The fungus Botrytis cinerea causes grey mould disease on a wide range of plant species, including fruits (strawberry, raspberry, grape), vegetables (lettuce, pepper, tomato) and ornamentals (roses). The fungus is commonly controlled by applying chemicals that reduce the disease, but these also affect human health and the environment. Numerous studies have been performed to understand how this fungus can attack so many plants. These studies revealed that Botrytis produces a spectrum of proteins and small molecules that can kill plant cells, which enables the fungus to invade a plant and consume dead tissue.
Almost ten years ago, Weiberg et al. (2013) reported an exciting observation that the fungus produces small RNAs (sRNAs, short RNA molecules that are usually 20-24 nucleotide in length) which can be transported into plant cells where they can silence plant immunity genes through RNA interference (RNAi). RNAi is a well-studied process by which sRNAs exploit nucleotide sequence similarity to trigger the destruction of messenger RNAs (mRNA), molecules that contain the instructions for producing proteins in a cell. The finding that sRNAs from Botrytis can instruct a plant to destroy specific mRNA molecules in a plant provided an exciting example of “cross-kingdom RNAi”.
We aimed to examine the contribution to disease development of sRNAs in the Botrytis – tomato interaction, in the hope of obtaining leads for developing crop protection methods for tomato against grey mould. We used high-throughput sequencing to identify sRNAs produced by Botrytis during early infection and to examine the expression of their predicted mRNA targets in tomato. We identified ~27,000 Botrytis sRNAs, of which 7000 were predicted to target >3,000 genes in tomato.
We performed several experiments to study causal relations between the production of sRNAs by Botrytis and the down-regulation of (predicted) target genes in tomato. One crucial experiment turned out to be conclusive. Using CRISPR technology, we edited key sRNA genes in the B. cinerea fungus to create “knock-out” mutants: both “Dicer-like genes”, which encode enzymes responsible for the production of sRNAs were deactivated. By sequencing the sRNA pools of Botrytis cultures, we observed a >99% reduction of sRNA production, however, the mutants remained able to infect tomato and three other plant species just as well as the wild type fungus (producing normal sRNA levels). Our results, contradictory to the previous publication, provide no evidence for a role of Botrytis sRNAs in cross-kingdom RNAi and in fungal virulence.
Si Qin, Javier Veloso, Mirna Baak, Britt Boogmans, Tim Bosman, Guido Puccetti, Xiaoqian Shi-Kunne, Sandra Smit, Robert Grant-Downton, Thomas Leisen, Matthias Hahn and Jan A. L. van Kan published this study in Molecular Plant Pathology:
TITLE IMAGE: Botrytis cinerea fungal isolates with deactivated Dicer-like genes (ΔBcdcl1/ΔBcdcl2) produce less sRNA molecules (with a length of 20-24 nucleotides), compared against sRNA molecules produced by wild-type isolates (graph on left). However, both wild-type (left side of leaf) and mutant (right side of leaf) fungi cause grey mould symptoms on tobacco (Nicotiana benthamiana). These results indicate that B. cinerea sRNAs do not have any detectable role in fungal virulence. All images used with permission of the author.
All images used with permission of the author.