OsPRMT6a-mediated arginine methylation of OsJAZ1 regulates jasmonate signaling and spikelet development in rice.

Although both protein arginine methylation (PRMT) and jasmonate (JA) signaling are crucial for regulating plant development, the relationship between these processes in the control of spikelet development remains unclear. In this study, we used the CRISPR/Cas9 technology to generate two OsPRMT6a loss-of-function mutants that exhibit various abnormal spikelet structures. Interestingly, we found that OsPRMT6a can methylate arginine residues in JA signal repressors OsJAZ1 and OsJAZ7. We showed that arginine methylation of OsJAZ1 enhances the binding affinity of OsJAZ1 with the JA receptors OsCOI1a and OsCOI1b in the presence of JAs, thereby promoting the ubiquitination of OsJAZ1 by the SCFOsCOI1a/OsCOI1b complex and degradation via the 26S proteasome. This process ultimately releases OsMYC2, a core transcriptional regulator in the JA signaling pathway, to activate or repress JA-responsive genes, thereby maintaining normal plant (spikelet) development. However, in the osprmt6a-1 mutant, reduced arginine methylation of OsJAZ1 impaires the interaction between OsJAZ1 and OsCOI1a/OsCOI1b in the presence of JAs. As a result, OsJAZ1 proteins become more stable, repressing JA responses, thus causing the formation of abnormal spikelet structures. Moreover, we discovered that JA signaling reduces the OsPRMT6a mRNA level in an OsMYC2-dependent manner, thereby establishing a negative feedback loop to balance JA signaling. We further found that OsPRMT6a-mediated arginine methylation of OsJAZ1 likely serves as a switch to tune JA signaling to maintain normal spikelet development under harsh environmental conditions such as high temperatures. Collectively, our study establishes a direct molecular link between arginine methylation and JA signaling in rice.

Rice requires a chromatin remodeler for Polymerase IV-small interfering RNA production and genomic immunity.

Transgenes are often spontaneously silenced, which hinders the application of genetic modifications to crop breeding. While gene silencing has been extensively studied in Arabidopsis (Arabidopsis thaliana), the molecular mechanism of transgene silencing remains elusive in crop plants. We used rice (Oryza sativa) plants silenced for a 35S::OsGA2ox1 (Gibberellin 2-oxidase 1) transgene to isolate five elements mountain (fem) mutants showing restoration of transgene expression. In this study, we isolated multiple fem2 mutants defective in a homolog of Required to Maintain Repression 1 (RMR1) of maize (Zea mays) and CLASSY (CLSY) of Arabidopsis. In addition to failing to maintain transgene silencing, as occurs in fem3, in which mutation occurs in NUCLEAR RNA POLYMERASE E1 (OsNRPE1), the fem2 mutant failed to establish transgene silencing of 35S::OsGA2ox1. Mutation in FEM2 eliminated all RNA POLYMERASE IV (Pol-IV)-FEM1/OsRDR2 (RNA-DEPENDENT RNA POLYMERASE 2)-dependent small interfering RNAs (siRNAs), reduced DNA methylation on genome-wide scale in rice seedlings, caused pleiotropic developmental defects, and increased disease resistance. Simultaneous mutation in 2 FEM2 homologous genes, FEM2-Like 1 (FEL1) and FEL2, however, did not affect DNA methylation and rice development and disease resistance. The predominant expression of FEM2 over FEL1 and FEL2 in various tissues was likely caused by epigenetic states. Overexpression of FEL1 but not FEL2 partially rescued hypomethylation of fem2, indicating that FEL1 maintains the cryptic function. In summary, FEM2 is essential for establishing and maintaining gene silencing; moreover, FEM2 is solely required for Pol IV-FEM1 siRNA biosynthesis and de novo DNA methylation.

DNA-dependent RNA polymerases in plants.

