AUTHOR=Zhang Qiulei , Li Yang , Zhang Yi , Wu Chuanbao , Wang Shengnan , Hao Li , Wang Shengyuan , Li Tianzhong 

TITLE=Md-miR156ab and Md-miR395 Target WRKY Transcription Factors to Influence Apple Resistance to Leaf Spot Disease

JOURNAL=Frontiers in Plant Science

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2017.00526

DOI=10.3389/fpls.2017.00526

ISSN=1664-462X

ABSTRACT=<p>MicroRNAs (miRNAs) are key regulators of gene expression that post-transcriptionally regulate transcription factors involved in plant physiological activities. Little is known about the effects of miRNAs in disease resistance in apple (<italic>Malus</italic>×<italic>domestica</italic>). We globally profiled miRNAs in the apple cultivar Golden Delicious (GD) infected or not with the apple leaf spot fungus <italic>Alternaria alternaria f. sp. mali</italic> (ALT1), and identified 58 miRNAs that exhibited more than a 2-fold upregulation upon ALT1 infection. We identified a pair of miRNAs that target protein-coding genes involved in the defense response against fungal pathogens; Md-miR156ab targets a novel WRKY transcription factor, MdWRKYN1, which harbors a TIR and a WRKY domain. Md-miR395 targets another transcription factor, MdWRKY26, which contains two WRKY domains. Real-time PCR analysis showed that Md-miR156ab and Md-miR395 levels increased, while <italic>MdWRKYN1</italic> and <italic>MdWRKY26</italic> expression decreased in ALT1-inoculated GD leaves; furthermore, the overexpression of Md-miR156ab and Md-miR395 resulted in a significant reduction in <italic>MdWRKYN1</italic> and <italic>MdWRKY26</italic> expression. To investigate whether these miRNAs and their targets play a crucial role in plant defense, we overexpressed <italic>MdWRKYN1</italic> or knocked down Md-miR156ab activity, which in both cases enhanced the disease resistance of the plants by upregulating the expression of the WRKY-regulated pathogenesis-related (PR) protein-encoding genes <italic>MdPR3-1, MdPR3-2, MdPR4, MdPR5, MdPR10-1</italic>, and <italic>MdPR10-2</italic>. In a similar analysis, we overexpressed <italic>MdWRKY26</italic> or suppressed Md-miR395 activity, and found that many PR protein-encoding genes were also regulated by <italic>MdWRKY26</italic>. In GD, ALT-induced Md-miR156ab and Md-miR395 suppress <italic>MdWRKYN1</italic> and <italic>MdWRKY26</italic> expression, thereby decreasing the expression of some PR genes, and resulting in susceptibility to ALT1.</p>