OsbHLH067, OsbHLH068, and OsbHLH069 redundantly regulate inflorescence axillary meristem formation in rice.

Rice axillary meristems (AMs) are essential to the formation of tillers and panicle branches in rice, and therefore play a determining role in rice yield. However, the regulation of inflorescence AM development in rice remains elusive. In this study, we identified no spikelet 1-Dominant (nsp1-D), a sparse spikelet mutant, with obvious reduction of panicle branches and spikelets. Inflorescence AM deficiency in nsp1-D could be ascribed to the overexpression of OsbHLH069. OsbHLH069 functions redundantly with OsbHLH067 and OsbHLH068 in panicle AM formation. The Osbhlh067 Osbhlh068 Osbhlh069 triple mutant had smaller panicles and fewer branches and spikelets. OsbHLH067, OsbHLH068, and OsbHLH069 were preferentially expressed in the developing inflorescence AMs and their proteins could physically interact with LAX1. Both nsp1-D and lax1 showed sparse panicles. Transcriptomic data indicated that OsbHLH067/068/069 may be involved in the metabolic pathway during panicle AM formation. Quantitative RT-PCR results demonstrated that the expression of genes involved in meristem development and starch/sucrose metabolism was down-regulated in the triple mutant. Collectively, our study demonstrates that OsbHLH067, OsbHLH068, and OsbHLH069 have redundant functions in regulating the formation of inflorescence AMs during panicle development in rice.

Rice and Arabidopsis homologs of yeast CHROMOSOME TRANSMISSION FIDELITY PROTEIN 4 commonly interact with Polycomb complexes but exert divergent regulatory functions.

Both genetic and epigenetic information must be transferred from mother to daughter cells during cell division. The mechanisms through which information about chromatin states and epigenetic marks like histone 3 lysine 27 trimethylation (H3K27me3) are transferred have been characterized in animals; these processes are less well understood in plants. Here, based on characterization of a dwarf rice (Oryza sativa) mutant (dwarf-related wd40 protein 1, drw1) deficient for yeast CTF4 (CHROMOSOME TRANSMISSION FIDELITY PROTEIN 4), we discovered that CTF4 orthologs in plants use common cellular machinery yet accomplish divergent functional outcomes. Specifically, drw1 exhibited no flowering-related phenotypes (as in the putatively orthologous Arabidopsis thaliana eol1 mutant), but displayed cell cycle arrest and DNA damage responses. Mechanistically, we demonstrate that DRW1 sustains normal cell cycle progression by modulating the expression of cell cycle inhibitors KIP-RELATED PROTEIN 1 (KRP1) and KRP5, and show that these effects are mediated by DRW1 binding their promoters and increasing H3K27me3 levels. Thus, although CTF4 orthologs ENHANCER OF LHP1 1 (EOL1) in Arabidopsis and DRW1 in rice are both expressed uniquely in dividing cells, commonly interact with several Polycomb complex subunits, and promote H3K27me3 deposition, we now know that their regulatory functions diverged substantially during plant evolution. Moreover, our work experimentally illustrates specific targets of CTF4/EOL1/DRW1, their protein-proteininteraction partners, and their chromatin/epigenetic effects in plants.

Structural Insight into DNA Recognition by CCT/NF-YB/YC Complexes in Plant Photoperiodic Flowering.

CONSTANS, CONSTANS-LIKE, and TIMING OF CAB EXPRESSION1 (CCT) domain-containing proteins are a large family unique to plants. They transcriptionally regulate photoperiodic flowering, circadian rhythms, vernalization, and other related processes. Through their CCT domains, CONSTANS and HEADING DATE1 (HD1) coordinate with the NUCLEAR FACTOR Y (NF-Y) B/C dimer to specifically target a conserved 'CCACA' motif within the promoters of their target genes. However, the mechanism underlying DNA recognition by the CCT domain remains unclear. Here we determined the crystal structures of the rice (Oryza sativa) NF-YB/YC dimer and the florigen gene Heading date 3a (Hd3a)-bound HD1CCT/NF-YB/YC trimer with resolutions of 2.0 Å and 2.55 Å, respectively. The CCT domain of HD1 displays an elongated structure containing two α-helices and two loops, tethering Hd3a to the NF-YB/YC dimer. Helix α2 and loop 2 are anchored into the minor groove of the 'CCACA' motif, which determines the specific base recognition. Our structures reveal the interaction mechanism among the CCT domain, NF-YB/YC dimer, and the target DNA. These results not only provide insight into the network between the CCT proteins and NF-Y subunits, but also offer potential approaches for improving productivity and global adaptability of crops by manipulating florigen expression.