DNA-dependent RNA polymerases (Pols) transfer the genetic information stored in genomic DNA to RNA in all organisms. In eukaryotes, the typical products of nuclear Pol I, Pol II, and Pol III are ribosomal RNAs, mRNAs, and transfer RNAs, respectively. Intriguingly, plants possess two additional Pols, Pol IV and Pol V, which produce small RNAs and long noncoding RNAs, respectively, mainly for silencing transposable elements. The five plant Pols share some subunits, but their distinct functions stem from unique subunits that interact with specific regulatory factors in their transcription cycles. Here, we summarize recent advances in our understanding of plant nucleus-localized Pols, including their evolution, function, structures, and transcription cycles.

Discovery of aphid-transmitted Rice tiller inhibition virus from native plants through metagenomic sequencing.

A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host uncharacterized endemic viruses with spillover potential to rice (Oryza sativa) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice (O. rufipogon) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector that is able to transmit RTIV and found that RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover.

The Rise and Fall of Billionaire siRNAs during Reproductive Development in Rice.

RNA polymerase IV-dependent siRNAs, usually 24 nt in length, function in the RNA-directed DNA methylation that is responsible for de novo methylation in plants. We analyzed 24 nt siRNAs in inflorescences and found that among the 20,200 24 nt siRNA clusters, the top 0.81% highly expressed clusters accounted for more than 68% of the 24 nt siRNA reads in inflorescences. We named the highly expressed siRNAs as billionaire siRNAs (bill-siRNAs) and the less-expressed siRNAs as pauper siRNAs (pau-siRNAs). The bill-siRNAs in inflorescences are mainly derived from the ovary. Female gametes produced more bill-siRNAs than male gametes. In embryos and seedlings developed from fertilized egg cells, the bill-siRNAs from gametes disappeared. The endosperm, which develops from the fertilized central cell, also contained no bill-siRNAs from gametes but did contain newly and highly expressed siRNAs produced in different regions. In contrast, bill-siRNAs from the ovaries were maintained in the seed coat. The biosynthesis of bill-siRNAs in various tissues and cells is dependent on OsRDR2 (RNA-dependent RNA polymerase 2) and Pol IV (DNA-dependent RNA polymerase IV). Similar to the pau-siRNAs, the first base of bill-siRNAs is enriched at adenine, and bill-siRNAs can direct DNA methylation in various tissues.

The warfare for plant highway: vascular plant-microbe interaction pinpoints lignin.

Plant vascular pathogens are one kind of destructive pathogens in agricultural production. However, mechanisms behind the vascular pathogen-recognition and the subsequent defense responses of plants are not well known. A recent pioneering study on plant vascular immunity discovered a conserved MKP1-MPK-MYB signaling cascade that activates lignin biosynthesis in vascular tissues to confer vascular resistance in both monocot rice and the dicot Arabidopsis. The breakthrough provides a novel view on plant immunity to vascular pathogens and offers a potential strategy for the future breeding of disease-resistant crops.

Reinforcement of CHH methylation through RNA-directed DNA methylation ensures sexual reproduction in rice.

DNA methylation is an important epigenetic mark that regulates the expression of genes and transposons. RNA-directed DNA methylation (RdDM) is the main molecular pathway responsible for de novo DNA methylation in plants. Although the mechanism of RdDM has been well studied in Arabidopsis (Arabidopsis thaliana), most mutations in RdDM genes cause no remarkable developmental defects in Arabidopsis. Here, we isolated and cloned Five Elements Mountain 1 (FEM1), which encodes RNA-dependent RNA polymerase 2 (OsRDR2) in rice (Oryza sativa). Mutation in OsRDR2 abolished the accumulation of 24-nt small interfering RNAs, and consequently substantially decreased genome-wide CHH (H = A, C, or T) methylation. Moreover, male and female reproductive development was disturbed, which led to sterility in osrdr2 mutants. We discovered that OsRDR2-dependent DNA methylation may regulate the expression of multiple key genes involved in stamen development, meiosis, and pollen viability. In wild-type (WT) plants but not in osrdr2 mutants, genome-wide CHH methylation levels were greater in panicles, stamens, and pistils than in seedlings. The global increase of CHH methylation in reproductive organs of the WT was mainly explained by the enhancement of RdDM activity, which includes OsRDR2 activity. Our results, which revealed a global increase in CHH methylation through enhancement of RdDM activity in reproductive organs, suggest a crucial role for OsRDR2 in the sexual reproduction of rice.

