Abdul Samad, Noraini

El-Sanousi, Omar Mussa

Ong, C. A.

Universiti Putra Malaysia Institutional Repository

UPM Research Report 1997-2000, Vol II, Section 2-Extended AbstractsBiological interactions of two satellites RNAs with some cucumber mosaiccucumovirusO.M. EI-5anouel, CA one and Noranl Audul 5amadFaculty of Science and Environmental StudiesUniversiti Putra Malaysia43400 UPM. Serdang, SelangorMalaysiaTelephone Number of Corresponding Author: 03-89466709E-mail ofCo"e.<pondingAuthornoraini@f.~a.<.upm.edu.myKey words: tomatoes, chillies, cucumber mosaic cucumovirus, satellite RNAsIntroductionCucumber mosaic virus (CMV) satellite-RNA (sat-RNA) often interferes with the replication of the helper virusgenome and affects viral concentration and symptom expression (Roossinck el al., 1992). The association of sat-RNAwith CMV generally reduced the virus accumulation (Mossop and Francki 1979; Takanami , 1981; Gonsalves etal.1982). The alteration of CMV symptoms by sat-RNA could be either an attenuation or an exacerbation. Waterworthet al. (1979) reported that Tabasco pepper infected with CMV-WT strain free from sat-RNA developed a severemosaic, stunting and necrosis of leaves, petioles and stem. The association of sat-RNA with this strain resulted inmild chlorosis to non-infection to this cultivar. The CMV disease symptoms in tomato could be attenuated by sat-RNA(Yoshida et al., 1985). Masuta et al. (1988) reported that sat-RNA of Y strain attenuated the CMV disease in severalplant species. The sat-RNA had been used to protect pepper plants against a virulent strain of CMV (Tien et al., 1987;Wu et al., 1989). The most prevalent example of disease exacerbation was the systemic necrosis of tomato which leadto collapse of the infected plants (Kaper and Waterworth, 1977; Waterworth et al., 1978; Nakashima and Ehar, 1989;Masuta et al., 1990; Kaper et al., 1995; White et al., 1995). Particular sat-RNA sometimes had different diseaseexpression with different strains of CMV. Masuta et al. (1988) reported that the association of sat-RNA Y with CMV-Y strain induced systemic necrosis on tomato but no necrosis occurred when it was associated with CMV -0 strain.Two sat-RNAs were found to be associated with CMV-6 (from infected purple cleome in Selangor) and CMV-7 (frominfected tobacco in Kelantan). They were designated as sat-RNA6 and sat-RNA7, respectively (EI-Sanousi et al. 1994;1996; 1998 ). The interaction of these sat-RNAs with their helper viruses and two other isolates and CMV-3 andCMV -4 are presented here by carrying out artificial introduction of the sat-RNA into each of the four CMV isolates.Materials and MethodsExtraction of Viral RNA Four virus isolates, CMV-3, CMV-4, CMV-6 and CMV-7, were purified from infected N.tabacum cv. White Burley leaves by the method of Mossop et al. (1976). The viral RNAs of each virus were isolatedfrom purified virus using the proteinase K and phenol-SDS procedure as described by Sambrook el al. (1989). In eacheppendorf tube 200 J.l1(I J.lg/J.lI)of virus was mixed with an equal volume of extraction buffer(0.03 M of KCI, 3 mMMgCI2, 0.01 M ofSDS, 0.02 M Tris HCI) and 50 J.lIof proteinase K (2 mg/ml) and incubated for 20 min at 500 C. Then80 J.l1of I M NaCI and one volume of equilibrated phenol (500 C) were added. The preparation was mixed vigorouslyand incubated for 5 min at 500 C. After centrifugation at 12,000 g for 5 min, the aqueous phase was removed andsubjected to another phenol extraction. The aqueous phase was removed and one volume of chloroform isoamylalcohol (24:1) was added and centrifuged at 12,000 g for 10 min at 40 C. The aqueous phase was removed andsubjected to two further chloroform isoamylalcohol extractions. The final aqueous phase was removed and twovolume of ethanol and one tenth volume of 3 molar sodium acetate were added and placed at _700 C. The precipitatewas collected by centrifugation at 12,000 g for IS min and washed once in 70 % ethanol dried and suspended in TEbuffer (10 mM Tris-HCI, I mM EDTA, pH 8.3) and stored at _200C.Recovery of Sat-RNA from the Gel: After electrophoresis, (sat-RNA) bands of CMV-6 and CMV-7 were cut outfrom the gel, and placed in sterile dialysis bags with 1-2 ml of TBE buffer. The current was run at 70 V for 1 hr; thepolarity was reversed for I min and the eluted sat-RNAs were reconcentrated by extraction with an equal volume of2-butanol until 0.5 ml of buffer phase was obtained. The RNA was finally precipitated with two volumes of ethanol aspreviously described for the virus RNA. The precipitate was collected by centrifugation at 12,000 g for 15 min andwashed once in 70% ethanol, dried, suspended in TE buffer and stored at _200 C.Inoculation with Sat-RNAEach sat-RNA was adjusted to 20 ug/rnl, mixed and inoculated with the virus genome ofeach CMV isolate to N. tabacum cv, White Burley. The presence of sat-RNA in each isolate was tested by further virus118Agricultural Sciencespurification followed by RNA extraction and RNA electrophoretic analysis as previously described. The response ofeach isolate to the sat-RNA was determined by hosts inoculation. The inoculum was prepared by homogenization ofinfected White Burley tobacco leaves in the presence of phosphate buffer.Elimination of Sat-RNA from Genomic RNA: To eliminate the sat-RNA from CMV-6 and CMV-7 the genomicRNAs 1-3 were cut and recovered from gels by electroelution, reconcentration and