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Segregation distorters (SDs) are genetic elements that distort the Mendelian segregation ratio to favor their own transmission and are able to spread even when they incur fitness costs on organisms carrying them. Depending on the biology of the host organisms and the genetic architecture of the SDs, the population dynamics of SDs can be highly variable. Inbreeding is considered an effective mechanism for inhibiting the spread of SDs in populations, and can evolve as a defense mechanism against SDs in some systems. However, we show that inbreeding in the form of selfing in fact promotes the spread of SDs acting as pollen killers in a toxin-antidote system in hermaphroditic plants by two mechanisms: (i) By reducing the effective recombination rate between killer and antidote loci in the two-locus system and (ii) by increasing the proportion of SD alleles in individual flowers, rather than in the general gene-pool. We also show that in rice (Oryza sativa L.), a typical hermaphroditic plant, all molecularly characterized SDs associated with pollen killing were involved in population hybridization and have introgressed across different species. Paradoxically, these loci, which are associated with hybrid incompatibility and can be thought of as Bateson-Dobzhansky-Muller incompatibility loci are expected to reduce gene-flow between species, in fact cross species boundaries more frequently than random loci, and may act as important drivers of introgression.

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21. These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield.

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.

Switchgrass (Panicum virgatum) is a bioenergy model crop valued for its energy efficiency and drought tolerance. The related monocot species rice (Oryza sativa) and maize (Zea mays) deploy species-specific, specialized metabolites as core stress defenses. By contrast, specialized chemical defenses in switchgrass are largely unknown. To investigate specialized metabolic drought responses in switchgrass, we integrated tissue-specific transcriptome and metabolite analyses of the genotypes Alamo and Cave-in-Rock that feature different drought tolerance. The more drought-susceptible Cave-in-Rock featured an earlier onset of transcriptomic changes and significantly more differentially expressed genes in response to drought compared to Alamo. Specialized pathways showed moderate differential expression compared to pronounced transcriptomic alterations in carbohydrate and amino acid metabolism. However, diterpenoid-biosynthetic genes showed drought-inducible expression in Alamo roots, contrasting largely unaltered triterpenoid and phenylpropanoid pathways. Metabolomic analyses identified common and genotype-specific flavonoids and terpenoids. Consistent with transcriptomic alterations, several root diterpenoids showed significant drought-induced accumulation, whereas triterpenoid abundance remained predominantly unchanged. Structural analysis verified select drought-responsive diterpenoids as oxygenated furanoditerpenoids. Drought-dependent transcriptome and metabolite profiles provide the foundation to understand the molecular mechanisms underlying switchgrass drought responses. Accumulation of specialized root diterpenoids and corresponding pathway transcripts supports a role in drought stress tolerance.

The recent advancement in image-based phenotyping platforms enables the acquisition of large-scale nondestructive crop phenotypes measured at frequent intervals. To further understand the underlying genetic basis over a physiological process and improve plant breeding programs, the question of how to efficiently utilize these time-series measurements in genome-enabled analysis including genomic prediction and genome-wide association studies (GWASs) should be considered. In this paper, a Bayesian random regression model with mixture priors is developed to introduce more meaningful biological assumptions to the analysis of longitudinal traits. The mixture prior for marker effects in Bayes Cπ is implemented in our developed model (RR-BayesC) for demonstration purpose. The estimation of single-nucleotide polymorphism-specific effects that are related to the dynamic performance of crops and the accuracy of genomic prediction by RR-BayesC were studied through both simulated and real rice (Oryza sativa L.) data. For genomic prediction, three predictive scenarios were studied. In the simulated study, RR-BayesC showed a significantly higher prediction accuracy than that obtained by single-trait analysis, especially for days when heritability were low. In real data analysis, RR-BayesC showed relatively high prediction accuracy when forecast is required for phenotypes at later period (e.g., from 0.94 to 0.98 for lines with observations at an earlier period and from 0.65 to 0.67 for lines without any observations). For GWASs, inference of single markers and inference of genomic windows were conducted. In the simulated study, RR-BayesC showed its promising ability to distinguish quantitative trait loci (QTL) that are invariant to temporal covariates and QTL that interact with time. An association study of real data was also presented to demonstrate the application of RR-BayesC in real data analysis. In this paper, we develop a Bayesian random regression model that is able to incorporate mixture priors to marker effects and show improved performance of genomic prediction and GWASs for longitudinal data analysis based on both simulated and real data. The software tool JWAS offers routines to perform our proposed random regression analysis.

Rice (Oryza sativa) was domesticated around 10,000 years ago and has developed into a staple for half of humanity. The crop evolved and is currently grown in stably wet and intermittently dry agro-ecosystems, but patterns of adaptation to differences in water availability remain poorly understood. While previous field studies have evaluated plant developmental adaptations to water deficit, adaptive variation in functional and hydraulic components, particularly in relation to gene expression, has received less attention. Here, we take an evolutionary systems biology approach to characterize adaptive drought resistance traits across roots and shoots. We find that rice harbors heritable variation in molecular, physiological, and morphological traits that is linked to higher fitness under drought. We identify modules of co-expressed genes that are associated with adaptive drought avoidance and tolerance mechanisms. These expression modules showed evidence of polygenic adaptation in rice subgroups harboring accessions that evolved in drought-prone agro-ecosystems. Fitness-linked expression patterns allowed us to identify the drought-adaptive nature of optimizing photosynthesis and interactions with arbuscular mycorrhizal fungi. Taken together, our study provides an unprecedented, integrative view of rice adaptation to water-limited field conditions.

The zygote, a totipotent stem cell, is crucial to the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells-the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Owing to technical challenges, the epigenetic changes that accompany the transition from differentiated gametes to totipotent zygote are poorly understood. Because siRNAs serve as both regulators and outputs of the epigenome, we characterized small RNA transcriptomes of zygotes from rice. Zygote small RNAs exhibit extensive maternal carryover and an apparent lack of paternal contribution, indicated by absence of sperm signature siRNAs. Zygote formation is accompanied by widespread redistribution of 24-nt siRNAs relative to gametes, such that ∼70% of the zygote siRNA loci do not overlap any egg cell siRNA loci. Newly detected siRNA loci in zygote are gene-proximal and not associated with centromeric heterochromatin, similar to canonical siRNAs, in sharp contrast to gametic siRNA loci that are gene-distal and heterochromatic. In addition, zygote but not egg siRNA loci are associated with high DNA methylation in the mature embryo. Thus, the zygote begins transitioning before the first embryonic division to an siRNA profile that is associated with future RdDM in embryogenesis. These findings indicate that, in addition to changes in gene expression, the transition to totipotency in the plant zygote is accompanied by resetting of the epigenetic reprogramming that occurred during gamete formation.