Genome-wide binding analysis of transcription factor Rice Indeterminate 1 reveals a complex network controlling rice floral transition.

RICE INDETERMINATE 1 (RID1) plays a critical role in controlling floral transition in rice (Oryza sativa). However, the molecular basis for this effect, particularly the target genes and regulatory specificity, remains largely unclear. Here, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) in young leaves at the pre-floral-transition stage to identify the target genes of RID1, identifying 2,680 genes associated with RID1 binding sites genome-wide. RID1 binding peaks were highly enriched for TTTGTC, the direct binding motif of the INDETERMINATE DOMAIN protein family that includes RID1. Interestingly, CACGTG and GTGGGCCC, two previously uncharacterized indirect binding motifs, were enriched through the interactions of RID1 with the novel flowering-promoting proteins OsPIL12 and OsTCP11, respectively. Moreover, the ChIP-seq data demonstrated that RID1 bound to numerous rice heading-date genes, such as HEADING DATE 1 (HD1) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (OsFKF1). Notably, transcriptome sequencing (RNA-seq) analysis revealed roles of RID1 in diverse developmental pathways. Genetic analysis combined with genome-wide ChIP-seq and RNA-seq results showed that RID1 directly binds to the promoter of OsERF#136 (a repressor of rice flowering) and negatively regulates its expression. Overall, our findings provide new insights into the molecular and genetic mechanisms underlying rice floral transition and characterize OsERF#136 as a previously unrecognized direct target of RID1.

RID1 sets rice heading date by balancing its binding with SLR1 and SDG722.

Rice (Oryza sativa) is a major crop that feeds billions of people, and its yield is strongly influenced by flowering time (heading date). Loss of RICE INDETERMINATE1 (RID1) function causes plants not to flower; thus, RID1 is considered a master switch among flowering-related genes. However, it remains unclear whether other proteins function together with RID1 to regulate rice floral transition. Here, we revealed that the chromatin accessibility and H3K9ac, H3K4me3, and H3K36me3 levels at Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) loci were significantly reduced in rid1 mutants. Notably, RID1 interacted with SET DOMAIN GROUP PROTEIN 722 (SDG722), a methyltransferase. We determined that SDG722 affects the global level of H3K4me2/3 and H3K36me2/3, and promotes flowering primarily through the Early heading date1-Hd3a/RFT1 pathway. We further established that rice DELLA protein SLENDER RICE1 (SLR1) interacted with RID1 to inhibit its transactivation activity, that SLR1 suppresses rice flowering, and that messenger RNA and protein levels of SLR1 gradually decrease with plant growth. Furthermore, SLR1 competed with SDG722 for interaction with RID1. Overall, our results establish that interplay between RID1, SLR1, and SDG722 feeds into rice flowering-time control.

The E3 Ubiquitin Ligase HAF1 Modulates Circadian Accumulation of EARLY FLOWERING3 to Control Heading Date in Rice under Long-Day Conditions.

The ubiquitin 26S proteasome system (UPS) is critical for enabling plants to alter their proteomes to integrate internal and external signals for the photoperiodic induction of flowering. We previously demonstrated that HAF1, a C3HC4 RING domain-containing E3 ubiquitin ligase, is essential to precisely modulate the timing of Heading Date1 accumulation and to ensure appropriate photoperiodic responses under short-day conditions in rice (Oryza sativa). However, how HAF1 mediates flowering under long-day conditions remains unknown. In this study, we show that OsELF3 (EARLY FLOWERING3) is the direct substrate of HAF1 for ubiquitination in vitro and in vivo. HAF1 is required for maintaining the circadian rhythm of OsELF3 accumulation during photoperiodic responses in rice. In addition, the haf1 oself3 double mutant headed as late as oself3 plants under long-day conditions. An amino acid variation (L558S) within the interaction domain of OsELF3 with HAF1 greatly contributes to the variation in heading date among japonica rice accessions. The japonica accessions carrying the OsELF3(L)-type allele are found at higher latitudes, while varieties carrying the OsELF3(S)-type allele are found at lower latitudes. Taken together, our findings suggest that HAF1 precisely modulates the diurnal rhythm of OsELF3 accumulation to ensure the appropriate heading date in rice.