precipitation as describedpreviously in recovery of sat-RNA from the the gel band except the current was run at 70 vol for 2 hrs. The genomicRNAs were then inoculated into the local lesion host, C. amaranticolor. Single local lesion from C. amaranticolor wasthen inoculated to individual White Burley tobacco plant. The satellite free genomic RNAs were then tested againsttomato seedlings by inoculation and further purified and isolated.Results and DiscussionAlthough there was no difficulty in introducing sat-RNA into the CMV isolates, but there was difficulty in eliminatingthese sat-RNAs from their natural CMV isolates. The attempts to free CMV isolates from sat-RNA was successfulwith CMV-7 but not with CMV-6. The association of both sat-RNAs with the four CMV isolates reduced the virusconcentration in White Burley tobacco by ten times.The interference of the sal-RNAs on the symptoms induced by CMV isolates on different hosts showed that theassociation of sat-RNA6 with the CMV-3, -4 and -7 isolates lead to severe systemic necrosis in L. esculetnum cv.Eggtomato and local necrosis in N. tabacum cv, White Burley. Although sat-RNA6 did not interfere with its naturalisolate (CMV-6) in infection ofMC4 chilli but it prevented infection by CMV-7 .CMV-1 free of sat-RNA produced mosaic symptoms in MC4 chilli. The association of sat-RNA7 with this isolateresulted in non-infection ofMC4 chilli. Association of sat-RNA7 with CMV-3 and CMV-4 resulted in reduction of thenumber of MC4 chilli plants infected by these isolates and to severe lethal necrosis in tomato. The results also showedthat sat-RNA 7 attenuated the symptom caused by CMV -4 from severe mosaic to mosaic in White Burley tobacco.The combination ofCMV-3, CMV-4, CMV-7 and CMV-6 with sat-RNA6 showed a much severe symptoms than sat-RNA7 with the respective CMV isolates in inducing necrosis in tomato. The artificial introduction of sat-7 intoCMV-3 and CMV-4 induced a more severe tomato necrosis as compared by its natural host, CMV-7 whichcaused a systemic necrosis to very few numbers of inoculated tomato plants. Sat-RNA 7 with CMV -3, -4 and CMV-7showed more reduction in numbers of infected plants of chilli MC4 than sat-RNA6 with the respective CMV isolates.The results also showed that the prevention of infection of MC4 chilli by CMV in the presence of sat-RNA was isolatedependent, since CMV-7 infection to MC4 chilli prevented by the presence of either sat-RNA-6 or-7ConclusionsThe presence of satellite RNA with the helper virus, CMV, showed mixed reaction in the tomato varieties tested. Thesatellite RNA-mediated resistance was only effective in certain varieties. Four of the varieties were resistant to ToMV.Only one variety showed positive satellite RNA-mediated resistance and also resistant to ToMV.Benefits from the studyProduction system and proper management of virus diseases for tomato growing in the tropical regions.Patent(s), if applicable:NilStage of Commercialization, if applicable:NilProject Publications in Refereed JournalsI.Abdul-Samad, N., M. Singh and K. Yusoff. 1996. Detection of tomato mosaic tobamovirus from Malaysia. Pertanika J. Trop.Agric, Sci. 19, 1-6.2. Abdul-Samad, N., T.K.Teh and K. Yusoff. 1996. Detection of tomato mosaic tobamovirus infection by double stranded RNAanalysis in infectedplants.MalaysianApplied Biology25,115-117.3. El-Sanousi, D., CA. Ong, K. YusofT,S. Napis and N. Abdul-Samad. 1998. Propertiesof four cucumber mosaic virus isolates fromWestMalaysia.Asia PacificJournal of MolecularBiology& Biotechnology6, 131-1374. El-Sanousi. 0..C.A. Ong, K. Yusof, S. Napis and N. Abdul-Samad. 2003. Biological interaction of rwo satellite RNAs withcucumber mosaic cucumovirus. Asia Pacific Journal of MolecularBiology& Biotechnology10,5-9119UPM Research Report 1997-2000. Vol II. Section 2-Extended AbstractsProject Publications In Conference ProceedingsI. El-Sanousi, 0., C.A Ong, S. Napis. K.Yusoff and N. Abdul-Samad. 1996. Serology and PCR identification of several isolates ofcucumber mosaic cucumovirus. Proc: Ninteeih Symposium of Malaysian Microbiolgy Society. Trengganu.2. EI-Sanousi, 0., A.A. Ong and N. Abdul-Samad. 1997.Satellite RNA from cucumber mosaic cucumovirus. Proc: Sixteenth ASIAN-Pacific Weed Science Society Conference. Kuala Lumpur.3. El-Sanousi, O. 1997.Differentiation and genetic studies of several isolates of cucumber mosaic virus. PhD Thesis. Universiti PutraMalaysia, Serdang.4 Quah, J.L and N. Abdul-Samad. 1998. Infectivity studies in tomatoes on the effect of cucumber mosaic virus in the presence andabsence of satellite RNA. Proc: Malaysian Society of Applied Biology Conference. Kuala Lumpur.s. El-Sanousi, 0., C.A. Ong, K. YusofT and N. Abdul-Samad. 1999. Properties of two satellite RNAs in cucumber mosaiccucumovirus isolates. Proc: Fifth International Conference on Plant Protection in the Tropics. Kuala Lumpur.Graduate Rescore"NameGraduateof Research Topic Field of Expertise Degree Awarded Graduation YearOmar El-Sanousi Differentiation and Virologygenetic studies ofseveral isolates ofcucumber mosaicvirusPhD 1997IRPA Project number: 01-02-04-0445UPM Research Cluster:AFF120

Biological interactions of two satellites RNAs with some cucumber mosaic cucumovirus

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Biological interactions of two satellites RNAs with some cucumber mosaic cucumovirus

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