Indian Journal of Experimental Biology Vol. 39, June 2001, pp 600-603

A systemic resistance inducing antiviral protein with N-glycosidase activity from Bougainvillea xbuttiana leaves

Sneh Narwal, A Balasubrahmanyam, P Sadhna, He Kapoor & ML Lodha*

Division of Biochemistry, Indian Agricultural Research Instit!lte, New Delhi 110 01:.., India

Received 16 May 2000; revised 9 February 2001

An antiviral protein from Bougainvillea xbuttiana leaves induced systemic resistance in host plants N. glutinosa and Cyamopsis tetragonoloba against TMV and SRV, respectively which was reversed by actinomycin D, when applied immediately or shortly after antiviral protein treatment. When the inhibitor was applied to the host plant leaves post inoculation, it was effective if applied upto 4 h after virus infection. It also delayed the expression of symptoms in systemic hosts of TMV. The inhibitor showed characteristic N- glycosidase activity on 25S rRNA of tobacco ribosomes, suggesting that it could also be interfering with virus mUltiplication through ribosome-inactivation process.

Plants belonging to different taxonomic families have been reported to contain substances, which when applied to other species, make them resistant to virus infection and are collectively referred as antiviral principles (A VP). The exact mechanism of action of these inhibitors is not known. Three modes of action 1

have been suggested for these inhibitors, these are (1) by acting directly on virus particles, (2) by acting on virus infection process, and (3) by altering host susceptibility. Bougainvillea species are known to contain virus inhibitors which are active against mechanical transmission of plant viruses. Virus inhibitors have been purified from .leaves2 and roots3

of B. spectabilis, of which the former has been found to possess ribosome-inactivating property (RIP). We have earlier reported4 the purification of virus inhibitor from the B. xbuttiana leaves and it was found to consist of two highly basic, lysine rich glycoproteins with molecular weights of 28,000 (BBP-28) and 24,000 (BBP-24). In the present investigation, we report that this inhibitor induces actinomycin D reversible systemic resistance and also possesses N-glycosidase activity on plant ribosomes.

Test plants, virus inoculation and test for antiviral activity -Nicotiana glutinosa and N. tabacum var. Samsun NN were used as local lesion hosts for tobacco mosaic virus (TMV) and Cyamopsis tetragonoloba for sunnhemp rosette virus (SRV) for conducting bioassay of the antiviral principle. Preparation of virus inoculum and inhibitor, raising of

*Correspondent author

test plants, inoculation procedures and purification of AVP were carried out as described before4

. Per cent inhibition was calculated using the formula : Per cent inhibition={ (C-T)/C} X 100, Where, C=average number of lesions on control plants; and T=average number of lesions on treated plants

Effect of actinomycin D (AMD)-AMD (20 ~g mrl in distilled water) was applied at different time intervals (0, 6 and 12 hr) following antiviral protein treatment, on both treated basal leaves and untreated top leaves of Cyamopsis tetragonoloba. An equal number of leaves in control sets were treated with AMD alone, A VP alone and buffer. All the leaves of test plants were inoculated with SRV after 24 hr and observed for lesion development.

Effect of antiviral principle after virus inoculation­N. glutinosa plants were first inoculated with TMV and then treated with purified virus inhibitor after different time intervals (0, 1, 2, 4 and 6 hr). Leaves were washed with distilled water for 10 min after the protein treatment. For control, the plants were treated with buffer after virus inoculation and per cent inhibition was calculated in each case.

Effect of virus inhibitor on systemic host-Systemic hosts (N. tabacum var. Samsun nn & N. tabacum var. NP 31) of TMV were treated with the purified virus inhibitor and then challange inoculated with the virus after 24 hr. For control, appropriate buffer was used and the plants were observed for the symptom development at regular intervals.

