Study On 2b Gene Of Cucumber Mosaic Virus And Its Interaction Between Satellite RNAs In Nicotiana.Spp

Cucumber mosaic virus (CMV) is a prevalent plant pathogen all over the world, and has an extremely large host range of over 1000 plant species. Because of its relatively simple genome organization, CMV was regareded as a model for study on the pathogenicity of RNA virus. In this study, we investigated 2b gene or satRNA of CMV and the interaction between 2b gene and satRNA in Nicotiana. spp. The results were shown as follow:1. Construction of infectious transcripts RNA for Cucumber mosaic virus Phy strainBy serological ELISA and SDS-PAGE electrophoresis analysis of virus particle, the results showed that chlorosis symptom on the tissues of Capsicum frutescens from Hangzhou suburb was induced by Cucumber mosaic virus (CMV), CMV-Phy, and TBE electrophoresis of CMV-Phy viral RNAs indicated that CMV-Phy contained a satellite RNA (satRNA). Similar symptom expression on the Nicotiana glutinosa plants inoculated with the sap from C. frutescens tissues at 14 dpi (days post-inoculatio, dpi).By touch-up RT-PCR, the full length of CMV-Phy genomic RNA1, RNA2 and RNA3 were amplified in a reaction, when using the CMV-Phy viral RNAs as the template. The PCR products of RNA1, RNA2 and RNA3 were ligated to pUC118 vector (pUC-P1, pUC-P2 and pUC-P3), and the ligated products were transformed into Escherichia coli. Comparison of transformation efficiency among DH5a, HB101, JM109, LE392, and NM522 E. coli competent cell was performed and the results suggested that only the HB101 was suitable to pUC-P1 transformation and but all the five kinds of E. coli were fit for pUC-P2 and pUC-P3 transformation. Sequence results showed the full length of CMV-Phy genomic RNA1, RNA2 and RNA3 was 3356 nt (nucleotide, nt), 3048 nt and 2220 nt, respectively (Accession Number: DQ402477, DQ412731 and DQ412732). Sequences aliment suggested that CMV-Phy belonged to Subgroup I but consisted of Subgroup IA RNA1, RNA2 and Subgroup IB RNA3. Thus, CMV-Phy was considered as a reassortant strain of CMV.cDNA clones of CMV-Phy genomic RNAs (pUC-P1, pUC-P2 and pUC-P3) were transcribed into RNAs and the infectivity of pUC-P1, pUC-P2 and pUC-P3 was analyzed. The results showed that in vitro transcription efficiency improved but the infectivity of transcripts RNA declined when two guanosines added to 5' terminal of pUC-P1, pUC-P2 and pUC-P3 clones. Biological assays of CMV-Phy infectious clone indicated that the symptom on the seedlings of Chenopodium amaranticolor and N. glutinosa induced by CMV-Phy transcripts RNA was the same as that induced by CMV-Phy virion. CMV-Phy transcripts RNA mixture plus Pz-satRNA, T1-satRNA, Rs-satRNA and Tsh-satRNA respectively, was co-inoculated on the tissues of N. glutinosa, and dsRNA detection results showed that CMV-Phy could support other CMV-satRNA accumulation in the host plants besides Pz-satRNA and that all the satRNA did not attenuate the symptom caused by CMV-Phy.2. Leu 55 of Cucumber mosaic virus 2b protein was genetic determinant for necrosis pathotype in N. glutinosaA tomato strain of CMV (CMV-CB7) induced top necrosis symptom on the seedling of N. glutinosa. Full-length cDNA sequence of genomic RNA1, RNA2, RNA3 was 3356 nt, 3045 nt and 2218 nt, respectively (Accession Number: EF216866, DQ785470 and EF216867). Infectious RNA transcripts from cDNA clones of CMV-CB7 were inoculated onto the tissues of N. glutinosa and the host plant expressed top necrosis symptom. Through pseudorecombination between CMV-CB7 and CMV-Fny genomic RNAs, we confirmed that the genetic determinant of necrosis phenotype was mapped to RNA2. Chimeric RNA2 consisting of CMV-CB7 and CMV-Fny was obtained by Overlapping PCR, and further assessments of pathogenidty for chimeric RNA2 of CMV-CB7 and CMV-Fny revealed that the 2b gene or 3' terminal of CMV-CB7 RNA2 was responsible for top necrotic