Role of satellite RNA of an Indian isolate of …nopr.niscair.res.in/bitstream/123456789/23994/1/IJEB 38(6...Indian Journal of Experimental Biology Vol. 38, June 2000, pp. 613-616

Indian Journal of Experimental Biology Vol. 38, June 2000, pp. 613-616

Role of satellite RNA of an Indian isolate of cucumber mosaic virus in inducing lethal necrosis of tobacco plants

S K Raj , A Srivastava, G Chandra & B P Singh

Plant Virus Laboratory, National Botanical Research Institute, Lucknow 226 00 I, India

Received 7 July 1999; revised 9 March 2000

Lethal necrosi s or systemic stem necrosis followed by death of Nicotiana benthamiana, severe leaf deformations of N. tabacum cv. white burley and blister formations on N. tabacum cv. samsun NN symptoms were induced by experimental in­oculations of CMV RNA preparations containing satellite RNA (sat-RNA). Inoculations of RNA preparations without sat­RNA did not induce that severe symptoms on these plants, only late mild mosaic was observed. It is suggested that sat-RNA of CMV isolate has a certain role for enhancing severity of symptoms in tobacco plants. Local and systemic lethal necrosis of N. benthamiana is due to sat-RNA present with genome of CMV isol ate. It is the first report of lethal necrosi s induced in

N. benthamiana by CMV satellite.

Cucumber mosaic virus (CMV) ?elongs to Cu­cumovirus group characterized by isometric particles of about 29 nm in diameter, three single-stranded positive sense genomic RNA species, and a fourth subgenomic RNA, which acts as messenger RNA for the coat protein (CP) of about 24 kDa (ref. 1 ). In ad­dition, some CMV isolates often support rep lication, encapsidation, and spread of an additional single­stranded RNA species of 330 to 391 nucleotides des­ignated as satellite RNA (sat-RNA) which is known to be involved in modulation of symptoms2

. However, some sate llite RNAs of CMV increase I exacerbate the pathogenicity in specific hosts3

. Lethal necrosis of tomato caused by CMV when a sat-RNA was associ­ated with CMV genome4

-6 are the examples of this

phenomenon. The yellow chlorosis of tobacco7 and white disease as well as yellow chlorosis of tomato8

·9

have also been reported to be associated with satellite RNAs ofCMV .

CMV isolates infecting tobacco, petunia, chrysan­themum, carnation and amaranth were isolated and characterized at National Botanical Research Insti­tute, Lucknow on the basis of aphid transmission in non-persistent manner, particle morphology, SDS­PAGE and serological relationships with other CMV strains 10

-14

• Some isolates of CMV were analyzed earlier 15

, among them CMV-C and CMV-T showed the presence of sat-RNA in their genome. CMV iso­late from amaranth (CMV -A) indicating presence of sat-RNA in its genome induced severe leaf deforma­tion in N. tabacum cv. white burley and Amaranthus

hypochondriacus14• Based on earlier evidences that

sat-RNA modulates the symptom expression we speculated that severity of symptoms and lethal ne­crosis in tobacco may be due to sat-RNA associated with CMV-Awar isolate. To prove this hypothesis, inoculations were done on three tobacco cultivars using RNA preparations with or without sat-RNA and symptoms that appeared on experimentally inoculated plants were analyzed.

Amaranth isolate of CMV (CMV -A) containing sat-RNA, isolated earlier14 was used as source of ini­tial inoculum for virus propagation. Virus was puri­fied from inoculated leaves (100 g) of N. tabacum cv. white burley as described 16and suspended in sterile water (1 mL). Virus preparations were observed by transmission electron microscope using uranyl acetate (pH 4.2; 20g/mL) as negative stain . The purified virus preparations were examined by UV spectrum and electron microscopy to assess the presence of CMV particles and by inoculations on Chenopodium ama­ranticolor (an indicator host of the isolate).

Viral nucleic acid was extracted by disrupting the purified particles of CMV with sodium dodecy l sul­phate (SDS; 10 g/L) and an equal volume of phe­nol/chloroform followed by ethanol precipitation 12

.

Nucleic acid preparations suspended in diethyl pyro­carbonate (DEPC) treated water were treated with DNase and RNase separately at 37°C for 30 min. and infectivity of treated and untreated nucleic acid preparations was checked by inoculating them on C. amaranticolor.