Plant TRANS-ACTING SIRNA3 (TAS3)-derived short interfering RNAs (siRNAs) include tasiR-AUXIN RESPONSE FACTORs (ARFs), which are functionally conserved in targeting ARF genes, and a set of non-tasiR-ARF siRNAs, which have rarely been studied. In this study, TAS3 siRNAs were systematically characterized in rice (Oryza sativa). Small RNA sequencing results showed that an overwhelming majority of TAS3 siRNAs belong to the non-tasiR-ARF group, while tasiR-ARFs occupy a diminutive fraction. Phylogenetic analysis of TAS3 genes across dicot and monocot plants revealed that the siRNA-generating regions were highly conserved in grass species, especially in the Oryzoideae. Target genes were identified for not only tasiR-ARFs but also non-tasiR-ARF siRNAs by analyzing rice Parallel Analysis of RNA Ends datasets, and some of these siRNA-target interactions were experimentally confirmed using tas3 mutants generated by genome editing. Consistent with the de-repression of target genes, phenotypic alterations were observed for mutants in three TAS3 loci in comparison to wild-type rice. The regulatory role of ribosomes in the TAS3 siRNA-target interactions was further revealed by the fact that TAS3 siRNA-mediated target cleavage, in particular tasiR-ARFs targeting ARF2/3/14/15, occurred extensively in rice polysome samples. Altogether, our study sheds light into TAS3 genes in plants and expands our knowledge about rice TAS3 siRNA-target interactions.

BackgroundOne of the ways genomes respond to stress is by producing extrachromosomal circular DNAs (eccDNAs). EccDNAs can contain genes and dramatically increase their copy number. They can also reinsert into the genome, generating structural variation. They have been shown to provide a source of phenotypic and genotypic plasticity in several species. However, whole circularome studies have so far been limited to a few model organisms. Fungal plant pathogens are a serious threat to global food security in part because of their rapid adaptation to disease prevention strategies. Understanding the mechanisms fungal pathogens use to escape disease control is paramount to curbing their threat.ResultsWe present a whole circularome sequencing study of the rice blast pathogen, Magnaporthe oryzae. We find that M. oryzae has a highly diverse circularome that contains many genes and shows evidence of large LTR retrotransposon activity. We find that genes enriched on eccDNAs in M. oryzae occur in genomic regions prone to presence-absence variation and that disease-associated genes are frequently on eccDNAs. Finally, we find that a subset of genes is never present on eccDNAs in our data, which indicates that the presence of these genes on eccDNAs is selected against.ConclusionsOur study paves the way to understanding how eccDNAs contribute to adaptation in M. oryzae. Our analysis also reveals how M. oryzae eccDNAs differ from those of other species and highlights the need for further comparative characterization of eccDNAs across species to gain a better understanding of these molecules.

BackgroundSmall 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.ResultsWe 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. Reproductive 21PHAS (21-nt phasiRNA-generating) and 24PHAS (24-nt phasiRNA-generating) precursors, which were commonly considered as noncoding RNAs, are bound by polysomes, and high-frequency cleavage of 21PHAS precursors by miR2118 and 24PHAS precursors 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.ConclusionsMBP 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.

Structural biology has the potential to illuminate the evolution of pathogen effectors and their commonalities that cannot be readily detected at the primary sequence level. Recent breakthroughs in protein structure modeling have demonstrated the feasibility to predict the protein folds without depending on homologous templates. These advances enabled a genome-wide computational structural biology approach to help understand proteins based on their predicted folds. In this study, we employed structure prediction methods on the secretome of the destructive fungal pathogen Magnaporthe oryzae. Out of 1,854 secreted proteins, we predicted the folds of 1,295 proteins (70%). We showed that template-free modeling by TrRosetta captured 514 folds missed by homology modeling, including many known effectors and virulence factors, and that TrRosetta generally produced higher quality models for secreted proteins. Along with sensitive homology search, we employed structure-based clustering, defining not only homologous groups with divergent members but also sequence-unrelated structurally analogous groups. We demonstrate that this approach can reveal new putative members of structurally similar MAX effectors and novel analogous effector families present in M. oryzae and possibly in other phytopathogens. We also investigated the evolution of expanded putative ADP-ribose transferases with predicted structures. We suggest that the loss of catalytic activities of the enzymes might have led them to new evolutionary trajectories to be specialized as protein binders. Collectively, we propose that computational structural genomics approaches can be an integral part of studying effector biology and provide valuable resources that were inaccessible before the advent of machine learning-based structure prediction.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The purpose of the current study was to assess the effects of substituting corn with ground brown rice on growth performance, immune status, and gut microbiota in weanling pigs. Seventy-two weanling pigs (28 d old with 6.78 ± 0.94 kg body weight [BW]) were randomly allotted to two dietary treatments with six pens and six pigs (three barrows and gilts) per pen within a randomized complete block design. The control pigs were fed a typical diet for weanling pigs based on corn and soybean meal diet (control diet: CON), and the other pigs were fed a formulated diet with 100% replacement of corn with ground brown rice for 35d (treatment diet: GBR). Growth performance, immune status, and gut microbiota of weanling pigs were measured. The substitution of corn with GBR did not affect growth performance or diarrhea frequency. Additionally, there were no differences in white blood cell number, hematocrit, cortisol, C-reactive protein, and serum tumor necrosis factor-alpha levels between pigs fed CON or GBR for the first 2 wk after weaning. However, weanling pigs fed GBR had lower (P < 0.05) serum transforming growth factor-beta 1 level than those fed CON. Furthermore, weanling pigs fed GBR had increased (P < 0.05) relative abundance of phylum Firmicutes and genus Lactobacillus and Streptococcus and decreased (P < 0.05) relative abundance of phylum Bacteroidetes and genus Clostridium and Prevotella in the gut microbiota compared with those fed CON. In conclusion, there was no significant difference in growth performance when corn was replaced with ground brown rice in diets for weanling pigs. Furthermore, the substitution of corn with ground brown rice in weaning diet modulated immune status and gut microbiota of pigs by increasing beneficial microbial communities and reducing harmful microbial communities. Overall, ground brown rice-based diet is a potential alternative to corn-based diet without negative effects on growth performance, immune status, and gut microbiota changes of weanling pigs.

