Plants have evolved efficient defence mechanisms to defend themselves from pathogen attack. Although many studies have focused on the transcriptional regulation of defence responses, less is known about the involvement of microRNAs (miRNAs) as post-transcriptional regulators of gene expression in plant immunity. This work investigates miRNAs that are regulated by elicitors from the blast fungus Magnaporthe oryzae in rice (Oryza sativa). Small RNA libraries were constructed from rice tissues and subjected to high-throughput sequencing for the identification of elicitor-responsive miRNAs. Target gene expression was examined by microarray analysis. Transgenic lines were used for the analysis of miRNA functioning in disease resistance. Elicitor treatment is accompanied by dynamic alterations in the expression of a significant number of miRNAs, including new members of annotated miRNAs. Novel miRNAs from rice are proposed. We report a new rice miRNA, osa-miR7695, which negatively regulates an alternatively spliced transcript of OsNramp6 (Natural resistance-associated macrophage protein 6). This novel miRNA experienced natural and domestication selection events during evolution, and its overexpression in rice confers pathogen resistance. This study highlights an miRNA-mediated regulation of OsNramp6 in disease resistance, whilst illustrating the existence of a novel regulatory network that integrates miRNA function and mRNA processing in plant immunity.

To prevent rereplication of genomic segments, the eukaryotic cell cycle is divided into two nonoverlapping phases. During late mitosis and G1 replication origins are "licensed" by loading MCM2-7 double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin-a "double fork stall" (DFS)-replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges, and G1-specific p53-binding protein 1 (53BP1) nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agreed with theoretical predictions of DFSs. The 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins were removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle.uring the eukaryotic cell cycle, the genome must be precisely duplicated with no sections left unreplicated and no sections replicated more than once. To prevent rereplication, the process is divided into two nonoverlapping phases: during late mitosis and G1 replication origins are "licensed" for subsequent use by loading MCM2-7 double hexamers, and during S phase DNAbound MCM2-7 is activated to form processive CMG (CDC45-MCM-GINS) helicases that drive replication fork progression. The prohibition of origin licensing during S phase and G2 ensures that rereplication of DNA cannot occur. However, the inability to license new origins after the onset of S phase provides a challenge for the cell to fully replicate the genome using its finite supply of licensed origins. Replication forks can irreversibly stall when they encounter unusual structures on the DNA, such as DNA damage or tightly bound protein-DNA complexes.When replication initiation occurs at a licensed replication origin the MCM2-7 double hexamer forms a pair of bidirectionally orientated CMG helicases (1-3). If one fork irreversibly stalls, the converging fork from a neighboring origin can compensate by replicating all of the DNA up to the stalled fork. However, if two converging forks both stall and there is no licensed origin between them-a "double fork stall" (DFS)-new replicative machinery cannot be recruited to replicate the intervening DNA (4). To compensate for this potential for underreplication, origins are licensed redundantly, with most (typically >70%) remaining dorm...

Formation of trans-acting small interfering RNAs (tasiRNAs) from the TAS3 precursor is triggered by the AGO7/miR390 complex, which primes TAS3 for conversion into double-stranded RNA by the RNA-dependent RNA polymerase RDR6 and SGS3. These ta-siRNAs control several aspects of plant development. The mechanism routing AGO7-cleaved TAS3 precursor to RDR6/SGS3 and its subcellular organization are unknown. We show that AGO7 accumulates together with SGS3 and RDR6 in cytoplasmic siRNA bodies that are distinct from P-bodies. siRNA bodies colocalize with a membrane-associated viral protein and become positive for stress-granule markers upon stress-induced translational repression, this suggests that siRNA bodies are membrane-associated sites of accumulation of mRNA stalled during translation. AGO7 congregates with miR390 and SGS3 in membranes and its targeting to the nucleus prevents its accumulation in siRNA bodies and ta-siRNA formation. AGO7 is therefore required in the cytoplasm and membranous siRNA bodies for TAS3 processing, revealing a hitherto unknown role for membrane-associated ribonucleoparticles in ta-siRNA biogenesis and AGO action in plants.

Eukaryotic RNA quality control (RQC) uses both endonucleolytic and exonucleolytic degradation to eliminate dysfunctional RNAs. In addition, endogenous and exogenous RNAs are degraded through post-transcriptional gene silencing (PTGS), which is triggered by the production of double-stranded (ds)RNAs and proceeds through short-interfering (si)RNA-directed ARGONAUTE-mediated endonucleolytic cleavage. Compromising cytoplasmic or nuclear 5′–3′ exoribonuclease function enhances sense-transgene (S)-PTGS in Arabidopsis, suggesting that these pathways compete for similar RNA substrates. Here, we show that impairing nonsense-mediated decay, deadenylation or exosome activity enhanced S-PTGS, which requires host RNA-dependent RNA polymerase 6 (RDR6/SGS2/SDE1) and SUPPRESSOR OF GENE SILENCING 3 (SGS3) for the transformation of single-stranded RNA into dsRNA to trigger PTGS. However, these RQC mutations had no effect on inverted-repeat–PTGS, which directly produces hairpin dsRNA through transcription. Moreover, we show that these RQC factors are nuclear and cytoplasmic and are found in two RNA degradation foci in the cytoplasm: siRNA-bodies and processing-bodies. We propose a model of single-stranded RNA tug-of-war between RQC and S-PTGS that ensures the correct partitioning of RNA substrates among these RNA degradation pathways.