| Research Abstract |
Viroids are small circular, single-stranded, non-coding RNAs, but, once infected into host plant cells, replicate autonomously depending on the host transcription machinery and caused severe to mild diseases on varieties of crop plants. To investigate the novel strategy to prevent plants from viorid infection, we have analyzed viroid-induced RNA silencing in relation to viroid pathogenicity and molecular evolution. First, by large-scale sequencing analysis using next generation sequencer on the biogenesis of PSTVd-specific small RNAs (srPSTVd), it was found that those derived from genomic strand was superior than those from anti-genomic strand, i.e., 9:1, in case of Rutgers tomato . a highly sensitive cultivar, but the ratio was almost 1:1 in Moneymaker tomato . a tolerant cultivar. In both cultivars, srPSTVds were derived in general from whole the molecule of both strands, but some specific regions . hotspots . produced more abundant srPSTVd. In the genomic strand, both cultivars show
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ed the similar hotspot pattern, however, the cultivar-specific pattern was observed in the anti-genomic strand, i.e., Moneymaker showed more variable hotspots. The results indicated that the srPSTVd biogenesis is host/cultivar-dependent. We have also compared the hotspot patterns among PSTVd variants with different pathogenicity, i.e., the Intermediate (severe) and the Dahlia (mild) strains, and found that both of the strains showed the similar hotspot pattern in Rutgers tomato in leaves and stem with distinct abundance depending on the individual hotspots.
To identify the host genes responsible to exhibit viroid-specific disease symptoms such as dwarf and/or leaf curling, we have then performed microarray analysis of gene expression in tomato cultivars (Rutgers and Moneymaker) with or without infecting PSTVd. Expression of the genes related to biosynthesis of most of the known plant hormones including gibberellin and auxin have more or less been altered by development of specific disease symptoms. Among them, we have surveyed genes containing a stretch of 21-nucleotide sequence homologous to PSTVd, and found that the gene encoding gibberellin β-hydroxylase contains a PSTVd-specific 21-nucleotide sequence and was down-regulated by PSTVd infection, suggesting that the gene could be a hypothetical target by PSTVd-induced RNA silencing. Several of the tomato microRNAs such as miR159 have also changed their expression levels.
We have created transgenic tomato and Nicotiana benthemiana lines constitutively expressing hairpin RNA derived from various regions of PSTVd or from host genes relating to RNA silencing, i.e., DCL2 and RDR6. At present, transgenic N. benthamiana T2 lines expressing near full-length hairpin PSTVd (PSTVd-ΔTL) and one of the srPSTVd hotspots (srPSTVd-257a) derived from lower central region of anti-genomic strand successfully exhibited resistance to PSTVd infection.
To investigate molecular evolution of viroid in relation to RNA silencing, we infected hops with natural Hop stunt viroid (HpSVd) isolates derived from four host species (hop, grapevine, plum and citrus). These plants were maintained for 15 years, then analyzed the HpSVd variants present. As the result, the variant originally found in cultivated grapevines gave rise to various combinations of mutations at positions 25, 26, 54, 193, and 281, and upon prolonged infection, these variants underwent convergent evolution resulting in a limited number of adapted mutants. Further analysis revealed that these five mutations did not give a merit upon the mutant to replicate either in the original host (grapevine), the adapted host (hops), and the third host (cucumber). However, the mutant accumulated less abundant srHpSVd in infected hops and cucumber, and especially the relative ratio of srHpSVd reads containing the five mutations per total srHpSVd reads were significantly downed, suggesting that the adapted mutant successfully escaped from viroid-induced RNA silencing by giving rise to the five mutations. Less
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