Parent-of-origin-dependent gene expression in mammals and flowering plants results from differing chromatin imprints (genomic imprinting) between maternally and paternally inherited alleles. Imprinted gene expression in the endosperm of seeds is associated with localized hypomethylation of maternally but not paternally inherited DNA, with certain small RNAs also displaying parent-of-origin-specific expression. To understand the evolution of imprinting mechanisms in Oryza sativa (rice), we analyzed imprinting divergence among four cultivars that span both japonica and indica subspecies: Nipponbare, Kitaake, 93-11, and IR64. Most imprinted genes are imprinted across cultivars and enriched for functions in chromatin and transcriptional regulation, development, and signaling. However, 4 to 11% of imprinted genes display divergent imprinting. Analyses of DNA methylation and small RNAs revealed that endosperm-specific 24-nt small RNA-producing loci show weak RNA-directed DNA methylation, frequently overlap genes, and are imprinted four times more often than genes. However, imprinting divergence most often correlated with local DNA methylation epimutations (9 of 17 assessable loci), which were largely stable within subspecies. Small insertion/deletion events and transposable element insertions accompanied 4 of the 9 locally epimutated loci and associated with imprinting divergence at another 4 of the remaining 8 loci. Correlating epigenetic and genetic variation occurred at key regulatory regions-the promoter and transcription start site of maternally biased genes, and the promoter and gene body of paternally biased genes. Our results reinforce models for the role of maternal-specific DNA hypomethylation in imprinting of both maternally and paternally biased genes, and highlight the role of transposition and epimutation in rice imprinting evolution.

XA21 encodes a rice immune receptor that confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo). XA21-mediated immunity is triggered by recognition of a small protein called RaxX-sY (required for activation of XA21-mediated immunity X, tyrosine-sulfated) secreted by Xoo. To identify components regulating XA21-mediated immunity, we generated and screened a mutant population of fast-neutron-mutagenized rice expressing Ubi:Myc-XA21 for those susceptible to Xoo. Here, we report the characterization of one of these rice mutants, named sxi2 (suppressor of XA21-mediated immunity-2). Whole-genome sequencing revealed that sxi2 carries a deletion of the PALADIN (PALD) gene encoding a protein with three putative protein tyrosine phosphatase-like domains (PTP-A, -B, and -C). Expression of PALD in the sxi2 genetic background was sufficient to complement the susceptible phenotype, which requires the catalytic cysteine of the PTP-A active site to restore resistance. PALD co-immunoprecipitated with the full-length XA21 protein, whose levels are positively regulated by the presence of the PALD transgene. Furthermore, we foundd that sxi2 retains many hallmarks of XA21-mediated immunity, similar to the wild type. These results reveal that PALD, a previously uncharacterized class of phosphatase, functions in rice innate immunity, and suggest that the conserved cysteine in the PTP-A domain of PALD is required for its immune function.

In angiosperms, endosperm development comprises a series of developmental transitions controlled by genetic and epigenetic mechanisms that are initiated after double fertilization. Polycomb repressive complex 2 (PRC2) is a key component of these mechanisms that mediate histone H3 lysine 27 trimethylation (H3K27me3); the action of PRC2 is well described in Arabidopsis thaliana but remains uncertain in cereals. In this study, we demonstrate that mutation of the rice (Oryza sativa) gene EMBRYONIC FLOWER2a (OsEMF2a), encoding a zinc-finger containing component of PRC2, causes an autonomous endosperm phenotype involving proliferation of the central cell nuclei with separate cytoplasmic domains, even in the absence of fertilization. Detailed cytological and transcriptomic analyses revealed that the autonomous endosperm can produce storage compounds, starch granules, and protein bodies specific to the endosperm. These events have not been reported in Arabidopsis. After fertilization, we observed an abnormally delayed developmental transition in the endosperm. Transcriptome and H3K27me3 ChIP-seq analyses using endosperm from the emf2a mutant identified downstream targets of PRC2. These included >100 transcription factor genes such as type-I MADS-box genes, which are likely required for endosperm development. Our results demonstrate that OsEMF2a-containing PRC2 controls endosperm developmental programs before and after fertilization.

Structural variants (SVs) are a largely unstudied feature of plant genome evolution, despite the fact that SVs contribute substantially to phenotypes. In this study, we discovered SVs across a population sample of 347 high-coverage, resequenced genomes of Asian rice (Oryza sativa) and its wild ancestor (O. rufipogon). In addition to this short-read data set, we also inferred SVs from whole-genome assemblies and long-read data. Comparisons among data sets revealed different features of genome variability. For example, genome alignment identified a large (∼4.3 Mb) inversion in indica rice varieties relative to japonica varieties, and long-read analyses suggest that ∼9% of genes from the outgroup (O. longistaminata) are hemizygous. We focused, however, on the resequencing sample to investigate the population genomics of SVs. Clustering analyses with SVs recapitulated the rice cultivar groups that were also inferred from SNPs. However, the site-frequency spectrum of each SV type-which included inversions, duplications, deletions, translocations, and mobile element insertions-was skewed toward lower frequency variants than synonymous SNPs, suggesting that SVs may be predominantly deleterious. Among transposable elements, SINE and mariner insertions were found at especially low frequency. We also used SVs to study domestication by contrasting between rice and O. rufipogon. Cultivated genomes contained ∼25% more derived SVs and mobile element insertions than O. rufipogon, indicating that SVs contribute to the cost of domestication in rice. Peaks of SV divergence were enriched for known domestication genes, but we also detected hundreds of genes gained and lost during domestication, some of which were enriched for traits of agronomic interest.

