How plant pri-miRNAs with complex secondary structures are recognized and processed has been unclear. A new study now suggests that unlike canonical processing of pri-miRNAs, terminal loop–branched pri-miRNAs can be processed by Dicer-like 1 (DCL1) complexes bidirectionally, either from the lower stem to the terminal loop or vice versa, resulting in productive and abortive processing of miRNAs, respectively. MicroRNAs (miRNAs) originate from primary transcripts (pri-miRNAs) with characteristic stem-loop structures, and their accurate processing is required for the production of functional miRNAs. Here, using the pri-miR-166 family in Arabidopsis thaliana as a paradigm, we report the crucial role of pri-miRNA terminal loops in miRNA biogenesis. We found that multibranched terminal loops in pri-miR-166s substantially suppress miR-166 expression in vivo. Unlike canonical processing of pri-miRNAs, terminal loop–branched pri-miRNAs can be processed by Dicer-like 1 (DCL1) complexes bidirectionally from base to loop and from loop to base, resulting in productive and abortive processing of miRNAs, respectively. In both cases, DCL1 complexes canonically cut pri-miRNAs at a distance of 16–17 bp from a reference single-stranded loop region. DCL1 also adjusts processing sites toward an internal loop through its helicase domain. These results provide new insight into the poorly understood processing mechanism of pri-miRNAs with complex secondary structures.

Plant viruses have evolved multiple strategies to overcome host defense to establish an infection. Here, we identified two components of a host mitogen-activated protein kinase (MAPK) cascade, MKK2 and MPK4, as bona fide targets of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNV). βC1 interacts with the kinase domain of MKK2 and inhibits its activity. In vivo, βC1 suppresses flagellin-induced MAPK activation and downstream responses by targeting MKK2. Furthermore, βC1 also interacts with MPK4 and inhibits its kinase activity. TYLCCNV infection induces the activation of the MAPK cascade, mutation in MKK2 or MPK4 renders the plant more susceptible to TYLCCNV, and can complement the lack of βC1. This work shows for the first time that a plant virus both activates and suppress a MAPK cascade, and the discovery of the ability of βC1 to selectively interfere with the host MAPK activation illustrates a novel virulence function and counter-host defense mechanism of geminiviruses. We identified a plant MAPK cascade that contributes to the defense against geminiviruses. Rapid activation of MAPKs was observed upon geminivirus infection. The βC1 protein of geminivirus betasatellite interacts with MKK2 and MPK4 and inhibits their kinase activity. Also, βC1 suppresses flagellin-induced MAPK activation and downstream responses. Knocking out two components of this cascade, MKK2 or MPK4 in tobacco renders the plant more susceptible to geminivirus infection and virus infection of geminivirus without betasatellite induced typical virus symptom in MKK2 or MPK4 knockout tobacco, indicating that MKK2 or MPK4 mutant can complement the lack of βC1 in virus infection. This study unveils MAPK cascades as novel players in anti-viral defense and illustrates how plant viruses have in turn evolved to suppress this pathway.

RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5’ products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC.