The key micronutrient copper orchestrates broad-spectrum virus resistance in rice.

Copper is a critical regulator of plant growth and development. However, the mechanisms by which copper responds to virus invasion are unclear. We previously showed that SPL9-mediated transcriptional activation of miR528 adds a previously unidentified regulatory layer to the established ARGONAUTE (AGO18)-miR528-L-ascorbate oxidase (AO) antiviral defense. Here, we report that rice promotes copper accumulation in shoots by inducing copper transporter genes, including HMA5 and COPT, to counteract viral infection. Copper suppresses the transcriptional activation of miR528 by inhibiting the protein level of SPL9, thus alleviating miR528-mediated cleavage of AO transcripts to strengthen the antiviral response. Loss-of-function mutations in HMA5, COPT1, and COPT5 caused a significant reduction in copper accumulation and plant viral resistance because of the increased SPL9-mediated miR528 transcription. Gain in viral susceptibility was mitigated when SPL9 was mutated in the hma5 mutant background. Our study elucidates the molecular mechanisms and regulatory networks of copper homeostasis and the SPL9-miR528-AO antiviral pathway.

A plant immune protein enables broad antitumor response by rescuing microRNA deficiency.

Cancer cells are featured with uncontrollable activation of cell cycle, and microRNA deficiency drives tumorigenesis. The RNA-dependent RNA polymerase (RDR) is essential for small-RNA-mediated immune response in plants but is absent in vertebrates. Here, we show that ectopic expression of plant RDR1 can generally inhibit cancer cell proliferation. In many human primary tumors, abnormal microRNA isoforms with 1-nt-shorter 3' ends are widely accumulated. RDR1 with nucleotidyltransferase activity can recognize and modify the problematic AGO2-free microRNA duplexes with mononucleotides to restore their 2 nt overhang structure, which eventually rescues AGO2-loading efficiency and elevates global miRNA expression to inhibit cancer cell-cycle specifically. The broad antitumor effects of RDR1, which can be delivered by an adeno-associated virus, are visualized in multiple xenograft tumor models in vivo. Altogether, we reveal the widespread accumulation of aberrant microRNA isoforms in tumors and develop a plant RDR1-mediated antitumor stratagem by editing and repairing defective microRNAs.

Suppression of rice miR168 improves yield, flowering time and immunity.

MicroRNA168 (miR168) is a key miRNA that targets Argonaute1 (AGO1), a major component of the RNA-induced silencing complex1,2. Previously, we reported that miR168 expression was responsive to infection by Magnaporthe oryzae, the causal agent of rice blast disease3. However, how miR168 regulates immunity to rice blast and whether it affects rice development remains unclear. Here, we report our discovery that the suppression of miR168 by a target mimic (MIM168) not only improves grain yield and shortens flowering time in rice but also enhances immunity to M. oryzae. These results were validated through repeated tests in rice fields in the absence and presence of rice blast pressure. We found that the miR168-AGO1 module regulates miR535 to improve yield by increasing panicle number, miR164 to reduce flowering time, and miR1320 and miR164 to enhance immunity. Our discovery demonstrates that changes in a single miRNA enhance the expression of multiple agronomically important traits.

Jasmonate Signaling Enhances RNA Silencing and Antiviral Defense in Rice.

Small RNA-mediated RNA silencing is an important antiviral mechanism in higher plants. It has been shown that RNA silencing components can be upregulated by viral infection. However, the mechanisms underlying the upregulation remain largely unknown. Here, we show that jasmonate (JA) signaling transcriptionally activates Argonaute 18 (AGO18), a core RNA silencing component that promotes rice antiviral defense through sequestering miR168 and miR528, which repress key antiviral defense proteins. Mechanistically, the JA-responsive transcription factor JAMYB directly binds to the AGO18 promoter to activate AGO18 transcription. Rice stripe virus (RSV) coat protein (CP) triggers JA accumulation and upregulates JAMYB to initiate this host defense network. Our study reveals that regulatory crosstalk exists between the JA signaling and antiviral RNA silencing pathways and elucidates a molecular mechanism for CP-mediated viral resistance in monocot crops.

Transcriptional Regulation of miR528 by OsSPL9 Orchestrates Antiviral Response in Rice.

Many microRNAs (miRNAs) are critical regulators of plant antiviral defense. However, little is known about how these miRNAs respond to virus invasion at the transcriptional level. We previously show that defense against Rice stripe virus (RSV) invasion entailed a reduction of miR528 accumulation in rice, alleviating miR528-mediated degradation of L-Ascorbate Oxidase (AO) mRNA and bolstering the antiviral activity of AO. Here we show that the miR528-AO defense module is regulated by the transcription factor SPL9. SPL9 displayed high-affinity binding to specific motifs within the promoter region of miR528 and activated the expression of miR528 gene in vivo. Loss-of-function mutations in SPL9 caused a significant reduction in miR528 accumulation but a substantial increase of AO mRNA, resulting in enhanced plant resistance to RSV. Conversely, transgenic overexpression of SPL9 stimulated the expression of miR528 gene, hence lowering the level of AO mRNA and compromising rice defense against RSV. Importantly, gain in RSV susceptibility did not occur when SPL9 was overexpressed in mir528 loss-of-function mutants, or in transgenic rice expressing a miR528-resistant AO. Taken together, the finding of SPL9-mediated transcriptional activation of miR528 expression adds a new regulatory layer to the miR528-AO antiviral defense pathway.

ROS accumulation and antiviral defence control by microRNA528 in rice.

MicroRNAs (miRNAs) are key regulators of plant-pathogen interactions. Modulating miRNA function has emerged as a new strategy to produce virus resistance traits1-5. However, the miRNAs involved in antiviral defence and the underlying mechanisms remain largely elusive. We previously demonstrated that sequestration by Argonaute (AGO) proteins plays an important role in regulating miRNA function in antiviral defence pathways6. Here we reveal that cleavage-defective AGO18 complexes sequester microRNA528 (miR528) upon viral infection. We show that miR528 negatively regulates viral resistance in rice by cleaving L-ascorbate oxidase (AO) messenger RNA, thereby reducing AO-mediated accumulation of reactive oxygen species. Upon viral infection, miR528 becomes preferentially associated with AGO18, leading to elevated AO activity, higher basal reactive oxygen species accumulation and enhanced antiviral defence. Our findings reveal a mechanism in which antiviral defence is boosted through suppression of an miRNA that negatively regulates viral resistance. This mechanism could be manipulated to engineer virus-resistant crop plants.