BackgroundThe circum-basmati group of cultivated Asian rice (Oryza sativa) contains many iconic varieties and is widespread in the Indian subcontinent. Despite its economic and cultural importance, a high-quality reference genome is currently lacking, and the group's evolutionary history is not fully resolved. To address these gaps, we use long-read nanopore sequencing and assemble the genomes of two circum-basmati rice varieties.ResultsWe generate two high-quality, chromosome-level reference genomes that represent the 12 chromosomes of Oryza. The assemblies show a contig N50 of 6.32 Mb and 10.53 Mb for Basmati 334 and Dom Sufid, respectively. Using our highly contiguous assemblies, we characterize structural variations segregating across circum-basmati genomes. We discover repeat expansions not observed in japonica-the rice group most closely related to circum-basmati-as well as the presence and absence variants of over 20 Mb, one of which is a circum-basmati-specific deletion of a gene regulating awn length. We further detect strong evidence of admixture between the circum-basmati and circum-aus groups. This gene flow has its greatest effect on chromosome 10, causing both structural variation and single-nucleotide polymorphism to deviate from genome-wide history. Lastly, population genomic analysis of 78 circum-basmati varieties shows three major geographically structured genetic groups: Bhutan/Nepal, India/Bangladesh/Myanmar, and Iran/Pakistan.ConclusionThe availability of high-quality reference genomes allows functional and evolutionary genomic analyses providing genome-wide evidence for gene flow between circum-aus and circum-basmati, describes the nature of circum-basmati structural variation, and reveals the presence/absence variation in this important and iconic rice variety group.

BACKGROUND:Whiteflies are a threat to cassava (Manihot esculenta), an important staple food in many tropical/subtropical regions. Understanding the molecular mechanisms regulating cassava's responses against this pest is crucial for developing control strategies. Pathogenesis-related (PR) protein families are an integral part of plant immunity. With the availability of whole genome sequences, the annotation and expression programs of the full complement of PR genes in an organism can now be achieved. An understanding of the responses of the entire complement of PR genes during biotic stress and to the defense hormones, salicylic acid (SA) and jasmonic acid (JA), is lacking. Here, we analyze the responses of cassava PR genes to whiteflies, SA, JA, and other biotic aggressors. RESULTS:The cassava genome possesses 14 of the 17 plant PR families, with a total of 447 PR genes. A cassava PR gene nomenclature is proposed. Phylogenetic relatedness of cassava PR proteins to each other and to homologs in poplar, rice and Arabidopsis identified cassava-specific PR gene family expansions. The temporal programs of PR gene expression in response to the whitefly (Aleurotrachelus socialis) in four whitefly-susceptible cassava genotypes showed that 167 of the 447 PR genes were regulated after whitefly infestation. While the timing of PR gene expression varied, over 37% of whitefly-regulated PR genes were downregulated in all four genotypes. Notably, whitefly-responsive PR genes were largely coordinately regulated by SA and JA. The analysis of cassava PR gene expression in response to five other biotic stresses revealed a strong positive correlation between whitefly and Xanthomonas axonopodis and Cassava Brown Streak Virus responses and negative correlations between whitefly and Cassava Mosaic Virus responses. Finally, certain associations between PR genes in cassava expansions and response to biotic stresses were observed among PR families. CONCLUSIONS:This study represents the first genome-wide characterization of PR genes in cassava. PR gene responses to six biotic stresses and to SA and JA are demonstrably different to other angiosperms. We propose that our approach could be applied in other species to fully understand PR gene regulation by pathogens, pests and the canonical defense hormones SA and JA.

Genomes of all characterized higher eukaryotes harbor examples of transposable element (TE) bursts-the rapid amplification of TE copies throughout a genome. Despite their prevalence, understanding how bursts diversify genomes requires the characterization of actively transposing TEs before insertion sites and structural rearrangements have been obscured by selection acting over evolutionary time. In this study, rice recombinant inbred lines (RILs), generated by crossing a bursting accession and the reference Nipponbare accession, were exploited to characterize the spread of the very active Ping/mPing family through a small population and the resulting impact on genome diversity. Comparative sequence analysis of 272 individuals led to the identification of over 14,000 new insertions of the mPing miniature inverted-repeat transposable element (MITE), with no evidence for silencing of the transposase-encoding Ping element. In addition to new insertions, Ping-encoded transposase was found to preferentially catalyze the excision of mPing loci tightly linked to a second mPing insertion. Similarly, structural variations, including deletion of rice exons or regulatory regions, were enriched for those with break points at one or both ends of linked mPing elements. Taken together, these results indicate that structural variations are generated during a TE burst as transposase catalyzes both the high copy numbers needed to distribute linked elements throughout the genome and the DNA cuts at the TE ends known to dramatically increase the frequency of recombination.

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

Little is known about the mechanism that regulates the core steps of antiviral RNA interference (RNAi) pathway in plants and animals. In this issue of Cell Host & Microbe, Yang et al. (2020) provide compelling evidence for the regulation of antiviral RNAi by the jasmonate hormone signaling in plants.

Systematic mappings of protein interactome networks have provided invaluable functional information for numerous model organisms. Here we develop PCR-mediated Linkage of barcoded Adapters To nucleic acid Elements for sequencing (PLATE-seq) that serves as a general tool to rapidly sequence thousands of DNA elements. We validate its utility by generating the ORFeome for Oryza sativa covering 2,300 genes and constructing a high-quality protein-protein interactome map consisting of 322 interactions between 289 proteins, expanding the known interactions in rice by roughly 50%. Our work paves the way for high-throughput profiling of protein-protein interactions in a wide range of organisms.

Rice (Oryza sativa) is one of the world's most important food crops, and is comprised largely of japonica and indica subspecies. Here, we reconstruct the history of rice dispersal in Asia using whole-genome sequences of more than 1,400 landraces, coupled with geographic, environmental, archaeobotanical and paleoclimate data. Originating around 9,000 yr ago in the Yangtze Valley, rice diversified into temperate and tropical japonica rice during a global cooling event about 4,200 yr ago. Soon after, tropical japonica rice reached Southeast Asia, where it rapidly diversified, starting about 2,500 yr BP. The history of indica rice dispersal appears more complicated, moving into China around 2,000 yr BP. We also identify extrinsic factors that influence genome diversity, with temperature being a leading abiotic factor. Reconstructing the dispersal history of rice and its climatic correlates may help identify genetic adaptations associated with the spread of a key domesticated species.

Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (

Knowledge of the genetic architecture of importantly agronomical traits can speed up genetic improvement in cultivated rice (Oryza sativa L.). Many recent investigations have leveraged genome-wide association studies (GWAS) to identify single nucleotide polymorphisms (SNPs), associated with agronomic traits in various rice populations. The reported trait-relevant SNPs appear to be arbitrarily distributed along the genome, including genic and nongenic regions. Whether the SNPs in different genomic regions play different roles in trait heritability and which region is more responsible for phenotypic variation remains opaque. We analyzed a natural rice population of 524 accessions with 3,616,597 SNPs to compare the genetic contributions of functionally distinct genomic regions for five agronomic traits, i.e., yield, heading date, plant height, grain length, and grain width. An analysis of heritability in the functionally partitioned rice genome showed that regulatory or intergenic regions account for the most trait heritability. A close look at the trait-associated SNPs (TASs) indicated that the majority of the TASs are located in nongenic regions, and the genetic effects of the TASs in nongenic regions are generally greater than those in genic regions. We further compared the predictabilities using the genetic variants from genic regions with those using nongenic regions. The results revealed that nongenic regions play a more important role than genic regions in trait heritability in rice, which is consistent with findings in humans and maize. This conclusion not only offers clues for basic research to disclose genetics behind these agronomic traits, but also provides a new perspective to facilitate genomic selection in rice.