Isolation of ribosomes and assay of N-glycosidase activity-Ribosomes were isolated from the leaves of

NOTES 601

N. tabacum var. Samsun nn employing the technique described by Turner et al5

• Enzyme assay was carried out using method of Girbes et a1.6

, with some modifications. Tobacco ribosomes (40 Jlg) were incubated with 6 Jlg of B. xbuttiana virus inhibitor for 15 min at 37°C in a reaction mixture of 100 Jll. Total RNA was extracted using phenol and SDS. RNA was subjected to aniline treatment and then analyzed on 5% polyacrylamide gel. After electrophoresis the gel was stained for 30 min with 0.5 Jlg mr' ethidium bromide in Ix TBE and observed under UV illuminator.

Systemic resistance inducers have been reported from certain plants like Clerodendrum aculeatum7

, C. inerme8 and they induce strong local and systemic resistance in susceptible hosts against different viruses. Treatment of lower leaves of host plants like Nicotiana tabacum Samsun NN, Cyamopsis tetragonoloba with these proteins resulted in complete protection of untreated upper leaves against viral infection. The resistance induced by all the systemic resistance inducers9-'I and a few localized resistance inducers like Chenopodium ambrosoides'2 and Celosia cristata13 is reported to be reversed by simultaneous application of AMD. The resistance induced by B. xbuttiana virus inhibitor was also found to be reversed when AMD was applied immediately after treatment with purified inhibitor, at both treated and untreated sites (Table 1). AMD is known to bind double helical DNA by intercalating between the bases and thereby · inhibiting DNA­directed RNA synthesis. This indicates that the active substances in all such plant extracts possibly induce the synthesis of a new virus interfering substance or

enhance the production of an existing one, thereby altering the cellular metabolism of the host.

Virus inhibitors from some other plants such as Bougainvillea spectabilis l4

, Pseuderanthemum bicolorl5

and Clerodendrum aculeatum7, have been reported to

result in the formation of new proteins called virus inhibitory agents (VIA) or antiviral agents (A V A) in the test plants. Preliminary studies done with B. xbuttiana antiviral principle 'have also indicated that there is a considerable increase in the production of some already existing proteins after 24 hr of treatment (results not shown). It is thus reasonable to speculate that a mobile signal may be induced in the treated leaves following binding of B. xbuttiana virus inhibitor on host plant surface. This mobile signal diffuses into the entire plant after sometime, where it enhances the production of certain proteins involved in defense response. All these observations in turn may suggest that the antiviral action of this virus inhibitor could be a host-mediated phenomenon.

The post-inoculation studies (Table 2) revealed that the lesion production was almost completely inhibited when the virus inhibitor was applied soon after virus inoculation. The inhibitory effect gradually decreased with increase in time between virus inoculation and A VP treatment and it was 46% after 4hr. Kalo and Taniguchi'6 and Baranwal and Vermal7 have also observed that the inhibitors from Spinach and Celosia cristata were active only up to 3 hr and 2 hr after virus inoculation, respectively.

The virus inhibitor also delayed the expression of symptoms in systemic hosts of TMV. Symptoms appeared 35-40 days after inoculation in N. tabacum var. Samsun nn and took 40-45 days in case of N.

Table I-Effect of actinomycin 0 (AMD) on the resistance induced by the purified virus inhibitor from B. xbuttiana in Cyamopsis tetragonoloba against sunnhemp rosette virus (SRV)

Treatment Average number of lesions + SE Per cent inhibition Treated Untreated Treated Untreated basal leaves top leaves basal leaves top leaves

Control 53.5±4.3 54.6±2.8 Purified antiviral

proteins (BBPs) 3.8;tO.4 14.6±1.2 93.0 73.3 AMD 50.2±3.6 53.2±3.0 6.2 2.6 BBPs+AMD

immediately 46.7±3.1 45.1±4.4 12.7 17.4 BBPs+AMD

after 6 hr 16.5±2.9 24.0±2.3 69.1 56.0 BBPs+AMD

after 12 hr 11.6+2,0 15.7+1.7 78.3 71.2

602 INDIAN J EXP BIOL, JUNE 2001

tabacul1l var. NP 31 as compared to 20-25 days in control. This delay in symptom expression might be due to reduced number of infectable sites available for the virus, as a result of which the virus took more time to cause systemic infection. Post-inoculation studies and effect on systemic host suggested that the virus inhibitor might also be acting on the establishment phase of the virus infection process by blocking the virus receptor sites on the leaf surface. This could be in addition to the systemic resistance induced in local lesion hosts.

