miRNA Discovery in Barley (Hordeum vulgare L.) and the Powdery Mildew Pathogen, Blumeria graminis

The interaction between barley (Hordeum vulgare L.) and the obligate biotroph Blumeria graminis f. sp. hordei (Bgh) potentially involves hundreds of secreted effector and host proteins. These effector proteins allow the fungus to suppress the host's innate immunity and to uptake nutrients from the plant. Barley has evolved an effective defense against Bgh through resistance genes (R genes) including Mildew resistance locus a (Mla), that recognize Bgh effector proteins and trigger immunity. Our group has recently shown that regulation of resistance through the Mla pathway is dependent on two barley miRNAs. miR9863 targets distinct Mla alleles to attenuate NLR-triggered disease resistance, whereas Mla itself controls miRNA398 and Cu/Zn-SOD to regulate Bgh-responsive cell death.

To identify additional small RNAs involved in Mla triggered resistance, we carried out Illumina small RNA sequencing of the CI 16151 (Mla6) progenitor and four fast-neutron-derived resistance signaling mutants. Three biological replications of a split-plot based design were inoculated with Bgh 5874 (AVR_a6), harvested at 0, 16, 20, 24, 32, and 48 hours, and small RNA (sRNA) libraries were prepared from the 90 barley-Bgh treatments for Illumina Hi-Seq 2500 sequencing. Several hundred conserved and novel putative sRNAs in barley and Bgh were identified from the resulting 2.1 billion reads (average 24.2 million reads/treatment) and differential expression analysis was carried out to look for significant differences among wild-type progenitor and mutant derivatives. Within and trans-kingdom target predictions were initiated for both barley and Blumeria, facilitating a genome-wide knowledge base of the sRNA players in Mla specified resistance.