."). The molecular weight of the CP was Table 1. Absorbance values (A405)of PLDMV in ELISA to anti-PLDMV IgGsa) IgGIgGAntigens papaya Cucumis 0.662 Infected papaya leaf 0.918 0.723 Infected papaya leaf antipainb) 0.712 0.401 Infected papaya leaf papain") 0.398 0.009 Healthy papaya leaf 0.017 Infected C. metuliferus leaf 0.060 0.335 0.221 Infected C. metuliferus leaf+antipainb) 0.029 0.140 , Infected C. metuliferus leaffpapain") 0.246 0.007 Healthy C. metuliferus leaf 0.007 a) Each value is the mean of three wells. b) Antigen was triturated with extraction buffer with 0.2% antipain. c) Antigen was triturated with extraction buffer with 2% papain.

+ +

Changes in Serological Reactivity of PLDMV

calculated on the basis of the location of the N-terminal amino acid sequences in the known sequence of PLDMVCP (DDBJ/EMBL/GenBank database accession No. D50082)") using the DNASIS system (version 7.0, Hitachi Software Engineering Co. Ltd., Tokyo, Japan). Direct sequencing on the SO-kDa band of CP-P yielded its Nterminal sequence, SSGSFI, which was located 64 to 69 residues from the N-terminus of intact CP"). The calculated molecular weight of the peptide removed by papain from the N-terminus of CP-C was 7012 Da. This agrees well with the difference between the molecular weights of intact CP and CP-P or papain-treated CP-C in SDSPAGE. These results clearly showed that the N-terminus of PLDMV-CP is degraded by papain during extraction. This digestion may be one reason for the need for a different purification method for papaya. The heterogeneity of CPs has been reported in several potyviruse^'^-^^).

Antisera against PLDMV from papaya or C. metuliferus were made by injecting purified viruses into rabbits. IgGs were purified from antisera using an Affi-Gel Protein A MAPS I1 Kit (Bio-Rad Laboratories) according to the manufacturer's instructions. Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was carried out by the method of Clark and Adams3).SSEMPAG (serologically specific electron microscopy using

A

91

protein A-gold) was performed as described by Maoka et al.'O). Gold-labeled grids were examined using a Hitachi H7000 electron microscope (Hitachi, Tokyo, Japan). Western blotting was performed as described by Usugi et al. 19). For serological tests, 0.01, 0.1, and 2% papain (w/v) were added to the buffers used for dilution or extraction of virus in SSEM-PAG, western blotting and ELISA, respectively. To inhibit the proteolytic activity of papain, 0.2% antipain (w/v, Transformation Research Inc., Framingham, USA)17)was added to the extraction buffer. Table 1 shows the results of ELISA. IgG-Cucumis reacted with all of the virus preparations tested. In contrast, IgG-papaya reacted with the virus preparations from papaya but did not react with any of the virus preparations from C. metuliferus except that treated with papain. The results of serological tests are summarized in Table 2. In SSEM-PAG, IgG-papaya reacted with virus particles purified or extracted from papaya but not with virus particles purified (Fig. 2A) or extracted from C. metuliferus. IgG- Cucumis reacted with virus particles purified or extracted from either host irrespective of treatment with papain or antipain. IgG-papaya reacted with virus particles purified (Fig. 2B) or extracted from C. metuliferus when they were treated with papain. The positive reaction of IgG-papaya to virus particles in leaf

B

Fig. 2. SSEM-PAG using an antibody raised against purified PLDMV from papaya. A, purified PLDMV from C. metuliferus ; B, purified PLDMV from C. metuliferus after treatment with papain. Bars represent 200 nm. ,

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Table 2. Serological reactivity of PLDMV in PaPaya and C. metuliferus to anti-PLDMV IaGs Antibodyb' IgG-papaya IgG-Cucumis

Method

Inoculated plant

Samplea)

Treatment

ELISA

papaya papaya C. metuliferus C. metuliferus papaya papaya C. metuliferus C. metuliferus papaya

LE LE LE LE LE, PV LE LE, PV LE, PV PV

no antipain no papain no antipain no papain no

+ +

papaya C. metuliferus C. metuliferus

LE LE LE

antipain no papain

-

SSEM-PAG

Western blotting

+ + + +-

+ + + + + + + + + + + -

Predicted degradation of CP Yes yesc) no Yes Yes no no

Yes no

no yesd)

a) LE, leaf extract ; PV, purified virus. b) , positive reaction ; - , negative reaction. c) Samples were incubated for 8 hr or overnight for ELISA. Antipain may have been inactivated during this long incubation time. d) Samples were boiled for 3 min at 100°Cafter adding SDS and mercaptoethanol.The electrophoreticpattern showed that CPs of PLDMV were completely digested by activated papain.

