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Wessjohann, L. A. (13)Wessjohann, L. (11)Kutchan, T. M. (9)Miersch, O. (9)Franke, K. (8)Strack, D. (8)Schmidt, J. (6)Tran, V. S. (6)Wasternack, C. (6)Hause, B. (5)Nguyen, T. H. A. (5)Nguyen, T. H. V. (5)Westermann, B. (5)Abel, S. (4)Braga, A. L. (4)Brandt, W. (4)Fester, T. (4)Feussner, I. (4)Kramell, R. (4)Ruijter, E. (4)Arnold, N. (3)Baumert, A. (3)Frick, S. (3)Lee, J. (3)Levy, M. (3)Milkowski, C. (3)Neuhaus, C. (3)Porzel, A. (3)Scheel, D. (3)Schliemann, W. (3)Schneider, P. H. (3)Abdala, G. (2)Ammer, C. (2)Becker, H. C. (2)Dörner, S. (2)Felix, G. (2)Göbel, C. (2)Hause, G. (2)Hornung, E. (2)Hüsken, A. (2)Jungblut, P. R. (2)Lübken, T. (2)Lüdtke, D. S. (2)Möllers, C. (2)Nimtz, M. (2)Nürnberger, T. (2)Paixão, M. W. (2)Rivera, D. G. (2)Rosahl, S. (2)Silveira, C. C. (2)Stenzel, I. (2)Sung, T. V. (2)Teichert, A. (2)Thuy, T. T. (2)Voigt, B. (2)Wray, V. (2)Zenk, M. H. (2)Ziegler, J. (2)Abbas, M. (1)Acotto, G. P. (1)Ahlfors, R. (1)Alemano, S. (1)Alves, E. F. (1)Andrade, A. (1)Apel, K. (1)Appelt, H. R. (1)Arbona, V. (1)Barth, A. (1)Bartsch, M. (1)Basso, A. (1)Bednarek, P. (1)Belbahri, L. (1)Besl, H. (1)Bevan, M. (1)Beveridge, A. (1)Bhat, R. (1)Bittner, F. (1)Boettcher, C. (1)Boller, T. (1)Botella, M. A. (1)Brosché, M. (1)Burns, S. A. (1)Cahill, D. J. (1)Carbonne, F. (1)Carsjens, J.-G. (1)Castresana, C. (1)Cenzano, A. (1)Chen, K. (1)Chu, I. K. (1)Clemens, S. (1)Clifford, A. J. (1)Colby, T. (1)Danon, A. (1)De la Peña, M. (1)De-Eknamkul, W. (1)Dhondt, S. (1)
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Publikation
Chrysanthemum chlorotic mottle viroid (CChMVd) RNA (398–401 nucleotides) can form hammerhead ribozymes that play a functional role in its replication through a rolling-circle mechanism. In contrast to most other viroids, which adopt rod-like or quasi-rod-like secondary structures of minimal free energy, the computer-predicted conformations of CChMVd and Peach latent mosaic viroid (PLMVd) RNAs are branched. Moreover, the covariations found in a number of natural CChMVd variants support that the same or a closely related conformation exists in vivo. Here we report that the CChMVd natural variability also supports that the branched conformation is additionally stabilized by a kissing-loop interaction resembling another one proposed in PLMVd from in vitro assays. Moreover, site-directed mutagenesis combined with bioassays and progeny analysis showed that: (1) single CChMVd mutants affecting the kissing loops had low or no infectivity at all, whereas infectivity was recovered in double mutants restoring the interaction; (2) mutations affecting the structure of the regions adjacent to the kissing loops reverted to wild type or led to rearranged stems, also supporting their interaction; and (3) the interchange between 4 nucleotides of each of the two kissing loops generated a viable CChMVd variant with eight mutations. PAGE analysis under denaturing and nondenaturing conditions revealed that the kissing-loop interaction determines proper in vitro folding of CChMVd RNA. Preservation of a similar kissing-loop interaction in two hammerhead viroids with an overall low sequence similarity suggests that it facilitates in vivo the adoption and stabilization of a compact folding critical for viroid viability.