Plant immune responses are triggered during the interaction with pathogens. The fungus Botrytis cinerea has previously been reported to use small RNAs (sRNAs) as effector molecules capable of interfering with the host immune response. Conversely, a host plant produces sRNAs that may interfere with the infection mechanism of an intruder. We used high-throughput sequencing to identify sRNAs produced by B. cinerea and Solanum lycopersicum (tomato) during early phases of interaction and to examine the expression of their predicted mRNA targets in the other organism. A total of 7042 B. cinerea sRNAs were predicted to target 3185 mRNAs in tomato. Of the predicted tomato target genes, 163 were indeed transcriptionally down-regulated during the early phase of infection. Several experiments were performed to study a causal relation between the production of B. cinerea sRNAs and the down-regulation of predicted target genes in tomato. We generated B. cinerea mutants in which a transposon region was deleted that is the source of c.10% of the fungal sRNAs. Furthermore, mutants were generated in which both Dicer-like genes (Bcdcl1 and Bcdcl2) were deleted and these displayed a >99% reduction of transposon-derived sRNA production.Neither of these mutants was significantly reduced in virulence on any plant species tested. Our results reveal no evidence for any detectable role of B. cinerea sRNAs in the virulence of the fungus.

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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 Solanum lycopersicum (tomato)-Botrytis cinerea 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 Bcspl1 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 Bcspl1 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 Bcspl1 transcript showed a transient decrease similar to wild type transcript, indicating that the tomato sRNA was not responsible for the downregulation of the Bcspl1 transcript. The virulence of the Bcspl1 target site mutant was identical to the wild type fungus.