pathotype. Northern blot results showed necrotic (CMV-CB7, CMV-Fny^F5C3) and non-necrotic (CMV-Fny, CMV-Fhy^C5F3) virus accumulated to similar levels in in host tissues. Therefore, the phenotype of CMV-CB7 on the N. glutinosa tissues was not related to its high genomic RNAs.2b genes of four different CMV isolates was replaced that of CMV-Fny respectively and the results from biological assays of these CMV-Fny mutants indicated that 2b gene led to the top necrosis phenotype on the seedlings of N. glunotisa infected by CMV-CB7. CMV-35# (isolated from Radish) induced slightly mosaic on the tissues of N. glunotisa, and but sequence homology was highest between 2b^CB7 and 2b^35#. Six chimeric 2b genes were constructed and results of pathogenicity for these CMV-Fny 2b gene mutants showed that Leu52 and Leu 55 of 2b protein might be responsible for the necrosis pathotype of CMV-CB7.2b protein was encoded CMV RNA2 subgenomic RNA and there was a overlapping region between N ' terminal of 2b and C ' terminal of 2a. Thus, 52 (L_cb7/F_35#) or 55 (L_Cb7/P_35#) in the 2b protein and 38 (F_Cb7/L_35#) or 52 (P_CB7/T_35#) were reciprocally mutated and all the eight mutants were inoculated onto the seedlings of N. glunotisa. The results indicated that 55 Leu of CMV-CB7 2b proteins was genetic determinant for top necrosis pathotype.The level of CMV-Fny2b^CB7, CMV-Fny2b^35#, CMV-Fny2b^CB7164 and CMV-Fny2b^35#164 genomic RNAs was nearly equal by Northern blot analysis. Therefore the top necrosis on N. glutinosa tissues induced by CMV-CB7 was associated with increased viral RNA accumulation, suggesting that other functions of the 2b protein are important in determining the necrosis hypervirulence in N. glutinosa.3. Genetic mapping of the satellite RNA of Cucumber mosaic virus for high accumulation in N. tabaccumAccording to the CMV-satRNA sequences deposited in the GenBank database, we designed CMV-satRNA full length sequence primers and obtained two satRNAs (Yi-satRNA and Yn12-satRNA) from the CMV-infected tomato and CMV-infected radish by RT-PCR, respectively. Full length of Yi-satRNA and Yn12-satRNA was 387 nt and 385 nt (Accession Number: DQ412733 and EF363688). Comparison of nucleotide sequences between Yi-satRNA and Ynl2-satRNA, there only differ in 3 positions: position 11(Yi-satRNA A, Yn12-satRNA U), position 269 (Yi-satRNA A, Yn12-satRNAU) and position 367-369 (Yi-satRNA UAU, Yn12-satRNAAΔΔ).When transcripts RNA from cDNA clone of Yi-satRNA and Yn12-satRNA plus CMV-Fny and CMV-CB7, respectively, was co-inoculated on the seedlings of N. tabacum, dsRNA detection results showed that CMV-Fny could save as helper virus for two satRNA and both two satRNA accumulated high level in the host plants, but CMV-CB7 only supported Yi-satRNA high accumulation. At 14 dpi, relative accumulation of CMV-CB7 and CMV-Fny plus Yi-satRNA or Yn12-satRNA genomic RNAs was quantitatively analyzed, and results indicated that the accumulation of CMV-CB7 genomic RNAs in presence of Yi-satRNA was 6-15.8%.Yi-satRNA and Yn12-satRNA reciprocal mutants plus CMV-CB7 was inoculated onto host plants. At 14 dpi, Northern blot showed that 11A and 269 A did not affect the accumulation of Yi-satRNA in host plant, but Yi-sat 367-369 AΔΔand Yi-sat 269T367-369 AΔΔmutants did not existed in the tissues of N. tabaccum. Therefore, high accumulation of Yi-satRNA related to its 367-369 UAU. When 11U and 367-369 AΔΔwas mutated to A and UAU receptively, CMV-CB7 could support high accumulation of Ynl2-satRNA. Six pseudorecombinant viruses between CMV-CB7 and CMV-Fny plus Ynl2-satRNA were co-inoculated into the host plants, and SatRNA in the systemic leaves detected by RT-PCR. The results indicated that only F1C2C3, F1C2F3 and F1F2C3 could harbor Yn12-satRNA in the seedlings of N. tabaccum. Therefore RNA1 of CMV-CB7 was determinant factor of defective accumulation of Yn12-satRNA in the host plants. Our results suggested that the absence of Yn12-satRNA in the tissues of N. tabaccum inoculated with CMV-CB7 plus Ynl2-satRNA might was due to its no replication or low replication. 