Publikationen - Molekulare Signalverarbeitung

Cachexia disease of citrus is caused by Hop stunt viroid (HSVd). In citrus, pathogenic and non-pathogenic strains differ by a “cachexia expression motif” of five to six nucleotides located in the variable domain of the proposed rod-like secondary structure. Here, site-directed mutants were generated to investigate if all these nucleotides were required for infectivity and/or symptom expression. Specifically an artificial cachexia inducing mutant M0 was generated by introducing the six nucleotides changes of the “cachexia expression motif” into a non-pathogenic sequence variant and M0 was used as a template to systematically restore some of the introduced changes. The resulting mutants in which specific changes introduced to generate M0, were restored presented a variety of responses: (i) M1, obtained by introducing two insertions forming a base-pair, was infectious but non-pathogenic; (ii) M2, obtained by introducing an insertion and restoring a substitution, presented low infectivity and the resulting progeny reverted to M0; (iii) M3, obtained by restoring a single substitution in the lower strand of the viroid secondary structure, was infectious but induced only mild cachexia symptoms; (iv) M4, obtained by restoring a single susbtitution in the upper strand of the viroid secondary structure, was non-infectious. These results confirm that the “cachexia expression motif” plays a major role in inciting cachexia symptoms, and that subtle changes within this motif affect symptom severity and may even suppress symptom expression.

Infection by viroids, non-protein-coding circular RNAs, occurs with the accumulation of 21–24 nt viroid-derived small RNAs (vd-sRNAs) with characteristic properties of small interfering RNAs (siRNAs) associated to RNA silencing. The vd-sRNAs most likely derive from dicer-like (DCL) enzymes acting on viroid-specific dsRNA, the key elicitor of RNA silencing, or on the highly structured genomic RNA. Previously, viral dsRNAs delivered mechanically or agroinoculated have been shown to interfere with virus infection in a sequence-specific manner. Here, we report similar results with members of the two families of nuclear- and chloroplast-replicating viroids. Moreover, homologous vd-sRNAs co-delivered mechanically also interfered with one of the viroids examined. The interference was sequence-specific, temperature-dependent and, in some cases, also dependent on the dose of the co-inoculated dsRNA or vd-sRNAs. The sequence-specific nature of these effects suggests the involvement of the RNA induced silencing complex (RISC), which provides sequence specificity to RNA silencing machinery. Therefore, viroid titer in natural infections might be regulated by the concerted action of DCL and RISC. Viroids could have evolved their secondary structure as a compromise between resistance to DCL and RISC, which act preferentially against RNAs with compact and relaxed secondary structures, respectively. In addition, compartmentation, association with proteins or active replication might also help viroids to elude their host RNA silencing machinery.

0