Introduction Materials and methods

Plant Science 154 2000 13 – 21 Purification and properties of growth stage-dependent antiviral proteins from the leaves of Celosia cristata A. Balasubrahmanyam a , V.K. Baranwal b , M.L. Lodha a , A. Varma b , H.C. Kapoor a, a Di6ision of Biochemistry, Indian Agricultural Research Institute, Pusa, New Delhi 110012 , India b Di6ision of Plant Pathology, Indian Agricultural Research Institute, Pusa, New Delhi 110012 , India Received 16 June 1999; received in revised form 21 September 1999; accepted 21 September 1999 Abstract Two antiviral glycoproteins, active against mechanical transmission of two tobamoviruses, tobacco mosaic virus and sunnhemp rosette virus, and citrus ring spot virus ungrouped, were purified from the dried leaves of Celosia cristata. These proteins, called CCP-25 and CCP-27, have M r 25 and 27 kDa, respectively. Their concentration was found to vary between the pre-flowering and post-flowering stages of C. cristata. Either of these proteins, obtained at different growth stages, inhibited by \ 90 lesion formation at a concentration of 20 – 30 mg ml − 1 . They were resistant to proteases in the native state, but were readily digested when denatured. Both of them imparted actinomycin D sensitive resistance by inhibiting local lesions on Nicotiana tabacum cv. Samsun NN by tobacco mosaic virus. Their application, individually, also resulted in high resistance in systemic hosts to sunnhemp rosette virus, and citrus ring spot virus, respectively. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords : Celosia cristata; Antiviral; Basic glycoproteins; Stage-dependent; Protein purification www.elsevier.comlocateplantsci

1. Introduction

A large number of higher plants possess potent inhibitors of plant viruses and a few have been purified and characterized [1,2]. They either inacti- vate virus in vitro [3], or interfere with mechanical transmission, e.g. those from spinach [4] and Mirabilis jalapa [5], and sometimes may induce systemic resistance [6] when applied at least some time prior to virus challenge. These antiviral prin- ciples are generally basic proteins, with or without a sugar moiety, with sizes ranging from 24 to 32 kDa and act against a range of plant viruses [1,7,8]. The leaf extract of Celosia cristata has been found to prevent lesion production by sunnhemp rosette virus SRV, tobacco mosaic virus TMV, and potato virus X PVX in several hosts [9]. Studies on partially purified inhibitor from this leaf extract indicated that it might interfere with virus establishment in the host [10]. The purifica- tion of the virus inhibitor to apparent homogene- ity is described here. Further evidence is presented that there could be two related glycoproteins whose levels might vary according to the growth stage in the leaf extract and both of which can impart a very high level of resistance to TMV and SRV on their respective local lesion hosts. They also provoke high resistance to SRV and citrus ring spot virus CRSV on their respective sys- temic hosts namely, Crotalaria juncea sunnhemp and Phaseolus 6ulgaris French bean. These proteins are referred as CCP-25 and CCP-27, based on their molecular weights. Corresponding author. Tel.: + 91-11-578-4038; fax: + 91-11-576- 6420. E-mail address : hck –biociari.ernet.in H.C. Kapoor 0168-945200 - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 9 2 - 2