VPB1 Encoding BELL-like Homeodomain Protein Is Involved in Rice Panicle Architecture.

Inflorescence architecture in rice (Oryza sativa) is mainly determined by spikelets and the branch arrangement. Primary branches initiate from inflorescence meristem in a spiral phyllotaxic manner, and further develop into the panicle branches. The branching patterns contribute largely to rice production. In this study, we characterized a rice verticillate primary branch 1(vpb1) mutant, which exhibited a clustered primary branches phenotype. Gene isolation revealed that VPB1 was a allele of RI, that it encoded a BELL-like homeodomain (BLH) protein. VPB1 gene preferentially expressed in the inflorescence and branch meristems. The arrangement of primary branch meristems was disturbed in the vpb1 mutant. Transcriptome analysis further revealed that VPB1 affected the expression of some genes involved in inflorescence meristem identity and hormone signaling pathways. In addition, the differentially expressed gene (DEG) promoter analysis showed that OsBOPs involved in boundary organ initiation were potential target genes of VPB1 protein. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter system further verified that VPB1 protein bound to the promoter of OsBOP1 gene. Overall, our findings demonstrate that VPB1 controls inflorescence architecture by regulating the expression of genes involved in meristem maintenance and hormone pathways and by interacting with OsBOP genes.

OsmiR167a-targeted auxin response factors modulate tiller angle via fine-tuning auxin distribution in rice.

Rice tiller angle determines plant growth density and further contributes grain production. Although a few genes have been characterized to regulate tiller angle in rice, the molecular mechanism underlying the control of tiller angle via microRNA is poorly understood. Here, we report that rice tiller angle is controlled by OsmiR167a-targeted auxin response factors OsARF12, OsARF17 and OsARF25. In the overexpression of OsMIR167a plants, the expression of OsARF12, OsARF17 and OsARF25 was severely repressed and displayed larger tiller angle as well as the osarf12/osarf17 and osarf12/ osarf25 plants. In addition, those plants showed compromised abnormal auxin distribution and less sensitive to gravity. We also demonstrate that OsARF12, OsARF17 and OsARF25 function redundantly and might be involved in HSFA2D and LAZY1-dependent asymmetric auxin distribution pathway to control rice tiller angle. Our results reveal that OsmiR167a represses its targets, OsARF12, OsARF17 and OsARF25, to control rice tiller angle by fine-tuning auxin asymmetric distribution in shoots.

Suppressor of rid1 (SID1) shares common targets with RID1 on florigen genes to initiate floral transition in rice.

The transition from vegetative to reproductive growth is a critical process in the life cycle of higher plants. Previously, we cloned Rice Indeterminate 1 (RID1), which acts as the master switch for the transition from the vegetative to reproductive phase in rice. Although the photoperiod pathway of RID1 inducing expression of the florigen genes Hd3a and RFT1 via Ehd1 has been established, the alternative pathways for the essential flowering transition need to be further examined. Here, we identified a Suppressor of rid1 (SID1), which rescues the never-flowering phenotype of rid1. SID1 encodes an INDETERMINATE DOMAIN (IDD) transcription factor. Mutation in SID1 showed the delayed flowering phenotype. Gain-of-function of SID1, OsIDD1, or OsIDD6 could restore the rid1 to flowering. Further analyses showed SID1 and RID1 directly target the promoter regions of Hd3a and RFT1, two florigen genes in rice. Taken together, our results reveal an autonomous flowering pathway might be mediated by RID1, thereby controlling the phase transition from vegetative to reproductive development in rice.

Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63.

Asian cultivated rice consists of two subspecies: Oryza sativa subsp. indica and O. sativa subsp. japonica Despite the fact that indica rice accounts for over 70% of total rice production worldwide and is genetically much more diverse, a high-quality reference genome for indica rice has yet to be published. We conducted map-based sequencing of two indica rice lines, Zhenshan 97 (ZS97) and Minghui 63 (MH63), which represent the two major varietal groups of the indica subspecies and are the parents of an elite Chinese hybrid. The genome sequences were assembled into 237 (ZS97) and 181 (MH63) contigs, with an accuracy >99.99%, and covered 90.6% and 93.2% of their estimated genome sizes. Comparative analyses of these two indica genomes uncovered surprising structural differences, especially with respect to inversions, translocations, presence/absence variations, and segmental duplications. Approximately 42% of nontransposable element related genes were identical between the two genomes. Transcriptome analysis of three tissues showed that 1,059-2,217 more genes were expressed in the hybrid than in the parents and that the expressed genes in the hybrid were much more diverse due to their divergence between the parental genomes. The public availability of two high-quality reference genomes for the indica subspecies of rice will have large-ranging implications for plant biology and crop genetic improvement.

