Abstract
RNA silencing is an important mechanism to regulate gene expression and antiviral defense in plants. Nevertheless, RNA silencing machinery in the important oil crop Brassica napus and function in resistance to the devastating fungal pathogen Sclerotinia sclerotiorum are not well-understood. In this study, gene families of RNA silencing machinery in B. napus were identified and their role in resistance to S. sclerotiorum was revealed. Genome of the allopolyploid species B. napus possessed 8 Dicer-like (DCL), 27 Argonaute (AGO), and 16 RNA-dependent RNA polymerase (RDR) genes, which included almost all copies from its progenitor species B. rapa and B. oleracea and three extra copies of RDR5 genes, indicating that the RDR5 group in B. napus appears to have undergone further expansion through duplication during evolution. Moreover, compared with Arabidopsis, some AGO and RDR genes such as AGO1, AGO4, AGO9, and RDR5 had significantly expanded in these Brassica species. Twenty-one out of 51 DCL, AGO, and RDR genes were predicted to contain calmodulin-binding transcription activators (CAMTA)-binding site (CGCG box). S. sclerotiorum inoculation strongly induced the expression of BnCAMTA3 genes while significantly suppressed that of some CGCG-containing RNA silencing component genes, suggesting that RNA silencing machinery might be targeted by CAMTA3. Furthermore, Arabidopsis mutant analyses demonstrated that dcl4-2, ago9-1, rdr1-1, rdr6-11, and rdr6-15 mutants were more susceptible to S. sclerotiorum, while dcl1-9 was more resistant. Our results reveal the importance of RNA silencing in plant resistance to S. sclerotiorum and imply a new mechanism of CAMTA function as well as RNA silencing regulation.
Highlights
RNA silencing refers to a variety of mechanisms whereby a small RNA molecule interferes with a given nucleotide sequence
The results showed that B. rapa genome contained 4 Dicerlike proteins (DCLs), AGO, and 6 RNA-dependent RNA polymerase (RDR) genes while B. oleracea genome carried 4 DCL, AGO, and 7 RDR genes (Table S1)
Napus genome expanded RDR5 genes compared with that of its progenitors B. oleracea and B. rapa and all genomes of three Brassica species expanded AGO1, AGO4, and AGO9 genes compared with the Arabidopsis genome
Summary
RNA silencing refers to a variety of mechanisms whereby a small RNA molecule interferes with a given nucleotide sequence It was first discovered in plants and occurs widely in eukaryotic organisms (Tijsterman et al, 2002; Ullu et al, 2004). DCLs undergo RNase III-type activities to process complementary double-strand RNAs into small RNAs with 21–26 nucleotides in length (Carmell and Hannon, 2004). These small RNAs are incorporated into AGO-containing RNA-induced silencing complexes (RISCs) to serve as the sequence specificity in RNA degradation, translational inhibition, or heterochromatin formation (Bologna and Voinnet, 2014). At the signal amplification stage, RDR enzymes are responsible for synthesis of dsRNAs from ssRNA templates to initiate a new round of RNA silencing (Sijen et al, 2001)
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