4. Effect of Cucumber mosaic virus 2b gene on satRNA accumulationRelative accumulation of Rs-satRNA in the seedlings of N. tabaccum, N. benthamiana and N. clevelandii infected by CMV-Fny plus Rs-satRNA was quantified by Realtime RT-PCR at 7 and 14 dpi. All the data showed that the level of Rs-satRNA was in a tendency of N. benthamiana>N. clevelandii>N. tabaccum. The results indicated that the accumulation of Rs-satRNA was related with the variety of host plants when Rs-satRNA plus CMV-Fny was co-inoculated onto the tissues of tobacco.The level of Rs-satRNA in the host plants inoculated with CMV-FnyΔ2b plus Rs-satRNA was equal to that inoculated with CMV-Fny plus Rs-satRNA at 7 and 14 dpi, and this suggested that 2b gene of CMV-Fny was not involved in the replication or accumulation of Rs-satRNA. When CMV-Fny, CMV-Fny2b^phy, CMV-Fny2b^35#and CMV-Fny2b^CB7 was used as helper virus of Rs-satRNA, respectively, the accumulation level of Rs-satRNA in the tissues of N. tabaccum, N. benthamiana and N. clevelandii plants was almost the same. Therefore, 2b gene exerted no effect on the accumulation of Rs-satRNA in the tobacco host plant. Our results suggested that the accumulation of Rs-satRNA was related with the variety of host plants and that 2b gene of CMV did not affect the level of Rs-satRNA in the seedlings of N. tabaccum, N. benthamiana and N. clevelandii.5. Satellite RNA-mediated reduction in accumulation of Cucumber mosaic virus genomic RNAs in N. tabacum related to 2b geneThe presence of Rs-satRNA apparently attenuated the symptoms induced by CMV-Fny on the seedlings of N. tabacum. At three post-inoculation points, relative accumulation of CMV-Fny and CMV-Fny plus Rs-satRNA genomic RNAs was quantitatively analyzed by Realtime RT-PCR, and the results showed that Rs-satRNA depressed the accumulation of CMV-Fny genomic RNAs.At 3 dpi, the accumulation of CMV-Fny1a, 2a, 2b, 3a, and was 7.6, 10.8, 9.8, 9.7 and 5.8 fold to that of CMV-Fsat respectively. At 10 dpi, the degree depressed by satRs was 2b> 2a >1a >3a> CP. The accumulation of CMV-Fsat1a, 2a, 2b, 3a and CP was 62.5%, 47.6%,34.5%, 30.3% and 52.6% that of CMV-Fny, respectively. Although the accumulation of CMV-Fny and CMV-Fsat was especially low at 21 dpi, CMV-Fny genomic RNAs, the accumulation of CMV-Fny 1a, 2a, 2b, 3a and CP was 11.6, 24.3, 37.6, 3.9 and 7.0 fold to that of CMV-Fsat in newly growing leaves, respectively. Therefore, the effect of Rs-satRNA on the accumulation of 2b gene was comparatively remarkable.CMV-Fny lacking 2b gene (CMV-FnyΔ2b) appeared mild mosaic ont the host tissues, similar to that of CMV-Fsat, and the presence of Rs-satRNA did not show further attenuation of the symptoms induced by CMV-FnyΔ2b. The level of CMV-FnyΔ2b genomic RNAs was only 0.5-5.0% that of CMV-Fny and Rs-satRNA did not reduce substantially the accumulation of CMV-FnyΔ2b genomic RNAs. It suggested that slightly mosaic symptoms on the tissues of N. tabaccum induced by CMV-FnyΔ2b was due to the low level its genomic RNAs and that Rs-satRNA did not affect the replication and /or accumulation of CMV-Fn when 2b gene was deleted.When the accumulation of CMV, 1a, 1b, 2b, 3a, and CP in both in inoculated leaves and systemic leaves were quantified by Realtime RT-PCR, the results suggested that the long distance movement of CMV-Fny in presence of Rs-satRNA declined.The 2b gene of different Cucumoviruses has been shown to have a major effect on viral long-distance movement and virus inducing RNA silencing. Our results indicated that the attenuation of CMV-Fny by Rs-satRNA was due to low accumulation of CMV-Fsat genomic RNAs and that Rs-satRNA might function in a way antagonistic to the function of the 2b gene, resulting in decrease of the accumulation of CMV-Fny genomic RNAs.