614 INDIAN J EXP BIOL, JUNE 2000

Table !-Symptoms appeared on various hosts* after inoculation of purified CMV, CMV- RNA with and without satellite RNA

Hosts Purified RNA 1,2,3 & 4 RNA 1,2, 3 &4 Sat-RNA particles (+)sat-RNA (-)sat-RNA alone

N. benthamiana LN, SN, D or SMSP LN, SN, D or SMSP LMM

N. tabacum LC&SM LC, BF&SM LMM cv. Samsun NN

Nicotiana tabacwn SM&LD SM&LD LMM cv. White burley

SM =Severe mosaic, LD =Leaf deformations, LMM =Late mild mosaic, LC = Leaf crinkle, BF = Blister formatio :;s, LN =Lethal ne­crosis, SN =Stem necrosis, D =Death of plant and SMSP =Severe mosaic in surviving plant and(-)= no symptoms. * 5 replicates of each plant species were taken for experiments.

For separation of RNA species, electrophoresis was carried out in agarose gel under non denaturing conditions as described earlier 17

. The gel was stained

with ethidium bromide (0.5 Jlg/mL) and observed un­der UV light. RNAI and 2, RNA3 , RNA4 (all to­gether) and RNA5 (sat-RNA) were eluted from LMP agarose gel (I 0 giL) electrophoresis under protected conditions of RNA and suspended in DEPC treated water. To investigate the role of sat-RNA, inocula­tions of RNA preparations with and without sat-RNA were done on N. tabacum cv. white burley, N. tabacum.cv. samsun NN and N. benthamiana plant species. Severity of symptoms was judged by visual observations of inoculated plants for 5-6 weeks. The back inoculation tests were also conducted on C. amaranticolor to know the presence of virus in

symptom expressing plants .

Isolated virus particles (200 Jlg/mL) which showed UV spectrum characteristic of nucleoprote in (with A 260/280 ratio of 1.58 ). Negatively stained samples of the preparation when observed under electron micro­scope reveal ed presence of 28 nm diam (typical of CMV) virus particles. Purified vi rus preparations were found infectious when inoculated on C. ama­raticolor.

Nucleic acid extracted from purified particles was also found infectious on C. amaranticor after being treated with DNAse but after treatment with RNAse infectivi ty was completely lost which indicated the presence of RNA as infectious entity. Electrophoresis of extracted nucleic acids revealed four di st inct bands on agarose gels ( 15 g/L) which were identified as RNA I and RNA2, RNA3, RNA4 and RNA5 (sat­RNA) while RNA I and 2 were very close and did not separate as two distinct bands. Pattern of all RNA species was similar to the one usually observed from

1 g. 2

3

4

5

Fig. 1-Agarose gel electrophoresis of RNA preparations ex­tracted from purified CMV particles. Lane I and 2 showing sepa­ration of four bands corresponding to RNA I & 2, RNA 3, RNA 4 and a satellite RNA (RNAS).

CMV 18. A sat-RNA of about 350 bases nearer to

bromophenol blue front was observed (Fig. I) as re-d I. 19

porte ear 1er .

Experimental inoculations of purified preparations and total RNA extracted from particles on N. bentha­miana plant ex hibited lethal necrosis in inoculated leaves after 5-6 days followed by systemic stem ne­crosis and death of plants after 15-20 days (Fig. 2). In a few surviving plants, lateral emerging branches ex­hibited severe systemic mosaic symptoms after 25-30 days . RNA preparation without sat-RNA did not pro­duce such severe symptoms, only late mild mosai c was observed on N. benthamiana. Results from Table I also revealed that N. tabacum cv. white burley and N. tabacum samsun NN plants developed severe mo-

NOTES 615

Fig. 2-Nicotiana benthamiana plants inoculated with total RNA preparations containing satellite RNA showing systemic stem necrosis (left) and healthy N. benthamiana plant (right).

saic, leaf deformation and blister formation symptoms after 15-20 days of inoculations with a RNA prepara­tion having sat-RNA, but no lethal necrosis .