The effect of RNA polymerase V on 24-nt siRNA accumulation depends on DNA methylation contexts and histone modifications in rice.

RNA-directed DNA methylation (RdDM) functions in de novo methylation in CG, CHG, and CHH contexts. Here, we performed map-based cloning of OsNRPE1, which encodes the largest subunit of RNA polymerase V (Pol V), a key regulator of gene silencing and reproductive development in rice. We found that rice Pol V is required for CHH methylation on RdDM loci by transcribing long noncoding RNAs. Pol V influences the accumulation of 24-nucleotide small interfering RNAs (24-nt siRNAs) in a locus-specific manner. Biosynthesis of 24-nt siRNAs on loci with high CHH methylation levels and low CG and CHG methylation levels tends to depend on Pol V. In contrast, low methylation levels in the CHH context and high methylation levels in CG and CHG contexts predisposes 24-nt siRNA accumulation to be independent of Pol V. H3K9me1 and H3K9me2 tend to be enriched on Pol V-independent 24-nt siRNA loci, whereas various active histone modifications are enriched on Pol V-dependent 24-nt siRNA loci. DNA methylation is required for 24-nt siRNAs biosynthesis on Pol V-dependent loci but not on Pol V-independent loci. Our results reveal the function of rice Pol V for long noncoding RNA production, DNA methylation, 24-nt siRNA accumulation, and reproductive development.

The SUMO E3 ligase SIZ1 partially regulates STOP1 SUMOylation and stability in Arabidopsis thaliana.

The zinc finger transcription factor STOP1 plays a crucial role in aluminum (Al) resistance and low phosphate (Pi) response. Al stress and low Pi availability do not affect STOP1 mRNA expression but are able to induce STOP1 protein accumulation by post-transcriptional regulatory mechanisms. We recently reported that STOP1 can be mono-SUMOylated at K40, K212, or K395 sites, and deSUMOylated by the SUMO protease ESD4. SUMOylation of STOP1 is important for the regulation of STOP1 protein function and Al resistance. In the present study, we further characterized the role of the SUMO E3 ligase SIZ1 in STOP1 SUMOylation, Al resistance and low Pi response. We found that mutation of SIZ1 reduced but not eliminated STOP1 SUMOylation, suggesting that SIZ1-dependent and -independent pathways are involved in the regulation of STOP1 SUMOylation. The STOP1 protein levels were decreased in siz1 mutants. Nevertheless, the expression of STOP1-target gene AtALMT1 was increased instead of reduced in siz1 mutants. The mutants showed enhanced Al resistance and low Pi response. Our results suggest that SIZ1 regulates Al resistance and low Pi response likely through the modulation of AtALMT1 expression.

Functional analysis of the cotton CLE polypeptide signaling gene family in plant growth and development.

The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)-RELATED (CLE) gene family encodes a large number of polypeptide signaling molecules involved in the regulation of shoot apical meristem division and root and vascular bundle development in a variety of plants. CLE family genes encode important short peptide hormones; however, the functions of these signaling polypeptides in cotton remain largely unknown. In the current work, we studied the effects of the CLE family genes on growth and development in cotton. Based on the presence of a conserved CLE motif of 13 amino acids, 93 genes were characterized as GhCLE gene family members, and these were subcategorized into 7 groups. A preliminary analysis of the cotton CLE gene family indicated that the activity of its members tends to be conserved in terms of both the 13-residue conserved domain at the C-terminus and their subcellular localization pattern. Among the 14 tested genes, the ectopic overexpression of GhCLE5::GFP partially mimicked the phenotype of the clv3 mutant in Arabidopsis. GhCLE5 could affect the endogenous CLV3 in binding to the receptor complex, comprised of CLV1, CLV2, and CRN, in the yeast two-hybrid assay and split-luciferase assay. Silencing GhCLE5 in cotton caused a short seedling phenotype. Therefore, we concluded that the cotton GhCLE gene family is functionally conserved in apical shoot development regulation. These results indicate that CLE also plays roles in cotton development as a short peptide hormone.

