Plants and their pathogens have constantly co-evolving mechanisms that determine infection success. Small RNAs (sRNAs) of 18-30 nucleotides can have a large effect regulating plant defense responses as well as fungal virulence factors. The goal of this project was to understand how sRNAs regulate gene expression both for species of origin, as well as trans-kingdom sRNA communication between barley and Blumeria graminis f. sp. hordei (Bgh), the causal agent of barley powdery mildew. To accomplish this goal we examined Bgh sRNA expression over a time course representing the key stages of Bgh infection of barley (appressorium formation, penetration of epidermal cells, and development of haustoria) in five barley lines including four fast-neutron derived immune-signaling mutants and their progenitor line CI 16151. The sRNA expression data was complemented by parallel analysis of RNA ends (PARE) analysis that confirms sRNA transcript cut sites with in vivo data. In barley, conserved and novel miRNAs were identified with predicted target transcripts enriched in the transcriptional regulation, signaling, and photosynthesis categories. Phasing siRNAs (phasiRNAs) were also identified in barley overlapping with protein coding genes including receptor-like kinases and resistance genes. Bgh micro RNA-like RNAs (milRNAs) were identified that are predicted to regulate transcripts encoding effectors, metabolic proteins, and translation-related proteins. A subset of effectors homologous to the AVRk1 and AVRa10 (EKA) family may be regulated by a sRNA-encoding hairpin that is overlapping and antiparallel to an EKA gene. These genes are heavily regulated by sRNAs, in contrast to most Bgh protein-coding genes. Potential trans-kingdom functional sRNAs were identified from both barley and Bgh. The predicted Bgh trans-kingdom sRNA are highly enriched in transcripts that function in non-species-specific defenses. The transcript targets encode proteins related to vesicle secretion, cell wall synthesis, protein turnover, transcriptional regulation, ROS response, and fungal cell wall breakdown. The potential barley trans-kingdom sRNAs are predicted to target Bgh transcripts including Bgh-specific effector proteins, ribosome synthesis/function, core transcription factors, and cell cycle regulators. Overall, these findings indicate that sRNAs are integral in regulation of gene expression during Bgh infection of barley leaves
