Xufeng Wang

Transposon silencing via RNA-directed DNA methylation (RdDM) is mediated primarily through 24-nucleotide (nt) small-interfering RNAs (siRNAs) and 2 plant-specific RNA polymerases, Pol IV and Pol V. We generated and characterized RNA-interference (RNAi) lines targeting the largest subunit of Pol IV and Pol V and the second-largest subunit shared by Pol IV and Pol V in soybean. Our analyses showed that the canonical roles of Pol IV and Pol V in RdDM as described in Arabidopsis, whereby Pol IV produces 24-nt siRNAs and Pol V recruits siRNAs to homologous loci to trigger DNA methylation, are conserved in soybean. Our analyses also uncovered previously unknown functions of Pol IV and Pol V in that they repress defense response genes en masse. These genes normally undergo RdDM and are silenced, but they are de-repressed when Pol IV and Pol V are knocked down. Furthermore, the de-repression of a set of defense-related genes channeled their RNAs into the RNAi pathway to produce 21- to 22-nt siRNAs. Knocking down Pol IV and Pol V either singly or together led to increased resistance against the oomycete pathogen Phytophthora sojae, suggesting that Pol IV- and Pol V-mediated gene silencing regulates plant immunity.

RNA silencing negatively regulates gene expression at the transcriptional and post-transcriptional levels through DNA methylation, histone modification, mRNA cleavage, and translational inhibition. Small interfering RNAs (siRNAs) of 21 to 24 nucleotides are processed from double-stranded RNAs by Dicer-like enzymes and play essential roles in RNA silencing in plants. Here, we demonstrated that ALTERED MERISTEM PROGRAM1 (AMP1) and its putative paralog LIKE AMP1 (LAMP1) impair RNA silencing by repressing the biogenesis of a subset of inverted repeat (IR)–derived siRNAs in Arabidopsis (Arabidopsis thaliana). AMP1 and LAMP1 inhibit Pol II-dependent IR gene transcription by suppressing ARGONAUTE 1 (AGO1) protein levels. Genetic analysis indicates that AMP1 acts upstream of RNA polymerase IV subunit 1 (NRPD1), RNA-dependent RNA polymerase 2 (RDR2), and Dicer-Like 4 (DCL4), which are required for IR-induced RNA silencing. We also show that AMP1 and LAMP1 inhibit siRNA-mediated silencing in a different mechanism from AGO4 and DCL3. Together, these results reveal two previously unknown players in siRNA biogenesis from IRs – AGO1, which promotes IR transcription, and AMP1, which inhibits IR transcription indirectly through the repression of AGO1 expression.

Summary: MicroRNA827 (miR827) is functionally conserved among different plant species and displays species‐specific characteristics, but the mechanisms by which miR827 regulates phosphate (Pi) starvation tolerance and maize development remain elusive. We found that miR827 selectively targets the Pi transporter genes SPX‐MFS1 and SPX‐MFS5. miR827 overexpression improved the Pi starvation tolerance, plant architecture and grain yield and quality, whereas miR827 suppression yielded a contrasting phenotype. In addition, we identified a specific long noncoding RNA (lncRNA767) that serves as a direct target and a facilitator of miR827 and can stabilize the SPX‐MFS1 and SPX‐MFS5 transcripts, leading to their translation inhibition. The orchestrated regulation of SPX‐MFS1 and SPX‐MFS5 modulates PHR1; 1 and PHR1; 2, which are critical transcription factors in Pi signalling, and thereby affects the expression of downstream Pi starvation‐induced genes. Together, these findings demonstrate that miR827, assisted by lncRNA767, enhances SPX‐MFS1 and SPX‐MFS5 suppression and thus exerts a significant impact on Pi homeostasis and several essential agronomic traits of maize.

The occurrence of NAD⁺ as a non-canonical RNA cap has been demonstrated in diverse organisms. TIR domain-containing proteins present in all kingdoms of life act in defense responses and can have NADase activity that hydrolyzes NAD⁺. Here, we show that TIR domain-containing proteins from several bacterial and one archaeal species can remove the NAM moiety from NAD-capped RNAs (NAD-RNAs). We demonstrate that the deNAMing activity of AbTir (from Acinetobacter baumannii) on NAD-RNA specifically produces a cyclic ADPR-RNA, which can be further decapped in vitro by known decapping enzymes. Heterologous expression of the wild-type but not a catalytic mutant AbTir in E. coli suppressed cell propagation and reduced the levels of NAD-RNAs from a subset of genes before cellular NAD⁺ levels are impacted. Collectively, the in vitro and in vivo analyses demonstrate that TIR domain-containing proteins can function as a deNAMing enzyme of NAD-RNAs, raising the possibility of TIR domain proteins acting in gene expression regulation.

Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S ribosomal RNA (rRNA) 3′ end maturation during late-stage 40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the genome-wide effects of eIF5B have not been studied at the single-nucleotide resolution in any organism, and 18S rRNA 3′ end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3′ end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3′ end maturation or metabolism. We quantitatively defined processing hotspots and identified adenylation as the prevalent nontemplated RNA addition at the 3′ ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNA interference to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3′ portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions and were not responsible for the 18S rRNA maturation or translation initiation defects in hot3 . Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late 40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, messenger RNA (mRNA) translation initiation, and siRNA biogenesis in plants.

