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    STUDIA BIOLOGIA - Issue no. 1 / 2013  
         
  Article:   ORAL PRESENTATION ABSTRACTS: THE EFFECT OF THE P19 SILENCING SUPPRESSOR ON ANTIVIRAL siRNA BIOGENESIS.

Authors:  LEVENTE KONTRA, EMESE SZABÓ XÓCHITL, JÓZSEF BURGYÁN.
 
       
         
  Abstract:   Here we showed that in CymRSV – Nicotiana benthamiana interaction DCL2 dependent 22nt small RNAs are generated in the greatest number. However this is masked by the 1 nucleotide shortening of the siRNA induced by P19. DCL2 and DCL4 drived small RNAs are crucial in mediating RNA silencing against RNA viruses. DCL2 and DCL 4 are capable of cleaving double stranded viral RNA into 22 and 21 nucleotide (nt) long viral small interfering RNAs (vsiRNA) respectably. And both can program RISC complexes to cleave the complementary viral RNA. P19, a viral protein capable of suppressing silencing, can invert the size ratio of vsiRNA in Cymbidium Ring Spot Virus (CymRSV) infected Nicotiana benthamiana (N.b.) plants. We know that a wild type CymRSV infected N.b. the amount of 21 nt long vsiRNAs is grater than 22nt vsiRNA’s, however in N.b. infected with P19 deficient CymRSV (Cym19S) it alters and 22 nt vsiRNA becomes the most common. We set our goal to shed some light on the under laying mechanism of the ratio change. We have proved that this can not be explained by the CymRSV’s ability to replicate in tissues where the Cym19S becomes silenced. By showing that DCL2 and DCL4 are both present in the assessed tissues and there is no significant difference in expression levels even when the viruses are present. We also investigated the accumulation of small RNAs in these tissues, with next generation sequencing and find that there is a small divergence of 21 and 22 nt vsiRNA ratio but the presence or absence of P19 distinguishes the samples the most. We discarded the possibility that the ratio sift is due to the P19’s putative ability of conserving the 21 nt based on the fact that P19 bind 21 and 22 nt siRNA in vitro with roughly the same affinity. We hypostatized that P19 is capable of inducing the shortening of the bound siRNSs by one nt. We had reported that when P19 was co-agroinfiltrated with GFP and its hairpin into N.b. we found that 22 nt siRNS could not been detected, and a grate number of 20 nt appeared on the northern blot, though none of the P19 independent small RNA (24nt siRNA, miRNA) shown any change after interaction with plant endogenous RNase. This hypothesis can also explain the enrichment of 20nt siRNA in our and in previously published small RNA sequencing. In silico maping of vsiRNA reads also supports this theory.  
         
     
         
         
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