Molecular Basis of Disease Resistance and Perspectives on Breeding Strategies for Resistance Improvement in Crops.

Crop diseases are major factors responsible for substantial yield losses worldwide, which affects global food security. The use of resistance (R) genes is an effective and sustainable approach to controlling crop diseases. Here, we review recent advances on R gene studies in the major crops and related wild species. Current understanding of the molecular mechanisms underlying R gene activation and signaling, and susceptibility (S) gene-mediated resistance in crops are summarized and discussed. Furthermore, we propose some new strategies for R gene discovery, how to balance resistance and yield, and how to generate crops with broad-spectrum disease resistance. With the rapid development of new genome-editing technologies and the availability of increasing crop genome sequences, the goal of breeding next-generation crops with durable resistance to pathogens is achievable, and will be a key step toward increasing crop production in a sustainable way.

Epigenetic memory marks determine epiallele stability at loci targeted by de novo DNA methylation.

It is generally assumed that DNA methylation changes at genomic regions targeted by the de novo RNA-directed DNA methylation (RdDM) pathway are unstable. Here, we show that RdDM targets in Arabidopsis can be classified into two groups on the basis of whether there is remethylation following the restoration of NRPD1 function in nrpd1 mutant plants-remethylable loci and non-remethylable loci. In contrast to the remethylable loci, the non-remethylable loci contain higher levels of the euchromatic marks of trimethylation at Lys 4 of histone H3 (H3K4me3), which interferes with the recruitment of the RdDM molecular machinery, and acetylation at Lys 18 of histone H3 (H3K18ac), which helps to recruit the DNA demethylase ROS1 to antagonize RdDM. Here, using targeted methylation erasure by CRISPR-dCas9-TET1, we demonstrate that methylated CG (mCG) and mCHG (where H represents A, C or T) are memory marks that are required for targeting the RdDM machinery to remethylable loci. Our results show that histone and DNA methylation marks are critical in determining the ability of RdDM target loci to form stable epialleles, and contribute to understanding the formation and transmission of epialleles.

Author Correction: Inducible overexpression of Ideal Plant Architecture1 improves both yield and disease resistance in rice.

In the Supplementary Information file originally published with this Article, the authors mistakenly omitted accompanying legends for Supplementary Figures 1-15; this has now been amended.

Inducible overexpression of Ideal Plant Architecture1 improves both yield and disease resistance in rice.

Breeding crops with resistance is an efficient way to control diseases. However, increased resistance often has a fitness penalty. Thus, simultaneously increasing disease resistance and yield potential is a challenge in crop breeding. In this study, we found that downregulation of microRNA-156 (miR-156) and overexpression of Ideal Plant Architecture1 (IPA1) and OsSPL7, two target genes of miR-156, enhanced disease resistance against bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo), but reduced rice yield. We discovered that gibberellin signalling might be partially responsible for the disease resistance and developmental defects in IPA1 overexpressors. We then generated transgenic rice plants expressing IPA1 with the pathogen-inducible promoter of OsHEN1; these plants had both enhanced disease resistance and enhanced yield-related traits. Thus, we have identified miR-156-IPA1 as a novel regulator of the crosstalk between growth and defence, and we have established a new strategy for obtaining both high disease resistance and high yield.

Four putative SWI2/SNF2 chromatin remodelers have dual roles in regulating DNA methylation in Arabidopsis.