Levels of gene expression underpin organismal phenotypes1,2, but the nature of selection that acts on gene expression and its role in adaptive evolution remain unknown1,2. Here we assayed gene expression in rice (Oryza sativa)3, and used phenotypic selection analysis to estimate the type and strength of selection on the levels of more than 15,000 transcripts4,5. Variation in most transcripts appears (nearly) neutral or under very weak stabilizing selection in wet paddy conditions (with median standardized selection differentials near zero), but selection is stronger under drought conditions. Overall, more transcripts are conditionally neutral (2.83%) than are antagonistically pleiotropic6 (0.04%), and transcripts that display lower levels of expression and stochastic noise7-9 and higher levels of plasticity9 are under stronger selection. Selection strength was further weakly negatively associated with levels of cis-regulation and network connectivity9. Our multivariate analysis suggests that selection acts on the expression of photosynthesis genes4,5, but that the efficacy of selection is genetically constrained under drought conditions10. Drought selected for earlier flowering11,12 and a higher expression of OsMADS18 (Os07g0605200), which encodes a MADS-box transcription factor and is a known regulator of early flowering13-marking this gene as a drought-escape gene11,12. The ability to estimate selection strengths provides insights into how selection can shape molecular traits at the core of gene action.

Gametes constitute a critical stage of the plant life cycle during which the genome undergoes reprogramming in preparation for embryogenesis. Here, we examined genome-wide distributions of small RNAs in the sperm and egg cells of rice. We found that 24-nt siRNAs, which are a hallmark of RNA-directed DNA methylation (RdDM) in plants, were depleted from heterochromatin boundaries in both gametes relative to vegetative tissues, reminiscent of siRNA patterns in DDM1-type nucleosome remodeler mutants. In sperm cells, 24-nt siRNAs were spread across heterochromatic regions, while in egg cells, 24-nt siRNAs were concentrated at a smaller number of heterochromatic loci throughout the genome, especially at loci which also produced siRNAs in other tissues. In both gametes, patterns of CHH methylation, typically a strong indicator of RdDM, were similar to vegetative tissues, although lower in magnitude. These findings indicate that the small RNA transcriptome undergoes large-scale redistribution in both male and female gametes, which is not correlated with recruitment of DNA methyltransferases in gametes and suggestive of unexplored regulatory activities of gamete small RNAs.

The extent to which sequence variation impacts plant fitness is poorly understood. High-resolution maps detailing the constraint acting on the genome, especially in regulatory sites, would be beneficial as functional annotation of noncoding sequences remains sparse. Here, we present a fitness consequence (fitCons) map for rice (Oryza sativa). We inferred fitCons scores (ρ) for 246 inferred genome classes derived from nine functional genomic and epigenomic datasets, including chromatin accessibility, messenger RNA/small RNA transcription, DNA methylation, histone modifications and engaged RNA polymerase activity. These were integrated with genome-wide polymorphism and divergence data from 1,477 rice accessions and 11 reference genome sequences in the Oryzeae. We found ρ to be multimodal, with ~9% of the rice genome falling into classes where more than half of the bases would probably have a fitness consequence if mutated. Around 2% of the rice genome showed evidence of weak negative selection, frequently at candidate regulatory sites, including a novel set of 1,000 potentially active enhancer elements. This fitCons map provides perspective on the evolutionary forces associated with genome diversity, aids in genome annotation and can guide crop breeding programs.

Rice (Oryza sativa L.) is more sensitive to drought stress than other cereals. To dissect molecular mechanisms underlying drought-tolerant yield in rice, we applied differential expression and co-expression network approaches to transcriptomes from flag-leaf and emerging panicle tissues of a drought-tolerant yield introgression line, DTY-IL, and the recurrent parent Swarna, under moderate reproductive-stage drought stress. Protein turnover and efficient reactive oxygen species scavenging were found to be the driving factors in both tissues. In the flag-leaf, the responses further included maintenance of photosynthesis and cell wall reorganization, while in the panicle biosynthesis of secondary metabolites was found to play additional roles. Hub genes of importance in differential drought responses included an expansin in the flag-leaf and two peroxidases in the panicle. Overlaying differential expression data with allelic variation in DTY-IL quantitative trait loci allowed for the prioritization of candidate genes. They included a differentially regulated auxin-responsive protein, with DTY-IL-specific amino acid changes in conserved domains, as well as a protein kinase with a DTY-IL-specific frameshift in the C-terminal region. The approach highlights how the integration of differential expression and allelic variation can aid in the discovery of mechanism and putative causal contribution underlying quantitative trait loci for drought-tolerant yield.

The production and N-glycosylation of recombinant human butyrylcholinesterase (BChE), a model highly glycosylated therapeutic protein, in a transgenic rice cell suspension culture treated with kifunensine, a strong α-mannosidase I inhibitor, was studied in a 5 L bioreactor. A media exchange was performed at day 7 of cultivation by removing spent sugar-rich medium (NB+S) and adding fresh sugar-free (NB-S) medium to induce the rice α-amylase 3D (RAmy3D) promoter to produce rice recombinant human BChE (rrBChE). Using a 1.25X-concentrated sugar-free medium together with an 80% reduced working volume during the media exchange led to a total active rrBChE production level of 79 ± 2 µg (g FW)-1 or 7.5 ± 0.4 mg L-1 in the presence of kifunensine, which was 1.5-times higher than our previous bioreactor runs using normal sugar-free (NB-S) media with no kifunensine treatment. Importantly, the amount of secreted active rrBChE in culture medium was enhanced in the presence of kifunensine, comprising 44% of the total active rrBChE at day 5 following induction. Coomassie-stained SDS-PAGE gel and Western blot analyses revealed different electrophoretic migration of purified rrBChE bands with and without kifunensine treatment, which was attributed to different N-glycoforms. N-Glycosylation analysis showed substantially increased oligomannose glycans (Man5/6/7/8) in rrBChE treated with kifunensine compared to controls. However, the mass-transfer limitation of kifunensine was likely the major reason for incomplete inhibition of α-mannosidase I in this bioreactor study.