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extracts from papaya was inhibited when the extraction was performed in a buffer containing 0.2%antipain. In western blotting, IgG-papaya did not react with virus proteins in extracts either from papaya or C. metuliferus. IgG- Cucumis reacted with virus proteins in extracts from C. metuliferus but not with virus proteins extracted from C. metuliferus with papain or those in extracts from papaya. It did react with virus proteins in extracts from papaya when antipain was added to the extraction buffer. Both IgGs reacted with CP of PLDMV purified from papaya. The present study revealed that IgG-papaya, which was raised against degraded CP of PLDMV, was reactive only to papain-degraded CP, while IgG- Cucumis was reactive to both intact and degraded CP. Hiebert et ~ 1 . and ~) Hiebert and McDonard4)reported that CP heterogeneity affected serological reactions. From the results of ELISA and SSEM-PAG, IgG-Cucumis consisted of an Nterminus- and core-region-specific antibodies, and IgGpapaya consisted of antibodies exclusive of any to the N-terminus. In the CP of Potyuirus, the N-terminus constituted the most immunodominant region, and crossreacting antibodies in polyclonal antisera were directed towards the homologous core protein regions15J6).However, neither of the IgGs cross-reacted with PRSV-P in any serological test (data not shown). Modified serological methods using antipain or papain were established to detect PLDMV. However, western blotting showed different results from those of ELISA and SSEM-PAG. IgG-Cucumis could not detect CPs when

samples were prepared in the presence of papain. IgGpapaya detected only CPs of purified PLDMV. Electrophoretic patterns showed that CP-C and CP-P were completely digested when the step to remove papain by ultracentrifugation with a sucrose cushion was omitted. The optimum temperature range for papain is in the range of 60-90"C2), and the enzyme is activated by mercaptoethanol'). In western blotting, samples were boiled for 3 minutes at 100°C after adding SDS and mercapt~ethanol'~). During this step, activated papain may digest CPs. Papain is a sulfhydryl protease that was isolated from the latex and the melonlike green fruit of papaya2). In ELISA and SSEM-PAG, papain made PLDMV from C. metuliferus reactive to IgG-papaya by degrading the Nterminus of CP. However, extreme care is needed when using papain for western blotting, as it digests PLDMVCP. Except for in ELISA, the digestion by papain was inhibited by adding antipain, a protease inhibitor that was isolated from Actin~mycetes'~). For ELISA, samples were incubated for 8 hr or overnight3), during which time antipain may have been inactivated. Suda et ul.17)reported that antipain is more specific to papain than other protease inhibitors such as leupeptin, pepstatin and chymostatin. It is remarkable that IgG- Cucumis reacted with the virus proteins extracted from papaya in western blotting when antipain was added to the extraction buffer. This report is the first to present a practical westernblotting technique to detect PLDMV in papaya plants.

Changes in Serological Reactivity of PLDMV

W e found that a n antiserum against a Japanese isolate of PRSV-P, which was raised against purified virus from C. metuliferus, did not react with PRSV-P in papaya extracts in western blotting (unpublished data). However, it did react with PRSV-P in extracts of papaya when antipain was added to the extraction buffer (unpublished data). Tennant et ~ 1 . ' ~observed ) CP degradation of PRSV-P in naturally infected papaya plants and in CPtransgenic papaya plants. Antipain would enable the intact CP in transgenic papaya plants to be detected in western blotting. The Potyvirus, which consists of 180 members, is the largest genus of plant viruses16).Because some of these viruses have properties inconsistent with each other, classification and diagnosis of potyviruses requires careful consideration. In this study, the antigenicity and molecular weights of the CP of PLDMV were found to differ depending on the host plant used for its propagation. This difference might cause a conflict in classification and diagnosis. The authors wish to thank Dr. K. Ishikawa for his useful suggestions about virus purification. We thank Drs. S. Kashiwazaki and H. Hibino for their helpful discussion and critical reading of the manuscript.

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