DNA methylation is a conserved epigenetic mark that is critical for many biological processes in plants and mammals. In Arabidopsis, the antagonistic activities of RNA-directed DNA methylation (RdDM) and ROS1-dependent active DNA demethylation are key for the dynamic regulation of locus-specific DNA methylation. However, the molecular factors that coordinate RdDM and active demethylation are largely unknown. Here we report that CLSY4 and its three paralogous SWI2/SNF2-type chromatin-remodeling proteins function in both RdDM and DNA demethylation in Arabidopsis. We initially identified CLSY4 in a genetic screen for DNA demethylation factors and subsequently demonstrated that it also is important in RdDM. Comprehensive genetic analyses using single and high order mutants of CLSY family proteins revealed their roles as double agents in the balance between methylation and demethylation reactions. The four CLSY proteins collectively are necessary for the canonical RdDM pathway; at the same time, each CLSY likely mediates DNA demethylation at specific loci where DNA methylation depends on RdDM. These results indicate that the four chromatin-remodeling proteins have dual functions in regulating genomic DNA methylation, and thus provide new insights into the dynamic regulation of DNA methylation in a model multicellular eukaryotic organism.

Plasma Membrane-Localized Calcium Pumps and Copines Coordinately Regulate Pollen Germination and Fertility in Arabidopsis.

Calcium plays an important role in plant growth, development, and response to environmental stimuli. Copines are conserved plasma membrane-localized calcium-binding proteins which regulate plant immune responses and development. In this study, we found that copine proteins BON2 and BON3, the paralogs of BON1, physically interact with calcium pumps ACA8 and ACA10 in Arabidopsis. Notably, ACA9, the closest homologue of ACA8 and ACA10 functioning in pollen tube growth, interacts with all three copines. This is consistent with the protein⁻protein interactions between the two protein families, the aca8, aca10, aca8/aca10, bon1/2/3 mutants as well as aca9 mutant exhibited defects on pollen germination and seed production. Taken together, plasma membrane-localized interacting calcium pumps and copines coordinately control pollen tube growth, likely through manipulating calcium efflux.

Rice Interploidy Crosses Disrupt Epigenetic Regulation, Gene Expression, and Seed Development.

Seed development in angiosperms requires a 2:1 maternal-to-paternal genome ratio (2m:1p) in the endosperm. When the ratio is disrupted, the seed development is impaired. Rice interploidy crosses result in endosperm failures, but the underlying molecular mechanisms remain unclear. Here, we report that the defective endosperm in rice interploidy crosses was associated with nonadditive expression of small RNAs and protein-coding genes. Interestingly, 24-nt small interfering RNAs were enriched in the 5' and 3' flanking sequences of nonadditively expressed genes in the interploidy crosses and were negatively associated with the expression of imprinted genes. Furthermore, some PRC2 family genes and DNA methylation-related genes including OsMET1b and OsCMT3a were upregulated in the 2×4 cross (pollinating a diploid "mother" with a tetraploid "father") but repressed in the reciprocal cross. These different epigenetic effects could lead to precocious or delayed cellularization during endosperm development. Notably, many endosperm-preferred genes, including starch metabolic and storage protein genes during grain filling, were found to be associated with DNA methylation or H3K27me3, which are repressed in both 2×4 and 4×2 crosses. WUSCHEL homeobox2 (WOX2)-like (WOX2L), an endosperm-preferred gene, was expressed specifically in the rice endosperm, in contrast to WOX2 expression in the Arabidopsis embryo. Disruption of WOX2L in transgenic rice by CRISPR/Cas9-mediated gene editing blocked starch and protein accumulation, resulting in seed abortion. In addition to gene repression, disrupting epigenetic process in the interploidy crosses also induced expression of stress-responsive genes. Thus, maintaining the 2m:1p genome ratio in the endosperm is essential for normal grain development in rice and other cereal crops.

The Arabidopsis Chromatin-Remodeling Factor CHR5 Regulates Plant Immune Responses and Nucleosome Occupancy.

