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Tight regulation of the interaction between Brassica napus and Sclerotinia sclerotiorum at the microRNA level

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Abstract

MicroRNAs (miRNAs) are multifunctional non-coding short nucleotide molecules. Nevertheless, the role of miRNAs in the interactions between plants and necrotrophic pathogens is largely unknown. Here, we report the identification of the miRNA repertoire of the economically important oil crop oilseed rape (Brassica napus) and those involved in interacting with its most devastating necrotrophic pathogen Sclerotinia sclerotiorum. We identified 280 B. napus miRNA candidates, including 53 novel candidates and 227 canonical members or variants of known miRNA families, by high-throughput deep sequencing of small RNAs from both normal and S. sclerotiorum-inoculated leaves. Target genes of 15 novel candidates and 222 known miRNAs were further identified by sequencing of degradomes from the two types of samples. MiRNA microarray analysis revealed that 68 miRNAs were differentially expressed between S. sclerotiorum-inoculated and uninoculated leaves. A set of these miRNAs target genes involved in plant defense to S. sclerotiorum and/or other pathogens such as nucleotide binding site-leucine-rich repeat (NBS-LRR) R genes and nitric oxygen and reactive oxygen species related genes. Additionally, three miRNAs target AGO1 and AGO2, key components of post-transcriptional gene silencing (PTGS). Expression of several viral PTGS suppressors reduced resistance to S. sclerotiorum. Arabidopsis mutants of AGO1 and AGO2 exhibited reduced resistance while transgenic lines over-expressing AGO1 displayed increased resistance to S. sclerotiorum in an AGO1 expression level-dependent manner. Moreover, transient over-expression of miRNAs targeting AGO1 and AGO2 decreased resistance to S. sclerotiorum in oilseed rape. Our results demonstrate that the interactions between B. napus and S. sclerotiorum are tightly regulated at miRNA level and probably involve PTGS.

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Abbreviations

AGO:

Argonaute

CMV:

Cucumber mosaic virus

DCL:

Dicer-like

MFEI:

Minimal folding free energies index

ETI:

Effector-triggered immunity

KMC:

K-Means Clustering

miRNA:

MicroRNA

NBS-LRR:

Nucleotide binding site-leucine-rich repeat

OE:

Overexpression

PDA:

Potato dextrose agar

Pst :

Pseudomonas syringae pv. tomato

PTGS:

Post-transcriptional gene silencing

PVX:

Potato virus X

qRT-PCR:

Quantitative real-time PCR

ROS:

Reactive oxygen species

TBSV:

Tomato bushy stunt virus

TCV:

Turnip crinkle virus

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Acknowledgments

We are grateful to Prof. Shou-Wei Ding (Department of Plant Pathology and Microbiology, University of California, Riverside, USA) for providing the Arabidopsis thaliana mutant ago1-27 and Prof. Yi-Jun Qi (Tsinghua-Peking Center for Life Sciences, and School of Life Sciences, Tsinghua University, China) for providing the A. thaliana mutants ago1-33 and ago2-1. We acknowledge Professor David Baulcombe (Sainsbury Laboratory, John Innes Centre) and Plant Bioscience Limited (Colney Lane, Norwich NR4 7UH, England) for providing the plasmids expressing silencing suppressors. This work was supported by the National Natural Science Foundation of China (31371892), the Special Fund for Agro-scientific Research in the Public Interest (201103016), the SRFDP (20110101110092) and the Fundamental Research Funds for the Central Universities (2016FZA6014).

Author contribution

Jia-Yi Cao performed miRNA idenfication and verification analyses. Li Zhao constructed AGO1-OE transgenic plants and evaluated their resistance. Jia-Yi Cao and Shuang-Sheng Li carried out the resistance evaluation analyses for ago mutants. Jia-Yi Cao and You-Ping Xu conducted gene expression and statistical analyses. Xin-Zhong Cai and You-Ping Xu conceived of the study and participated in its design and coordination. Xin-Zhong Cai and Jia-Yi Cao prepared the manuscript.

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Authors and Affiliations

  1. Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou, 310058, China

    Jia-Yi Cao, Li Zhao, Shuang-Sheng Li & Xin-Zhong Cai

  2. Centre of Analysis and Measurement, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou, 310058, China

    You-Ping Xu

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  1. Jia-Yi Cao
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Correspondence to Xin-Zhong Cai.

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Cao, JY., Xu, YP., Zhao, L. et al. Tight regulation of the interaction between Brassica napus and Sclerotinia sclerotiorum at the microRNA level. Plant Mol Biol 92, 39–55 (2016). https://doi.org/10.1007/s11103-016-0494-3

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