Results depicted in Fig. 1 clearly shows that the aniline-treatment of rRNA extracted from antiviral principle treated ribosomes resulted in generation of a specific RNA fragment (lane b) due to its N­glycosidase activity. The position of 25S rRNA was also shifted and its mobility became slightly faster than that of control (lane a) . The fragment released was diagnostic for Ribosome-inactivating protein (RIP)-catalysed depurination and therefore, B. xbuttiaTla virus inhibitor was a ribosome-inactivating protein. Antiviral proteins isolated from Phy folacca

• 18 M' b·t· . I 19 Cl I I allleriCana, Ira I IS Ja opa, ero(' el1(' rum inerme

20 and few other plants have also been reported to have N-glycosidase activities. Taylor et ol. 21 have shown a positive correlation between RIP-catalyzed depurination of tabacco ribosomes and antiviral activities of pokeweed antiviral protein and dianthin 32. These observations, therefore, supported the hypothesis that the antiviral proteins also work through ribosome-inactivation and thus indirectly prevent the mUltiplication of the virus particles. However, recent studies with transgenic plants expressing active PAP and PAP- II have shown that RIP property by itself is not responsible for antiviral action. It may help to generate a signal other than salicylic acid that renders the plant resistant to virus infection22

•23

• Turner et al. 5 based on their studies on C-terminal deletion mutants of PAP, have concluded that the antiviral activity of PAP can be dissociated

from its toxicity. Based on these findings, it can be suggested that

the mode of action of B. xbuttiana antiviral principle might involve interference with virus establishment process as well as interaction with the host plant resulting in the induction of systemic resistance.

The senior author thanks Director, Indian Agricultural Research Institute, New Delhi for

a L

Fig. \-Testing N-glycosidase aCllvlty of A VP on tobacco ribosomes. [Lane (a)- control (no A VP treatment); and lane (b)­ribosomes treated with antiviral proteins]

Table 2 - Effect of B. xbuftialla inhibitor on TMV infection after virus inoculation in N. giutillosa plants.

Time of application A verage number of Per cent of virus inhibitor lesions ± SE inhibition after inoculation (hr)

Immediately 5.6 ± 1.5 94.4

1 18.6± 3.7 81.3

2 30.5 ±3.3 69.4

4 53.5 ± 4.7 46.3

6 83.6 + 10.2 16.1

NOTES 603

awarding Senior Research Fellowship. The support provided under lCAR Centre of Advanced Studies in Biochemistry is gratefully acknowledged.

References Verma HN, Varsha & Baranwal VK, Agricultural role of endogenous antiviral substances of plant origin, in Antiviral proteins ill higher plants, edited by M Chessin, D DeBorde and A Zipf (CRC Press, Boca Raton, Florida) 1995,23.

2 Bolognesi A, Polioto L, Olivieri F, Valbonesi P, Barbieri L, Baueli MG, Carusi MV, Benvenuto E, Blanco FDV, Maro AD, Parente AL & Stirpe F, New ribosome-inactivating proteins with polynucleotide : Adenosine glycosidase and antiviral activities from Basella rubra L. and Bougainvillea spectabilis Wild, Planta, 203 (1997) 422.

3 Balasaraswathi R, Sadasivam S, Ward M & Walker JM, An antiviral protein from Bougainvillea spectabilis roots; purification and characterization, Phytochemistry, 47 (1998) 1561.

4 Narwal Sneh, Balasubrahmanyam A, Lodha ML & Kapoor HC. Purification and properties of antiviral proteins from the leaves of BOllgainvillea xbuttialla, Indian J Biochem Biophys,(In press) (2001).