CMV genomic RNA (RNA 1, 2, 3 and 4) with sat­RNA could induce only mild mosaic symptoms on N. tabacum cv. white burley, N. tabacum samsun NN and N. benthamiana plants. Satellite RNA alone did not induce any of the symptoms on these plants . However, when the inoculum contained both genomic and sat-RNA together, the symptoms became severe, resulting in lethal necrosis, leal deformation and stem necrosis or death of the plants (Table 1) . These re­sults indicate that sat-RNA is a major cause for en­hancement of severity of symptoms in inoculated to­bacco plants. Moreover, other Solanaceous species like Lycopersicon esculentum, Solanum melongena and Capsicum qnnum did not show local and systemic symptoms when inoculated with a purified CMV preparation (data not included) .

Satellite RNAs are the parasites of plant viruses which depend upon their helper viruses for their'F_ep­lication and encapsidation. Usually sat-RNAs reduce the level of helper virus accumulation and auenuate disease symptoms induced by helper virus3

. However, some sat-RNAs of CMV intensify the viral disease symptoms i.e. yellow7

, white8 or systemic necrosis20

in some hosts . Satellite RNA has been reported as causal agent for tomato necrosis2

·5

·6

·2

' · , its role in eti­ology of tomato fruit necrosis in Italy has been ob­served earlier22

.

Kaper et a/. 23 have reported that CMV sat-RNA induced necrosis in . tomato plants and suggested that symptom modulation depends upon trilateral interac­tion among sat-RNA, virus and host plant during in­fection but it was not clear whether these factors are of host or satellite origin . 1he, cell death (necrosis) and severe yellowing (chlorosis) in tomato induced by cucumber mosaic virus (CMV) supporting par­ticular satellite RNAs have been reported recentl/ 4

.

They have also determined whether CMV RNA se­quences are needed to induce necrosis or chlorosis and they infected tomato seedlings with potato virus X (PVX) vector expressing either a necrosis or chlo­rosis inducing sat-RNA of CMV. Infected plants de­veloped necrosis but only all or part of a 335 nucleo­tide necrogenic sat-RNA was expressed in ( ~) polar­ity; ie, the strand not packaged in virus particles. Plants often respond to virus infection with the devel­opment of hypersensitive reaction in which virus is confined or restrict by a localized necrosis but it is unusual that necrosis spreads through out the plant resulting in systemic lethal necrosis. It has been ob­served that alteration in either pathogen genes25or plant genes26 can affect the extent of cell death.

The observations of earlier workers clearly explain that sat-RNA somehow, enhances severity of CMV symptoms in infected plants. Chandra 10 has observed severe necrosis in N. tabacum cv white burley and suggested that the severe necrosis in tobacco is due to sat-RNA present in CMV-T isolate. We have demon­strated that sat-RNA associated with some isolates of CMV genome has a role in enhancing severity of symptoms inN. tabacum cv white burley, N. tabacum cv samsun NN, leading to death of N. benthamiana plants and exclusion of sat-RNA from the RNA preparation resulted in complete abolition of lethal necrotic/severity of symptoms in these plants . How­ever, we could not study the CMV accumulation with and without sat-RNA in these plants species at differ­ent intervals of post inoculation . We described the existence of a sat-RNA in an Indian isolate of CMV which causes systemic lethal necrosis of tobacco. Furthermore, the lethal necrosis induced in N. benthamiana by ;;at-RNA associated with CMV ama­ranth isolate would be a first report.

Thanks are due to DBT, India for financial support and CSIR, New Delhi for fellowship to the author (AS) .

616 INDIAN J EXP BIOL, JUNE 2000

References I Palukaitis P, Roossinck M J, Dietzgen R G & Francki RIB,

Cucumber Mosaic Virus. Adv Virus Res, 41 (1992) 281. 2 Kaper J M & Waterworth H E, Cucumber Mosaic virus asso­

ciated RNA 5: Causal agent for tomato necrosis. Science, 196 (1977) 429.

3 Roossinck M J, Sleat D & Palukaitis P, Satellite RNAs of plant viruses: Structures and biological effects. Microbial Rev, 56 (1992) 265.

4 Osaka, Y. Hanada, K. Fukunishi, T & Tochihara, H, Cucum­ber mosaic virus isolate causing tomato necrosis disease in Kyoto prefecture. Ann Phytopathol Soc JPN, 55 ( 1989) 229.

5 Jorda C, Alfaro A, Aranda M A, Moriones E & Garcia­Arena! F, Epidemic of cucumber mosaic virus plus satellite RNA in tomatoes in Eastern Spain. Plant Dis, 76 (1992) 363 .