ATP-dependent chromatin-remodeling factors use the energy of ATP hydrolysis to alter the structure of chromatin and are important regulators of eukaryotic gene expression. One such factor encoded by CHR5 (Chromatin-Remodeling Factor 5) in Arabidopsis (Arabidopsis thaliana) was previously found to be involved in regulation of growth and development. Here we show that CHR5 is required for the up-regulation of the intracellular immune receptor gene SNC1 (SUPPRESSOR OF npr1-1, CONSTITUTIVE1) and consequently the autoimmunity induced by SNC1 up-regulation. CHR5 functions antagonistically with another chromatin-remodeling gene DDM1 (DECREASED DNA METHYLATION 1) and independently with a histone mono-ubiquitinase HUB1 (HISTONE MONOUBIQUITINATION 1) in SNC1 regulation. In addition, CHR5 is a positive regulator of SNC1-independent plant immunity against the bacterial pathogen Pseudomonas syringae. Furthermore, the chr5 mutant has increased nucleosome occupancy in the promoter region relative to the gene body region at the whole-genome level, suggesting a global role for CHR5 in remodeling nucleosome occupancy. Our study thus establishes CHR5 as a positive regulator of plant immune responses including the expression of SNC1 and reveals a role for CHR5 in nucleosome occupancy which probably impacts gene expression genome wide.

Calcium Pumps and Interacting BON1 Protein Modulate Calcium Signature, Stomatal Closure, and Plant Immunity.

Calcium signaling is essential for environmental responses including immune responses. Here, we provide evidence that the evolutionarily conserved protein BONZAI1 (BON1) functions together with autoinhibited calcium ATPase10 (ACA10) and ACA8 to regulate calcium signals in Arabidopsis. BON1 is a plasma membrane localized protein that negatively regulates the expression of immune receptor genes and positively regulates stomatal closure. We found that BON1 interacts with the autoinhibitory domains of ACA10 and ACA8, and the aca10 loss-of-function (LOF) mutants have an autoimmune phenotype similar to that of the bon1 LOF mutants. Genetic evidences indicate that BON1 positively regulates the activities of ACA10 and ACA8. Consistent with this idea, the steady level of calcium concentration is increased in both aca10 and bon1 mutants. Most strikingly, cytosolic calcium oscillation imposed by external calcium treatment was altered in aca10, aca8, and bon1 mutants in guard cells. In addition, calcium- and pathogen-induced stomatal closure was compromised in the aca10 and bon1 mutants. Taken together, this study indicates that ACA10/8 and BON1 physically interact on plasma membrane and function in the generation of cytosol calcium signatures that are critical for stomatal movement and impact plant immunity.

Both maternally and paternally imprinted genes regulate seed development in rice.

Genetic imprinting refers to the unequal expression of paternal and maternal alleles of a gene in sexually reproducing organisms, including mammals and flowering plants. Although many imprinted genes have been identified in plants, the functions of these imprinted genes have remained largely uninvestigated. We report genome-wide analysis of gene expression, DNA methylation and small RNAs in the rice endosperm and functional tests of five imprinted genes during seed development using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated gene9 (CRISPR/Cas9) gene editing technology. In the rice endosperm, we identified 162 maternally expressed genes (MEGs) and 95 paternally expressed genes (PEGs), which were associated with miniature inverted-repeat transposable elements, imprinted differentially methylated loci and some 21-22 small interfering RNAs (siRNAs) and long noncoding RNAs (lncRNAs). Remarkably, one-third of MEGs and nearly one-half of PEGs were associated with grain yield quantitative trait loci. Most MEGs and some PEGs were expressed specifically in the endosperm. Disruption of two MEGs increased the amount of small starch granules and reduced grain and embryo size, whereas mutation of three PEGs reduced starch content and seed fertility. Our data indicate that both MEGs and PEGs in rice regulate nutrient metabolism and endosperm development, which optimize seed development and offspring fitness to facilitate parental-offspring coadaptation. These imprinted genes and mechanisms could be used to improve the grain yield of rice and other cereal crops.