BackgroundFlowering time is one of the most important agronomic characteristics that ultimately determine yield potential and eco-geographical adaptation in crops. Ghd8 and Ghd7, two major flowering genes, have similar functions and large pleiotropic effects in controlling the heading date, plant height and grain yield of rice. However, these gene interactions at the genetic and molecular levels have not been determined to date.ResultsIn this study, we investigated the genetic interaction between Ghd8 and Ghd7 by using a set of near-isogenic lines and a panel of natural germplasm accessions in rice. We found that Ghd8 affected multiple agronomic traits in a functional Ghd7-dependent manner. Both functional Ghd8 and Ghd7 are pivotal for rice photoperiod sensitivity controlled by Hd1 and Hd3a. GHD8 could form a heterotrimeric complex with HD1 and OsHAP5b to activate the transcription of Ghd7 by binding directly to the promoter region of Ghd7, which contains the CCAAT-box motif.ConclusionsThe results of this study help to elucidate the genetic and molecular bases of Ghd8 and Ghd7 interactions, indicating that Ghd8 acts upstream of Ghd7 to activate its transcription, which inhibits Hd3a expression and thus affects flowering time and rice adaptation.

BackgroundSalt stress, a major plant environmental stress, is a critical constraint for rice productivity. Dissecting the genetic loci controlling salt tolerance in rice for improving productivity, especially at the flowering stage, remains challenging. Here, we conducted a genome-wide association study (GWAS) of salt tolerance based on exome sequencing of the Thai rice accessions.ResultsPhotosynthetic parameters and cell membrane stability under salt stress at the flowering stage; and yield-related traits of 104 Thai rice (Oryza sativa L.) accessions belonging to the indica subspecies were evaluated. The rice accessions were subjected to exome sequencing, resulting in 112,565 single nucleotide polymorphisms (SNPs) called with a minor allele frequency of at least 5%. LD decay analysis of the panel indicates that the average LD for SNPs at 20 kb distance from each other was 0.34 (r2), which decayed to its half value (~ 0.17) at around 80 kb. By GWAS performed using mixed linear model, two hundred loci containing 448 SNPs on exons were identified based on the salt susceptibility index of the net photosynthetic rate at day 6 after salt stress; and the number of panicles, filled grains and unfilled grains per plant. One hundred and forty six genes, which accounted for 73% of the identified loci, co-localized with the previously reported salt quantitative trait loci (QTLs). The top four regions that contained a high number of significant SNPs were found on chromosome 8, 12, 1 and 2. While many are novel, their annotation is consistent with potential involvement in plant salt tolerance and in related agronomic traits. These significant SNPs greatly help narrow down the region within these QTLs where the likely underlying candidate genes can be identified.ConclusionsInsight into the contribution of potential genes controlling salt tolerance from this GWAS provides further understanding of salt tolerance mechanisms of rice at the flowering stage, which can help improve yield productivity under salinity via gene cloning and genomic selection.

BackgroundThe availability of thousands of complete rice genome sequences from diverse varieties and accessions has laid the foundation for in-depth exploration of the rice genome. One drawback to these collections is that most of these rice varieties have long life cycles, and/or low transformation efficiencies, which limits their usefulness as model organisms for functional genomics studies. In contrast, the rice variety Kitaake has a rapid life cycle (9 weeks seed to seed) and is easy to transform and propagate. For these reasons, Kitaake has emerged as a model for studies of diverse monocotyledonous species.ResultsHere, we report the de novo genome sequencing and analysis of Oryza sativa ssp. japonica variety KitaakeX, a Kitaake plant carrying the rice XA21 immune receptor. Our KitaakeX sequence assembly contains 377.6 Mb, consisting of 33 scaffolds (476 contigs) with a contig N50 of 1.4 Mb. Complementing the assembly are detailed gene annotations of 35,594 protein coding genes. We identified 331,335 genomic variations between KitaakeX and Nipponbare (ssp. japonica), and 2,785,991 variations between KitaakeX and Zhenshan97 (ssp. indica). We also compared Kitaake resequencing reads to the KitaakeX assembly and identified 219 small variations. The high-quality genome of the model rice plant KitaakeX will accelerate rice functional genomics.ConclusionsThe high quality, de novo assembly of the KitaakeX genome will serve as a useful reference genome for rice and will accelerate functional genomics studies of rice and other species.

By the year 2100, the number of people on Earth is expected to increase by ∼50%, placing increasing demands on food production in a time when a changing climate is predicted to compromise crop yields. Feeding this future world requires scientifically informed innovations in agriculture. Here, we describe how a rice gene conferring tolerance to prolonged submergence has helped farmers in South and Southeast Asia mitigate rice crop failure during floods. We discuss how planting of this new variety benefited socially disadvantaged groups. This example indicates that investment in agricultural improvement can protect farmers from risks associated with a changing climate.

Flooding due to extreme weather threatens crops and ecosystems. To understand variation in gene regulatory networks activated by submergence, we conducted a high-resolution analysis of chromatin accessibility and gene expression at three scales of transcript control in four angiosperms, ranging from a dryland-adapted wild species to a wetland crop. The data define a cohort of conserved submergence-activated genes with signatures of overlapping cis regulation by four transcription factor families. Syntenic genes are more highly expressed than nonsyntenic genes, yet both can have the cis motifs and chromatin accessibility associated with submergence up-regulation. Whereas the flexible circuitry spans the eudicot-monocot divide, the frequency of specific cis motifs, extent of chromatin accessibility, and degree of submergence activation are more prevalent in the wetland crop and may have adaptive importance.

Power calculation prior to a genetic experiment can help investigators choose the optimal sample size to detect a quantitative trait locus (QTL). Without the guidance of power analysis, an experiment may be underpowered or overpowered. Either way will result in wasted resource. QTL mapping and genome-wide association studies (GWAS) are often conducted using a linear mixed model (LMM) with controls of population structure and polygenic background using markers of the whole genome. Power analysis for such a mixed model is often conducted via Monte Carlo simulations. In this study, we derived a non-centrality parameter for the Wald test statistic for association, which allows analytical power analysis. We show that large samples are not necessary to detect a biologically meaningful QTL, say explaining 5% of the phenotypic variance. Several R functions are provided so that users can perform power analysis to determine the minimum sample size required to detect a given QTL with a certain statistical power or calculate the statistical power with given sample size and known values of other population parameters.

