Intron-capture RNA-seq reveals the landscape of intronic RNAs in Arabidopsis.

Intronic RNAs have been overlooked for a long time: They are functional, but treated as "junk." In this work, we designed a new sequencing strategy to investigate intronic RNAs. By using intron-capture RNA-seq, we systematically analyzed the intronic RNAs in Arabidopsis by zooming into the intronic regions an order of magnitude deeper than in previous work. Our key findings include: (1) Intron-capture RNA-seq is a much more efficient approach to analyze intronic RNAs than total RNA-seq and mRNA-seq. (2) We identified three types of intronic RNAs, and found that the GC pattern differs significantly between the introns with and without intronic RNAs. (3) We detected many hidden elements in introns, including circular RNAs, splice junctions, and transcripts that have previously been overlooked. (4) The expression of these intronic RNAs varies during the time course of pathogen infection, which indicates that an unknown mechanism may exist for these RNAs. (5) We also demonstrated that most of intronic RNAs are detectable in both Arabidopsis and rice, suggesting that these non-coding molecules are conserved. Taken together, this work proposes an efficient strategy to analyze intronic RNAs, and provides an unprecedented view of this essential component in biological pathways.

An OsPRMT5-OsAGO2/miR1875-OsHXK1 module regulates rice immunity to blast disease.

Rice ARGONAUTE2 (OsAGO2) is a core component of the rice RNA-induced silencing complex (RISC), which is repressed by Magnaporthe oryzae (M. oryzae) infection. Whether and how OsAGO2-mediated gene silencing plays a role in rice blast resistance and which sRNAs participate in this process are unknown. Our results indicate that OsAGO2 is a key immune player that manipulates rice defense responses against blast disease. OsAGO2 associates with the 24-nt miR1875 and binds to the promoter region of HEXOKINASE1 (OsHXK1), which causes DNA methylation and leads to gene silencing. Our multiple genetic evidence showed that, without M. oryzae infection, OsAGO2/miR1875 RISC promoted OsHXK1 promoter DNA methylation and OsHXK1 silencing; after M. oryzae infection, the reduced OsAGO2/miR1875 led to a relatively activated OsHXK1 expression. OsHXK1 acts as a positive regulator of blast disease resistance that OsHXK1-OE rice exhibited enhanced resistance, whereas Cas9-Oshxk1 rice showed reduced resistance against M. oryzae infection. OsHXK1 may function through its sugar sensor activity as glucose induced defense-related gene expression and reactive oxygen species (ROS) accumulation in Nipponbare and OsHXK1-OE but not in Cas9-Oshxk1 rice. OsAGO2 itself is delicately regulated by OsPRMT5, which senses M. oryzae infection and attenuates OsAGO2-mediated gene silencing through OsAGO2 arginine methylation. Our study reveals an OsPRMT5-OsAGO2/miR1875-OsHXK1 regulatory module that fine tunes the rice defense response to blast disease.

OsAPX1 Positively Contributes to Rice Blast Resistance.

Ascorbate peroxidases (APXs) maintain cellular reactive oxygen species (ROS) homeostasis through their peroxidase activity. Here, we report that OsAPX1 also promotes ROS production such that a delicate cellular ROS homeostasis is achieved temporally after Magnaporthe oryzae infection. OsAPX1 specifically induces ROS production through increasing respiratory burst oxidase homologs (OsRBOHs) expression and can be inhibited by DPI, a ROS inhibitor. The time-course experiment data show that the simultaneous induction of OsAPX1 and OsRBOHs leads to ROS accumulation at an early stage; whereas a more durable expression of OsAPX1 leads to ROS scavenging at a later stage. By the temporal switching between ROS inducer and eliminator, OsAPX1 triggers an instant ROS burst upon M. oryzae infection and then a timely elimination of ROS toxicity. We find that OsAPX1 is under the control of the miR172a-OsIDS1 regulatory module. OsAPX1 also affects salicylic acid (SA) synthesis and signaling, which contribute to blast resistance. In conclusion, we show that OsAPX1 is a key factor that connects the upstream gene silencing and transcription regulatory routes with the downstream phytohormone and redox pathway, which provides an insight into the sophisticated regulatory network of rice innate immunity.