5 Turner, NE, Hwang D & Bonness M, C-terminal deletion mutant of pokeweed antiviral protein inhibits viral infection but does not depurinate host ribosomes, Proc Natl Acad Sci USA, 94 (1997) 3866.

6 Girbes T, Citores L, Iglesias R, Ferreras 1M, Munoz R, Rojo MA, Arias FJ, Gracia JR, Mendez E & Colonge M, Ebulin I, A non-toxic novel type-2 ribosome-inactivating protein from Sambucus ebuius L, J Bioi Chem, 268 (1993) 18195.

7 Verma HN, Srivastava S, Varsha & Kumar D, Induction of systemic resistance in plants against viruses by a basic protein from Clerodendrum aculeatum leaves, Phytopathology, 86 (1996) 485.

8 Prasad V, Srivastava S, Varsha & Verma HN, Two basic proteins isolated from Clerodendrum inerme are inducers of systemic antiviral resistance in susceptible plants, Plant Sci, 110 (1995) 73.

9 Verma H N, Chowdhury B & Rastogi P, Antiviral activity in leaf extracts of different Clerodendrum species, Z Pjlanzenkr Pjlanzenschutz, 91 (1984) 34.

10 Kubo S, Ikeda T, Imaizumi S, Takanami Y & Mikami Y, A potent virus inhibitor found in Mirabilis jalapa L, Ann Phytopath Soc Japan, 56 (1990) 481.

11 Verma HN & Khan MMAA, Occurrence of a strong virus interfering agent in susceptible plants sprayed with Pseuderanthemum atropurpureum tricolor leaf extract, Indian J Virol, I (1985) 26.

12 Verma HN & Baranwal VK, Antiviral activity and the physical properties of the leaf extract of Chenopodium ambrosoides L., Proc Indian Acad Sci (Plant Sci), 92 (1983) 461.

13 Balasubrahmanyam A, Baranwal VK, Lodha ML, Varma A & Kapoor HC, Purification and properties of growth stage­dependent antiviral proteins from the leaves of Celosia cristata, Plant Sci, 154 (2000) 13.

14 Verma HN & Dwivedi SD. Properties of an virus inhibiting agent, isolated from plants which have been treated with leaf extracts from Bougainvillea spectabilis, Physiol Plant Pathol, 23 (1984) 93.

15 Khan MMAA & Verma HN, Partial characterization of an induced virus inhibitory protein, associated with systemic resistance in Cyamopsis tetragonoloba (L.) Taub plants, Ann Appl Bioi, 117 (1990) 617.

16 Kalo F & Tanguchi T, Properties of a virus inhibitor from spinach leaves and mode of action, Ann Phytopath Soc Japan, 53 (1987) 159.

17 Baranwal VK & Verma HN, Localised resistance against virus infection by leaf extract of Celosia cristata, Plam Pathol, 41 (1992) 633.

18 Taylor BE & Irvin JD, Depurination of plant ribosomes by pokeweed antiviral protein, FEBS Lett, 273 (1990) 144.

19 Kataoka J, Habuka N, Miyano M, Masuta C & Koiwai A, Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP), Plant Mol Bioi, 20 (1992) 1111.

20 Olivieri F, Prasad V, Valbonesi P, Srivastava S, Ghosal­Chowdhury P, Barbieri L, Bolognesi A & Stirpe F, A systemic antiviral resistance-inducing protein isolated from Clerodendrum illenne Gaertn. is a polynucleotide : Adenosine glycosidase (ribosome-inactivating protein), FEBS Lett, 396 (1996) 132.

21 Taylor S, Massiah A, Lomonossoff G, Roberts LM, Lord JM & Hartely M, Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection, Plant J, 5 (1994) 827.

22 Smirnov S, Shulaev V & Turner NE, Expression of pokeweed antiviral protein in transgenic plants induces virus resistance in grafted wild type plants independently of salicylic acid accumulation .and pathogenesis related proteins synthesis, Plant Physiol, 114 (1997) 1113.

23 Wang P, Zoubenko 0 & Turner NE, Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II, Plant Mol Bioi, 38 (1998) 957.