Genomic prediction benefits hybrid rice breeding by increasing selection intensity and accelerating breeding cycles. With the rapid advancement of technology, other omic data, such as metabolomic data and transcriptomic data, are readily available for predicting breeding values for agronomically important traits. In this study, the best prediction strategies were determined for yield, 1000 grain weight, number of grains per panicle, and number of tillers per plant of hybrid rice (derived from recombinant inbred lines) by comprehensively evaluating all possible combinations of omic datasets with different prediction methods. It was demonstrated that, in rice, the predictions using a combination of genomic and metabolomic data generally produce better results than single-omics predictions or predictions based on other combined omic data. Best linear unbiased prediction (BLUP) appears to be the most efficient prediction method compared to the other commonly used approaches, including least absolute shrinkage and selection operator (LASSO), stochastic search variable selection (SSVS), support vector machines with radial basis function and epsilon regression (SVM-R(EPS)), support vector machines with radial basis function and nu regression (SVM-R(NU)), support vector machines with polynomial kernel and epsilon regression (SVM-P(EPS)), support vector machines with polynomial kernel and nu regression (SVM-P(NU)) and partial least squares regression (PLS). This study has provided guidelines for selection of hybrid rice in terms of which types of omic datasets and which method should be used to achieve higher trait predictability. The answer to these questions will benefit academic research and will also greatly reduce the operative cost for the industry which specializes in breeding and selection.

Unfavourable environmental conditions, including soil salinity, lead to decreased rice (Oryza sativa L.) productivity, especially at the reproductive stage. In this study, we examined 30 rice varieties, which revealed significant differences in the photosynthetic performance responses under salt stress conditions during the reproductive stage, which ultimately affected yield components after recovery. In rice with a correlation between net photosynthetic rate (PN) and intercellular CO2 concentration (Ci) under salt stress, PN was found to be negatively correlated with filled grain number after recovery. Applying stringent criteria, we identified 130,317 SNPs and 15,396 InDels between two "high-yield rice" varieties and two "low-yield rice" varieties with contrasting photosynthesis and grain yield characteristics. A total of 2,089 genes containing high- and moderate-impact SNPs or InDels were evaluated by gene ontology (GO) enrichment analysis, resulting in over-represented terms in the apoptotic process and kinase activity. Among these genes, 262 were highly expressed in reproductive tissues, and most were annotated as receptor-like protein kinases. These findings highlight the importance of variations in signaling components in the genome and these loci can serve as potential genes in rice breeding to produce a variety with salt avoidance that leads to increased yield in saline soil.

Epigenetic reprogramming is required for proper regulation of gene expression in eukaryotic organisms. In Arabidopsis, active DNA demethylation is crucial for seed viability, pollen function, and successful reproduction. The DEMETER (DME) DNA glycosylase initiates localized DNA demethylation in vegetative and central cells, so-called companion cells that are adjacent to sperm and egg gametes, respectively. In rice, the central cell genome displays local DNA hypomethylation, suggesting that active DNA demethylation also occurs in rice; however, the enzyme responsible for this process is unknown. One candidate is the rice REPRESSOR OF SILENCING 1a (ROS1a) gene, which is related to DME and is essential for rice seed viability and pollen function. Here, we report genome-wide analyses of DNA methylation in wild-type and ros1a mutant sperm and vegetative cells. We find that the rice vegetative cell genome is locally hypomethylated compared with sperm by a process that requires ROS1a activity. We show that many ROS1a target sequences in the vegetative cell are hypomethylated in the rice central cell, suggesting that ROS1a also demethylates the central cell genome. Similar to Arabidopsis, we show that sperm non-CG methylation is indirectly promoted by DNA demethylation in the vegetative cell. These results reveal that DNA glycosylase-mediated DNA demethylation processes are conserved in Arabidopsis and rice, plant species that diverged 150 million years ago. Finally, although global non-CG methylation levels of sperm and egg differ, the maternal and paternal embryo genomes show similar non-CG methylation levels, suggesting that rice gamete genomes undergo dynamic DNA methylation reprogramming after cell fusion.

The rice XA21-mediated immune response is activated on recognition of the RaxX peptide produced by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The 60-residue RaxX precursor is post-translationally modified to form a sulfated tyrosine peptide that shares sequence and functional similarity with the plant sulfated tyrosine (PSY) peptide hormones. The 5-kb raxX-raxSTAB gene cluster of Xoo encodes RaxX, the RaxST tyrosylprotein sulfotransferase, and the RaxA and RaxB components of a predicted type I secretion system. To assess raxX-raxSTAB gene cluster evolution and to determine its phylogenetic distribution, we first identified rax gene homologues in other genomes. We detected the complete raxX-raxSTAB gene cluster only in Xanthomonas spp., in five distinct lineages in addition to X. oryzae. The phylogenetic distribution of the raxX-raxSTAB gene cluster is consistent with the occurrence of multiple lateral (horizontal) gene transfer events during Xanthomonas speciation. RaxX natural variants contain a restricted set of missense substitutions, as expected if selection acts to maintain peptide hormone-like function. Indeed, eight RaxX variants tested all failed to activate the XA21-mediated immune response, yet retained peptide hormone activity. Together, these observations support the hypothesis that the XA21 receptor evolved specifically to recognize Xoo RaxX.

Transposable elements (TEs) shape genome evolution through periodic bursts of amplification. In this study prior knowledge of the mPing/Ping/Pong TE family is exploited to track their copy numbers and distribution in genome sequences from 3,000 accessions of domesticated Oryza sativa (rice) and the wild progenitor Oryza rufipogon. We find that mPing bursts are restricted to recent domestication and is likely due to the accumulation of two TE components, Ping16A and Ping16A_Stow, that appear to be critical for mPing hyperactivity. Ping16A is a variant of the autonomous element with reduced activity as shown in a yeast transposition assay. Transposition of Ping16A into a Stowaway element generated Ping16A_Stow, the only Ping locus shared by all bursting accessions, and shown here to correlate with high mPing copies. Finally, we show that sustained activity of the mPing/Ping family in domesticated rice produced the components necessary for mPing bursts, not the loss of epigenetic regulation.