2. Materials and methods

Preparations of virus inoculum and inhibitor antiviral principle, testing the activity, raising of test plants and inoculation procedures were car- ried out as described before [9]. The different virus-host local lesion host combinations used in the current study are Nicotiana glutinosa, N. tabacum cv. Samsun NN against TMV, and Cyamopsis tetragonoloba against SRV, depending on availability of test plants and seasons. The percentage inhibition was calculated by the for- mula C − TC × 100, where C is the number of lesions in control and T is the number on treated leaves. 2 . 1 . Preparation of purified 6irus inoculum Purified preparations of TMV and SRV were obtained using polyethylene glycol PEG and dif- ferential centrifugation as described by Noordam [11]. CRSV was purified by extraction with seven volumes of phosphate buffer 0.05 M, pH 7.8 containing 0.005 M DIECA, 0.01 M EDTA and 0.02 M sodium sulphite from virus infected French bean tissue followed by treatment with chloroform 10, vv. The suspension was centrifuged at 10 000 × g for 10 min and the supernatant was subjected to two cycles of high speed 40 000 × g, 2.5 h and low speed 10 000 × g, 5 min centrifu- gation. The supernatant obtained after final low speed centrifugation was used as purified virus preparation. 2 . 2 . Optimum pH for extraction of maximum anti6iral acti6ity Leaf extract of C. cristata was prepared using different buffers of varying pH and applied on to the well-developed leaves of the test plants. The extraction buffers used were: i acetate buffer 20 mM, pH 5.2; ii phosphate buffers 20 mM with pH a 6.0 and b 7.0; and iii Tris – HCl buffer 20 mM, pH 8.6. 2 . 3 . Purification of 6irus inhibitor 2 . 3 . 1 . Extraction The leaves of C. cristata were harvested sepa- rately at three different stages of plant growth, i.e. pre-flowering, flowering and post-flowering 20 – 25 days after flowering. They were washed with tap water and dried at room temperature or at 40 – 45°C. This material 40 g was homogenized with 7 volumes of extraction buffer 0.1 M sodium acetate buffer, pH 5.2, containing 12 mM b-mer- captoethanol and 20 mg of polyvinyl polypyrol- lidone in a waring blender. The slurry was filtered through muslin cloth and centrifuged at 12 000 × g for 15 min. The clear supernatant was collected for further purification. 2 . 3 . 2 . Ammonium sulphate precipitation The precipitation of protein by ammonium sul- phate was initially carried out at 25, 60, 80 and 100 saturation. The protein obtained at each step was centrifuged 12 000 × g, 10 min and su- pernatant was used for further precipitation pro- cess. The pellet obtained at each step was suspended in buffer A 20 mM sodium phosphate, pH 6.2, 10 mM NaCl and dialyzed against the same buffer. The clear sample was tested for the antiviral activity on the test hosts. Since activity was found in two protein fractions precipitates between 25 and 60, and between 60 and 80 ammonium sulphate saturation, all of the protein precipitation between 25 and 80 saturation was used for further purification. 2 . 3 . 3 . Ion exchange chromatography 2 . 3 . 3 . 1 . DEAE-cellulose chromatography. Treat- ment of DEAE-cellulose was carried out as de- scribed [10]. The active protein 18.8 mg obtained after ammonium sulphate precipitation was loaded on a DEAE-cellulose column 15 × 1.5 cm and washed with buffer A. Fractions of 10 ml were collected from the column until no protein was found in the eluant. The bound protein was eluted with 0.5 M NaCl in the same buffer. Both frac- tions were tested for their antiviral activity. The active unadsorbed fractions were pooled and sub- jected to cation exchange chromatography. 2 . 3 . 3 . 2 . CM-sepharose chromatography. The active DEAE-cellulose unadsorbed protein 9.3 mg was fractionated on a CM-sepharose Sigma, St. Louis, MO column 35 × 1 cm equilibrated with buffer B 20 mM sodium acetate, pH 5.2, containing 10 mM NaCl and 0.01 sodium azide. After wash- ing the column with four to five bed volumes of buffer B, the bound protein was eluted with a discontinuous gradient of NaCl 0.1 and 0.2 M prepared in the same buffer. About four to five bed volumes of buffer were used at each step and fractions 3.0 ml were collected on an LKB frac- tion collector system at a flow rate of 30 ml h − 1 . Protein elution was simultaneously monitored at 280 nm and signals were traced on to a printer. Fractions falling within the protein peak were pooled and tested for antiviral activity. 2 . 3 . 4 . Gel filtration chromatography The pooled protein sample 1.9 mg was either lyophilized at − 20°C or concentrated in a dialy- sis bag on a sucrose pad at 4°C. Later it was chromatographed on a Sephadex G-75 Pharma- cia Fine Chemicals, Uppsala, Sweden column 61 × 1.5 cm equilibrated with buffer C 20 mM sodium acetate, pH 5.2 containing 0.01 sodium azide. The protein was eluted at a constant flow rate of 40 ml h − 1 and fractions 3.1 ml were monitored at 280 nm. Fractions falling within the protein peak were pooled and tested for antiviral activity. 2 . 4 . Protein and carbohydrate estimation Protein estimation was done by the Bradford method [12] using bovine serum albumin BSA as a standard. The presence of carbohydrate was tested by the Molisch test [13] and the estimation of total neutral sugars was done by the phenol- sulfuric acid method [14]. 2 . 5 . Protein characterization SDS-PAGE was performed in a 12 separating gel according to Laemmli [15]. The proteins were also analyzed in a 10 – 20 linear gradient gel. The gradient was prepared using an LKB gradi- ent maker. The proteins were visualised with Coomassie blue or by silver staining [16]. Glyco- proteins were detected on polyacrylamide gels us- ing the periodic acid-Schiff reagent PAS [17]. Ovalbumin was used as a positive control on the gel, while BSA served as the negative control. The molecular weights of the purified proteins were determined under native conditions by cali- brated gel-permeation chromatography using MW GF-70 kit contains aprotinin, 6.5 kDa; cy- tochrome c, 12.4 kDa; carbonic anhydrase, 29 kDa and bovine serum albumin, 66 kDa Sigma in a Sephadex G-75 column. The R f of molecular marker and purified proteins were plotted against the molecular weight on a semi-log graph paper. The molecular weights of the purified proteins were also determined under denatured conditions in a 12 polyacrylamide gel and a 10 – 20 linear gradient gel by SDS-PAGE using MW SDS-70 kit. 2 . 6 . Stability to protease digestion The purified proteins, obtained by the above mentioned purification procedure, were incubated at 37°C for 12 h with trypsin, chymotrypsin, Staphylococcus aureus protease XVII-B or papain. The incubation mixture contained purified protein and protease in a ratio of 25:1 ww in 50 mM Tris – HCl buffer, pH 6.8. The mixture was then applied onto test plants to determine its antiviral activity. Both proteins, after incubation, were also monitored by SDS-PAGE. The antivi- ral proteins were also denatured in Cleveland buffer [18] and digested with each of the above proteases for 15 or 30 min and subjected to SDS- PAGE. 2 . 7 . Effect on systemic hosts The purified protein was tested for its ability to impart resistance in different systemic host-virus combinations. About 60 mg of purified protein in 1 ml was mixed with each of the purified viruses TMV, SRV and CRSV, and the mixture was applied on carborundum dusted leaves of three test plants each of tobacco cv NP33, sunnhemp and French bean, respectively. The symptoms were observed after 25-35 days of virus infection in case of TMV and SRV, and after 6 days in case of CRSV. Back-inoculation or electron mi- croscopy or both were carried out to detect the virus in the treated systemic host plant.

3. Results