|Yokomi, Raymond - Ray|
Submitted to: Citrograph
Publication Type: Popular publication
Publication Acceptance Date: 7/10/2012
Publication Date: 8/15/2011
Citation: Yokomi, R.K., Saponari, M., Doddapaneni, H. 2011. Investigation of seedling yellows cross protection by mild components of the Dekopon strain of Citrus tristeza virus. Citrograph. 3(4):38-43. Interpretive Summary: Cross protection has been used as a control strategy against virulent stem pitting strains of Citrus tristeza virus. Although it has been shown that CTV cross protection only works against CTV isolates of the same genotype, its underlying mechanism remains unknown. Cross protection can break down over time, typically as a consequence of challenge by a new virulent strain of CTV. This study of cross protection was undertaken with a model system where a parental CTV isolate called Dekopon was a mixture of three genetically different CTV strains. The Dekopon isolate protected sour orange seedlings from expression of seedling yellows (SY) symptoms which is significant because SY is associated with virulence of CTV. The parental Dekopon mixture was separated by aphid transmission and the resultant sub-isolates induced different symptoms, as expected based on individual genotypes; namely, the VT- and T3- genotypes induced strong SY in seedlings of sour orange and Duncan grapefruit; whereas the NS genotype was very mild. Various combinations of these sub-isolates failed to show cross protection or at least at the level expressed by the parental strain. Thus, the Dekopon isolate contained the essential component(s) of symptom amelioration. Deep sequencing using the Illumina platform was undertaken to analyze small interfering (si) RNAs profiles in sour orange severely affected by SY and in cross protected sour orange. An accumulation of virus-derived siRNAs mapping to the 3’-end of the CTV genome was found to be common in all three strains. Contigs from these datasets identified sequences homologous to the 282 Kb Ctv resistance locus of Poncirus trifoliata, thus, providing evidence that this region is involved in siRNA pathways. A total of 50 micro (mi) RNA (regulators of gene expression) families had significant frequency of which 8 miRNA families showed differential expression after CTV infection. Analysis of putative mRNA (chemical blueprint resulting in a protein product) targets in the EST database revealed 433 were common to sour orange plants showing protection or strong SY symptoms. Other mRNA targets were specific to cross protected plants (873) or sour orange showing severe SY (753). The different mRNA targets identified suggested that different CTV strains induced different miRNA-mRNA interactions. The experimental procedures undertaken in this research were designed to explore the siRNA pathway involved in the Citrus-CTV interactions and to identify the siRNAs and miRNAs associated with host response. The understanding gained should provide a new insights on mechanisms of CTV disease expression and cross protection.
Technical Abstract: Virulent strains of Citrus tristeza virus (CTV) can be controlled by pre-infection by mild strains of CTV which is called cross protection. However, the mode of action of cross protection is unknown and its durability unpredictable. RNA silencing is a regulatory mechanism to maintain genome integrity and defends against plant viruses. The objective of this study is to examine if RNA interference (i) by mild CTV strains can induce cross protection against virulent CTV strains. Sour orange (SO) and Duncan grapefruit (DGF) were infected with a CTV field isolate called Dekopon which contains a mixture of three viral genotypes (VT, T3 and non-standard) versus each genotype strain separately. The Dekopon isolate and the non-standard (NS) genotype showed no severe symptoms in SO and DGF; whereas plants with VT or T3 genotype strains showed strong seedling yellows (SY). Virus replication was assessed by real time RT-PCR assay with strain-specific probes designed in the P20 gene. After 12 months, plants with the parental isolate showed cross protection although all strains were replicating at the same level. SO plants inoculated simultaneously with each individual genotype expressed mild stunting and mild SY but DGF had strong SY. Plants inoculated with the NS genotype and challenged after one month with VT or T3 strains showed no cross protection and plants contained similar titers of each genotype as those protected by the parental isolate. Thus, cross protection was not induced by the NS sub-isolate. Therefore, cross protection observed must involve the CTV quasispecies population in parental source. Since mediators of RNA interference are 21-24 bp small interfering RNAs (siRNAs), profiles of viral (v) siRNAs from cross protected and SY-symptomatic SO and DGF were assessed by deep sequencing. Short reads (ca. 8-10 million) were processed and SO had prevalence of 21 and 24 nucleotide (nt) size classes (24% and 41%, respectively); whereas healthy SO was dominated by 24 nt. CTV-derived reads 21 to 24nt were mapped on CTV reference genomes and all three CTV genotypes showed similar patterns and most were located in the 3’ end region encompassing the genes P18, P13, P20, P23 and 3’UTR. In general, CTV-siRNAs were 60% sense versus 40% antisense and predominantly in the 21 and 22nt size classes which suggested that Dicer-like proteins (DCL), homologues to Arabidopsis thaliana DCL-2 and DCL-4, may play a key role in the biogenesis of CTV-derived siRNAs. The terminal nt for the 5’ vsiRNA for both sense and antisense strands showed preference for U which is known to direct the RNA silencing-related protein Argonaute 1 (AGO1) in A. thaliana. Blast analysis of the host-derived short reads showed that all datasets contained 21-24nt sequences highly homologous to the 282 Kb Ctv resistance locus of Poncirus trifoliata, providing evidence that this region is involved in the siRNA pathways, although no significant differences were found between the CTV-infected and non-infected host. A total of 50 known miRNA families were found to have significant sequence presence, of which 8 miRNA families showed differential expression. Analysis of Citrus mRNA (EST) targets for these miRNAs in the cross protected SO plant and the SO showing strong SY and healthy untreated SO control showed that there were 433 targets common to both the treated plants but absent in healthy control; 873 targets that are only in the cross protected SO plant; and 753 targets in the SO showing strong SY. This suggested dynamic rewiring of the miRNA-mRNA interaction network.