Molecular characterization of cross-kingdom RNA interference in by tomato small RNAs.

Previous studies have suggested that plants can modulate gene expression in pathogenic fungi by producing small RNAs (sRNAs) that can be translocated into the fungus and mediate gene silencing, which may interfere with the infection mechanism of the intruder. We sequenced sRNAs and mRNAs in early phases of the (tomato)- interaction and examined the potential of plant sRNAs to silence their predicted mRNA targets in the fungus. Almost a million unique plant sRNAs were identified that could potentially target 97% of all fungal genes. We selected three fungal genes for detailed RT-qPCR analysis of the correlation between the abundance of specific plant sRNAs and their target mRNAs in the fungus. The fungal gene, which had been reported to be important for the fungal virulence, showed transient down-regulation around 20 hours post inoculation and contained a unique target site for a single plant sRNA that was present at high levels. In order to study the functionality of this plant sRNA in reducing the transcript level, we generated a fungal mutant that contained a 5-nucleotide substitution that would abolish the interaction between the transcript and the sRNA without changing the encoded protein sequence. The level of the mutant transcript showed a transient decrease similar to wild type transcript, indicating that the tomato sRNA was not responsible for the downregulation of the transcript. The virulence of the target site mutant was identical to the wild type fungus.