Molecular characterization reveals no functional evidence for naturally occurring cross‐kingdom RNA interference in the early stages of Botrytis cinerea–tomato interaction

Abstract

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.

Botrytis cinereasmall RNAs are associated with tomato AGO1 and silence tomato defense-related target genes supporting cross-kingdom RNAi

“…At least one of the Bc-sRNA#3 tomato targets, SISKP2, was down-regulated after infection (Figure 1C). Silencing of a Bc-sRNA#5 target SIRLK was also detected by Qin et al in Figure 4 of Qin et al (2022). Thus, we demonstrated that cross-kingdom RNAi is evident between B. cinerea and tomato.…”

“…There are contradictory results and interpretations in Qin et al (2022). Deleting the ms3003 element certainly did not certainly lead to a drastic loss of sRNA production, as suggested by Qin et al (2022).…”

“…Thus, the extremely high number of tomato target genes predicted by Qin et al (2022) was most likely overestimated and thus many of their predicted target genes in tomato may not be true Bc-sRNA targets. Importantly, even true sRNA/mRNA targets will not be functional unless at the very least they are present in the same cell and within a silencing complex.…”

“…Thus, using these data to determine the expression levels of Bc-sRNAs targeted mRNAs is most likely inaccurate. Qin et al (2022) decided to generate ms3003 deletion mutant strains because "approximately 10% of the total sRNAs produced by B. cinerea strain B05.10 originated from a retrotransposon region on Chr14 annotated as ms3003". Several misleading interpretations were made here: First, estimating sRNA production per retrotransposon (RT) locus based on total sRNA read counts makes no sense because many sRNAs map multiple times and to different RT loci, leading to a doubling of sRNA read counts.…”

“…In this response letter, we address the questions raised in this study. This response is not intended to dismiss the argument of Qin and colleagues but to use experimental evidence and additional bioinformatics analysis of the datasets produced by Qin et al (2022) to identify the key reasons why a discrepancy with previously published findings occurred. Here, we also provided additional experimental evidence to support the presence of cross-kingdom RNAi between tomato and B. cinerea.…”