Inhibition of Cell Growth and Shoot Development by a Specific Nucleotide Sequence in a Noncoding Viroid RNA

Abstract

Viroids are small noncoding and infectious RNAs that replicate autonomously and move systemically throughout an infected plant. The RNAs of the family Pospiviroidae contain a central conserved region (CCR) that has long been thought to be involved in replication. Here, we report that the CCR of Potato spindle tuber viroid (PSTVd) also plays a role in pathogenicity. A U257A change in the CCR converted the intermediate strain PSTVd Int to a lethal strain that caused severe growth stunting and premature death of infected plants. PSTVd with nucleotide U257 changed to C or G did not cause such symptoms. The pathogenic effect of the U257A substitution was abolished by a C259U substitution in the same RNA. Analyses of the pathogenic effects of the U257A substitution in three other PSTVd variants established A257 as a new pathogenicity determinant that functions independently and synergistically with the classic pathogenicity domain. The U257A substitution did not alter PSTVd secondary structure, replication levels, or tissue tropism. The stunted growth of PSTVd Int U257A-infected tomato plants resulted from restricted cell expansion but not cell division or differentiation. This was correlated positively with the downregulated expression of an expansin gene, LeExp2 . Our results demonstrate that specific nucleotides in a noncoding, pathogenic RNA have a profound effect in altering distinct cellular responses, which then lead to well-defined alterations in plant growth and developmental patterns. The feasibility of correlating viroid RNA sequence/structure with the altered expression of specific host genes, cellular processes, and developmental patterns makes viroid infection a valuable system in which to investigate host factors for symptom expression and perhaps also to characterize the mechanisms of RNA regulation of gene expression in plants.

On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites

“…This ruled out the possibility that the symptoms are due to the generation of infectious PSTVd. The symptoms in hpPSTVd plants did not appear to be as strong as those described for plants infected with the natural RG1 strain (6,15). This is probably due to lower levels of siRNA being produced from the hpPSTVd transgenes than from replicating PSTVd.…”

“…Intriguingly, some viroids and satellites can induce unique, highly host species-specific disease symptoms despite their exceedingly small size and lack of mRNA activity. Previous studies have shown that one, or a few, nucleotide changes in their RNA genomes can dramatically alter the virulence of these subviral RNAs or the host-plant specificity of the disease symptoms (5)(6)(7). Despite intensive investigation, major questions remain as to how these minor sequence variations modulate viroid and satellite pathology and how host plants develop symptoms in response to specific sequences.…”

A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation

“…These two strains, which differ by only one nucleotide (U versus A at genomic position 257 in the central region; Fig. 1A and B), cause intermediate and lethal symptoms, respectively (66). We were interested in determining whether small RNA profiles from strains of different pathogenicities would differ.…”

Resistencia a viroides inducida por ribozimas de cabeza de martillo y RNAs interferentes

“…El dominio C contiene el sitio donde los RNAs viroidales multiméricos de polaridad (+) son cortados y ligados (Baumstark et al, 1997;Gas et al, 2007). El dominio P se asoció con los efectos patogénicos en el PSTVd y otros viroides relacionados, aunque trabajos posteriores mostraron que la expresión de síntomas también está regulada por determinantes discretos situados en otros dominios (Sano et al, 1992;Reanwarakorn y Semancik, 1998;Qi y Ding, 2003b). Por último, el dominio TR parece estar implicado en fenómenos de recombinación (Keese y Symons, 1985) y el TL en modular la replicación o acumulación (Sano e Ishiguro, 1998).…”

“…Es un motivo de RNA mantenido por interacciones no canónicas, al que podría unirse alguna proteína involucrada en la replicación o el transporte intranuclear . Trabajos recientes proponen un papel para el bucle E en la replicación del PSTVd (Baumstark et al, 1997;Zhong et al, 2006) y del viroide de la exocortis de los cítricos (Citrus exocortis viroid, CEVd) Gas et al, 2007), en la especificidad de huésped (Zhong et al, 2006) y en la patogénesis (Qi y Ding, 2003b), a la vez que otros han confirmado su existencia in vivo (Eiras et al, 2007;Wang et al, 2007). Recientemente, se ha caracterizado un elemento similar de estructura terciaria en el viroide cloroplástico PLMVd (Hernández et al, 2006).…”