Profile of small RNAs and preliminary epigenetic studies in Citrus plants upon citrus tristeza virus infection

Citrus tristeza virus is one of the agent of devastating cultivated citrus trees especially if grafted on sour orange (Citrus aurantium), which appears to be the most susceptible species. The trifoliate orange (Poncirus trifoliata) appears to be resistant to the disease compared to the sour orange, the sweet orange and the grapefruit, while, the Citrange carrizo, derived from cross to C. sinensis L. x P. trifoliate, appears to be tolerant. In Citrange carrizo, the virus replicates and spreads throughout the tolerant plant without showing any symptoms. On the contrary in the resistant plants of Poncirus, the virus replication is not blocked, but it seems likely that it acts by preventing cell to cell and/or long distance moviment (Karasev et al., 2010). The resistance to the virus is due to the presence of a locus, called locus CTV, which is available an accurate genetic map (Yang et al., 2001; Rai, 2006). Within this region are present 22 genes seven of which (Ctv.4, Ctv.7, Ctv.8, Ctv.11, Ctv.17, Ctv.18 and Ctv.21), are denoted R(1-7) genes, since they show a high homology with Arabidopsis resistance genes encoding to CC-NBS-LRR proteins (coil-coil-nucleotide binding site-leucine rich repeat) (Deng et al., 2000; Yang et al., 2003). In addition, six other genes, with a high homology sequence to known function genes of other species are present. In particolar the Ctv.20 showed homology with a plant virus movement-like protein (Karasev and Hilf 2010). This membrane protein, through the formation of channels in plasmodesma, is involved in the transfer of proteins and viral RNA from cell to cell. Ctv.20 was predicted to contain three open reading frames (ORFs) by GenScan Web server (Burge and Karlin, 1997), but BLAST searches indicated that both the first and the third ORFs were highly homologous with Petunia vein-clearing virus (PVCV) ORF1 (Yang et al. 2003). Yang and colleagues (2003), performed the northern hybridization analyses using DNA fragments from the first and the third ORFs and both these fragment hybridized with the same band of about 9 kb, that presented a size compatible with the GenScan prediction (data not shown). Northern hybridization indicated that Ctv.20 and its orthologous are highly expressed in P. trifoliata and sweet orange leaves and in P. trifoliata bark tissues, but are relatively lowly expressed in the phloem of sweet orange (data not shown). The ortholog of Ctv.20 in sweet orange is about 8.5 kb, which is slightly smaller than Ctv.20 (9 kb) in P. trifoliata. CTV tends to accumulate in phloem tissue of infected plants, which suggests that Ctv.20 could also be considered as a candidate gene for Ctv resistance (Yang et al., 2003). The aim of the present research was to carry out different siRNA 21:24 nucleotides libraries by Illumina sequencing, from susceptible plants of sour orange and tolerant of Citrange carrizo ones, both healthy and infected with different strains of CTV virus. The presence of a homologous locus in susceptible species such as Citrus aurantium suggests that the mechanisms of resistance and therefore of regulating the expression of the genes present in the locus cannot be fully explained by DNA sequence alone. On this basis we moved to verify the role of siRNA through an epigenetic regulation of the methylation status of ctv locus with particular reference to Ctv.20 gene. The difference between methylated and unmethylated condition in tolerant and susceptible plants was performed by PCR analysis on DNA digested by sensitive and unsensitive enzymes to cytosine methylation.