The macronutrient phosphorus is essential for plant growth and development. Plants have evolved multiple strategies to increase the efficiency of phosphate (Pi) acquisition to protect themselves from Pi starvation. However, the crosstalk between Pi homeostasis and plant development remains to be explored. Here, we report that overexpressing microRNA399 (miR399) in maize (Zea mays) is associated with premature senescence after pollination. Knockout of ZmPHO2 (Phosphate 2), a miR399 target, resulted in a similar premature senescence phenotype. Strikingly, we discovered that INDETERMINATE1 (ID1), a floral transition regulator, inhibits the transcription of ZmMIR399 genes by directly binding to their promoters, alleviating the repression of ZmPHO2 by miR399 and ultimately contributing to the maintenance of Pi homeostasis in maize. Unlike ZmMIR399 genes, whose expression is induced by Pi deficiency, ID1 expression was independent of the external inorganic orthophosphate status, indicating that ID1 is an autonomous regulator of Pi homeostasis. Furthermore, we show that ZmPHO2 was under selection during maize domestication and cultivation, resulting in a more sensitive response to Pi starvation in temperate maize than in tropical maize. Our study reveals a direct functional link between Pi-deprivation sensing by the miR399-ZmPHO2 regulatory module and plant developmental regulation by ID1.

MicroRNA156 (miR156) and miR529 have high sequence similarity and recognize overlapping sites in the same target genes, SQUAMOSA promoter binding protein-like (SPL or SBP box) genes, making it difficult to accurately distinguish their roles in regulatory networks that affect numerous biological functions. Here, we collected data about miR156 and miR529 family members from representative land plants and performed sequence comparisons, phylogenetic analysis, small RNA sequencing, and parallel analysis of RNA ends (PARE) analysis to dissect their evolutionary and functional differences. Although miR156 and miR529 are highly similar, there are differences in their mismatch-sensitive regions, which are essential for target recognition. In land plants, miR156 precursors are conserved mainly within the hairpin region, whereas miR529 precursors are conserved outside the hairpin region, including both the 5’ and 3’ arms. Phylogenetic analysis showed that MIR156 and MIR529 evolved independently, through divergent evolutionary patterns. The two genes also exhibit different expression patterns, with MIR529 preferentially expressed in reproductive tissues and MIR156 in other tissues. PARE analysis revealed that miR156 and miR529 possess specific targets in addition to common targets in maize, pointing to functional differences between them. Based on our findings, we developed a method for the rapid identification of miR529 and miR156 family members and uncovered the evolutionary divergence of these families, providing insights into their different regulatory roles in plant growth and development.

Significance: Some RNAs in both prokaryotes and eukaryotes were recently found to contain the NAD ⁺ cap, indicating a novel mechanism in gene regulation through noncanonical RNA capping. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry has been used to label NAD ⁺ -capped RNAs (NAD-RNAs) for their identification. However, copper-caused RNA fragmentation/degradation interferes with the analysis. We developed the NAD tagSeq II method for transcriptome-wide NAD-RNA analysis based on copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry. This method was used to compare NAD-RNA and total transcriptome profiles in Escherichia coli . Our study reveals genome-wide alterations in E. coli RNA NAD ⁺ capping in different growth phases and indicates that NAD-RNAs could be the primary form of transcripts from some genes under certain environments.

Significance: The m ⁷ G cap is the canonical RNA cap in eukaryotes, but other noncanonical RNA caps exist, including the NAD ⁺ cap. NAD captureSeq has been widely used to profile NAD ⁺ -capped RNAs (NAD-RNAs) in prokaryotes and eukaryotes. However, NAD captureSeq reacts at a low level with m ⁷ G-RNAs and introduces copper ions that cause RNA fragmentation, resulting in reduced sensitivity and loss of full-length sequence information. To address these issues, we developed the copper-free SPAAC-NAD-seq, which utilizes the strain-promoted azide–alkyne cycloaddition reaction to capture NAD-RNAs followed by high-throughput sequencing. Compared to NAD captureSeq, SPAAC-NAD-seq is more sensitive and retains full-length sequence information. This optimized technique provides a useful tool to profile NAD-RNAs in prokaryotes and, when combined with m ⁷ G-RNA depletion, in eukaryotes.

Background: Small RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) serve as core players in gene silencing at transcriptional and post-transcriptional levels in plants, but their subcellular localization has not yet been well studied, thus limiting our mechanistic understanding of sRNA action.Results: We investigate the cytoplasmic partitioning of sRNAs and their targets globally in maize (Zea mays, inbred line “B73”) and rice (Oryza sativa, cv. “Nipponbare”) by high-throughput sequencing of polysome-associated sRNAs and 3′ cleavage fragments, and find that both miRNAs and a subset of 21-nucleotide (nt)/22-nt siRNAs are enriched on membrane-bound polysomes (MBPs) relative to total polysomes (TPs) across different tissues. Most of the siRNAs are generated from transposable elements (TEs), and retrotransposons positively contributed to MBP overaccumulation of 22-nt TE-derived siRNAs (TE-siRNAs) as opposed to DNA transposons. Widespread occurrence of miRNA-mediated target cleavage is observed on MBPs, and a large proportion of these cleavage events are MBP-unique. Reproductive21PHAS(21-nt phasiRNA-generating) and24PHAS(24-nt phasiRNA-generating) precursors, which were commonly considered as noncoding RNAs, are bound by polysomes, and high-frequency cleavage of21PHASprecursors by miR2118 and24PHASprecursors by miR2275 is further detected on MBPs. Reproductive 21-nt phasiRNAs are enriched on MBPs as opposed to TPs, whereas 24-nt phasiRNAs are nearly completely devoid of polysome occupancy.Conclusions: MBP overaccumulation is a conserved pattern for cytoplasmic partitioning of sRNAs, and endoplasmic reticulum (ER)-bound ribosomes function as an independent regulatory layer for miRNA-induced gene silencing and reproductive phasiRNA biosynthesis in maize and rice.