6 Skoric D, Krajacic M, Barbarossa L, Cillo F, Grieco F, Saric A & Gallitelli D, Occurrence of cucumber mosaic virus with satellite RNA in lethal necrosis affected tomatoes in Croatia. J Phytopatlw l, 44 ( 1996) 593.

7 Takanami Y, A striking change in symptoms on cucumber mosaic virus - infected tobacco plants induced by a satellite RNA. Virology, I 09 ( 1981) 120.

8 Gonsalves D, Providenti R & Edwards M C, Tomato white leaf the relation of an apparent satellite RNA and cucumber mosaic virus. Phytopathology, 72 (1982) 1533.

9 Palukaitis P, Pathogenicity regulation by satellite RNA of Cucumber mosaic virus: Minor nucleotide sequence changes alter host responses. Mol Plant-Microb Interact, I ( 1988) 175.

10 Chandra G, Ph .D. Thesis, Kanpur University, Kanpur (199 1).

II Srivastava A, Chandra G, Raizada R K, Srivastava K M & Singh B P, Characterization of a virus causing necrosis in Petunia hybrida and establishment of its relationship usi ng ELISA. Indian J Exp Bioi, 29 ( 1991) 591.

12 Srivastava K M, Raj S K & Singh B P, Properties of a cu­cumber mosaic vi rus strain naturally infecting chrysanthe­mum in India. Plant Dis, 76 (1992) 474.

13 Raj S K, Aminuddin, Srivastava K M & Singh B P, Natural infection of Cucumber mosaic virus on Dianthus barbatus in India. Plant ?athol. 42 ( 1993) 811.

14 Raj S K, Arninuddin, Singh B P & Pal M, Characterization of a cucumber mosaic virus isolate causing leaf crinkle and

severe mosaic of Amaranthus in India. Can J Plant ?athol, 19 (1996) 97.

15 Raj S K, Chandra G & Singh B P, Some Indian strains of cucumber mosaic virus lacking satellite RNA. Indian J Exp Bioi, 35 (1997) 1128.

16 Lot H, Marrou 1 B & Esvan C, Contribution a !'etude du virus de Ia rnosaique du comcornbre (CMV) II. Ann Phyto­pathol, 4 (1972) 25.

17 Sarnbrook J, Fritsche E F & Maniatis T, Molecular cloning -A Laboratory Manual, 2nd Edition Cold Spring Harbor Laboratory Press, New York) 1989.

18 Francki R I B, Mossop D W & Hatta T, Description of plant viruses vol. 213 (Common Wealth Mycology Institute Asso­ciation Applied Biologists; Kew, England) 1979.

19 Ohashi M & Karniunten H, Mosaic disease of grain amaranth (A. hypochondriacus L.) Ann Phytopalhol Soc JPN, 60 (1994) 119.

20 Sleat D E & Palukaitis P. A single nucleotide change within a plant virus satellite RNA alters the host specificity of dis­ease induction. Plant J, 2 (1992) 43.

21 Gallitelli D, Di F(anco A, Vovlas C and Kaper J M, Mixed infecti on of CMY and potyviruses in vegetable crops in southern Italy. lnf Fitopathol, 38 ( 1988) 57.

22 Crescenzi A, Barbarossa L, Cillo F, Di Franco A, Vovlas N & Gallitelli D, Role of cucumber mosaic virus and its satel­lite RNA in etiology of tomato fruit necrosis in Italy. Arch Viral, 131 (1993) 321.

23 Kaper 1M, Geletka L M, Wu G S & Tousignant M E. Effect of temperature on Cucumber mosaic virus satellite - induced lethal tomato necrosis is helper virus strain dependent. Arch Viral, 140 (1995) 65.

24 Taliansky M E. Ryabov E V, Robinson D 1 & Palukaitis P, Tomato cell death mediated by complementary pl ant viral satellite RNA sequences. Mol Plant-Microb Interact, II (1998) 1214.

25 Kim C H & Palukaitis, P, The plant defence response to cucumber mosaic virus in cowpea elicited by viral polymer­ase gene and affects virus accumulation in single cells. EMBO J, 16 ( 1997) 4060.

26 Mur LA 1, Bi Y-M, Darby R M, Firek S & Draper J, Corn­promising early salicylic acid accumulation delays the hyper sensitive response and increases viral dispersal during lesion establishment in TMV infected tobacco. Plant J, 12 ( 1997) 1113.