Transcription factors (TFs) are an important class of regulatory molecules. Despite their importance, only a small number of genes encoding TFs have been characterized in Oryza sativa (rice), often because gene duplication and functional redundancy complicate their analysis. To address this challenge, we developed a web-based tool called the Rice Transcription Factor Phylogenomics Database (RTFDB) and demonstrate its application for predicting TF function. The RTFDB hosts transcriptome and co-expression analyses. Sources include high-throughput data from oligonucleotide microarray (Affymetrix and Agilent) as well as RNA-Seq-based expression profiles. We used the RTFDB to identify tissue-specific and stress-related gene expression. Subsequently, 273 genes preferentially expressed in specific tissues or organs, 455 genes showing a differential expression pattern in response to 4 abiotic stresses, 179 genes responsive to infection of various pathogens and 512 genes showing differential accumulation in response to various hormone treatments were identified through the meta-expression analysis. Pairwise Pearson correlation coefficient analysis between paralogous genes in a phylogenetic tree was used to assess their expression collinearity and thereby provides a hint on their genetic redundancy. Integrating transcriptome with the gene evolutionary information reveals the possible functional redundancy or dominance played by paralog genes in a highly duplicated genome such as rice. With this method, we estimated a predominant role for 83.3% (65/78) of the TF or transcriptional regulator genes that had been characterized via loss-of-function studies. In this regard, the proposed method is applicable for functional studies of other plant species with annotated genome.

Variations in the gene promoter play critical roles in the evolution of important adaptive traits in crops, but direct links of the regulatory mutation to the adaptive change are not well understood. Here, we examine the nucleotide variations in the promoter region of a transcription factor (Ghd8) that control grain number, plant height and heading date in rice. We find that a dominant promoter type of subspecies japonica displayed a high activity for Ghd8 expression in comparison with the one in indica. Transgenic analyses revealed that higher expression levels of Ghd8 delayed heading date and enhanced cold tolerance in rice. Furthermore, a single-nucleotide polymorphism (T1279G) at the position -1279 bp that locates on the potential GA-responsive motif in the Ghd8 promoter affected the expression of this gene. The 1279 T variant has elevated expression of Ghd8, thus conferring increased cold tolerance of rice seedlings. Nucleotide diversity analysis revealed that the approximately 25-kb genomic region surrounding Ghd8 in the subspecies japonica was under significant selection pressure. Our findings demonstrate that the join effects of the regulatory and coding variants largely contribute to the divergence of japonica and indica and increase the adaptability of japonica to the cold environment.

Abstract Background Events of gene fusion have been reported in several organisms. However, the general role of gene fusion as part of new gene origination remains unknown. Results We conduct genome-wide interrogations of four Oryza genomes by designing and implementing novel pipelines to detect fusion genes. Based on the phylogeny of ten plant species, we detect 310 fusion genes across four Oryza species. The estimated rate of origination of fusion genes in the Oryza genus is as high as 63 fusion genes per species per million years, which is fixed at 16 fusion genes per species per million years and much higher than that in flies. By RNA sequencing analysis, we find more than 44% of the fusion genes are expressed and 90% of gene pairs show strong signals of purifying selection. Further analysis of CRISPR/Cas9 knockout lines indicates that newly formed fusion genes regulate phenotype traits including seed germination, shoot length and root length, suggesting the functional significance of these genes. Conclusions We detect new fusion genes that may drive phenotype evolution in Oryza. This study provides novel insights into the genome evolution of Oryza.

Rice nucleolin (OsNUC1), consisting of two isoforms, OsNUC1-L and OsNUC1-S, is a multifunctional protein involved in salt-stress tolerance. Here, OsNUC1-S's function was investigated using transgenic rice lines overexpressing OsNUC1-S. Under non-stress conditions, the transgenic lines showed a lower yield, but higher net photosynthesis rates, stomatal conductance, and transpiration rates than wild type only in the second leaves, while in the flag leaves, these parameters were similar among the lines. However, under salt-stress conditions at the booting stage, the higher yields in transgenic lines were detected. Moreover, the gas exchange parameters of the transgenic lines were higher in both flag and second leaves, suggesting a role for OsNUC1-S overexpression in photosynthesis adaptation under salt-stress conditions. Moreover, the overexpression lines could maintain light-saturation points under salt-stress conditions, while a decrease in the light-saturation point owing to salt stress was found in wild type. Based on a transcriptome comparison between wild type and a transgenic line, after 3 and 9 days of salt stress, the significantly differentially expressed genes were enriched in the metabolic process of nucleic acid and macromolecule, photosynthesis, water transport, and cellular homeostasis processes, leading to the better performance of photosynthetic processes under salt-stress conditions at the booting stage.

BackgroundCalmodulin (CaM) is an important calcium sensor protein that transduces Ca2+ signals in plant stress signaling pathways. A previous study has revealed that transgenic rice over-expressing the calmodulin gene OsCam1-1 (LOC_Os03g20370) is more tolerant to salt stress than wild type. To elucidate the role of OsCam1-1 in the salt stress response mechanism, downstream components of the OsCam1-1-mediated response were identified and investigated by transcriptome profiling and target identification.ResultsTranscriptome profiling of transgenic 'Khao Dawk Mali 105' rice over-expressing OsCam1-1 and wild type rice showed that overexpression of OsCam1-1 widely affected the expression of genes involved in several cellular processes under salt stress, including signaling, hormone-mediated regulation, transcription, lipid metabolism, carbohydrate metabolism, secondary metabolism, photosynthesis, glycolysis, tricarboxylic acid (TCA) cycle and glyoxylate cycle. Under salt stress, the photosynthesis rate in the transgenic rice was slightly lower than in wild type, while sucrose and starch contents were higher, suggesting that energy and carbon metabolism were affected by OsCam1-1 overexpression. Additionally, four known and six novel CaM-interacting proteins were identified by cDNA expression library screening with the recombinant OsCaM1. GO terms enriched in their associated proteins that matched those of the differentially expressed genes affected by OsCam1-1 overexpression revealed various downstream cellular processes that could potentially be regulated by OsCaM1 through their actions.ConclusionsThe diverse cellular processes affected by OsCam1-1 overexpression and possessed by the identified CaM1-interacting proteins corroborate the notion that CaM signal transduction pathways compose a complex network of downstream components involved in several cellular processes. These findings suggest that under salt stress, CaM activity elevates metabolic enzymes involved in central energy pathways, which promote or at least maintain the production of energy under the limitation of photosynthesis.