Panicle Morphology Mutant 1 (PMM1) determines the inflorescence architecture of rice by controlling brassinosteroid biosynthesis.

BACKGROUND: Panicle architecture is one of the main important agronomical traits that determine branch number and grain number in rice. Although a large number of genes involved in panicle development have been identified in recent years, the complex processes of inflorescence patterning need to be further characterized in rice. Brassinosteroids (BRs) are a class of steroid phytohormones. A great understanding of how BRs contribute to plant height and leaf erectness have been reported, however, the molecular and genetic mechanisms of panicle architecture influenced by BRs remain unclear. RESULTS: Here, we identified PMM1, encoding a cytochrome P450 protein involved in BRs biosynthesis, and characterized its role in panicle architecture in rice. Three alleles of pmm1 were identified from our T-DNA insertional mutant library. Map-based cloning revealed that a large fragment deletion from the 2nd to 9th exons of PMM1 was responsible for the clustered primary branch morphology in pmm1-1. PMM1 is a new allele of DWARF11 (D11) PMM1 transcripts are preferentially expressed in young panicles, particularly expressed in the primordia of branches and spikelets during inflorescence development. Furthermore, overexpression of OsDWARF4 (D4), another gene encoding cytochrome P450, completely rescued the abnormal panicle phenotype of pmm1-1. Overall, it can be concluded that PMM1 is an important gene involved in BRs biosynthesis and affecting the differentiation of spikelet primordia and patterns of panicle branches in rice. CONCLUSIONS: PMM1 is a new allele of D11, which encodes a cytochrome P450 protein involved in BRs biosynthesis pathway. Overexpression of D4 could successfully rescue the abnormal panicle architecture of pmm1 plants, indicating that PMM1/D11 and D4 function redundantly in BRs biosynthesis. Thus, our results demonstrated that PMM1 determines the inflorescence architecture by controlling brassinosteroid biosynthesis in rice.

The RING-Finger Ubiquitin Ligase HAF1 Mediates Heading date 1 Degradation during Photoperiodic Flowering in Rice.

The photoperiodic response is one of the most important factors determining heading date in rice (Oryza sativa). Although rhythmic expression patterns of flowering time genes have been reported to fine-tune the photoperiodic response, posttranslational regulation of key flowering regulators has seldom been elucidated in rice. Heading date 1 (Hd1) encodes a zinc finger transcription factor that plays a crucial role in the photoperiodic response, which determines rice regional adaptability. However, little is known about the molecular mechanisms of Hd1 accumulation during the photoperiod response. Here, we identify a C3HC4 RING domain-containing E3 ubiquitin ligase, Heading date Associated Factor 1 (HAF1), which physically interacts with Hd1. HAF1 mediates ubiquitination and targets Hd1 for degradation via the 26S proteasome-dependent pathway. The haf1 mutant exhibits a later flowering heading date under both short-day and long-day conditions. In addition, the haf1 hd1 double mutant headed as late as hd1 plants under short-day conditions but exhibited a heading date similar to haf1 under long-day conditions, thus indicating that HAF1 may determine heading date mainly through Hd1 under short-day conditions. Moreover, high levels of Hd1 accumulate in haf1. Our results suggest that HAF1 is essential to precise modulation of the timing of Hd1 accumulation during the photoperiod response in rice.

Replication protein A2c coupled with replication protein A1c regulates crossover formation during meiosis in rice.

Replication protein A (RPA) is a conserved heterotrimeric protein complex comprising RPA1, RPA2, and RPA3 subunits involved in multiple DNA metabolism pathways attributable to its single-stranded DNA binding property. Unlike other species possessing a single RPA2 gene, rice (Oryza sativa) possesses three RPA2 paralogs, but their functions remain unclear. In this study, we identified RPA2c, a rice gene preferentially expressed during meiosis. A T-DNA insertional mutant (rpa2c) exhibited reduced bivalent formation, leading to chromosome nondisjunction. In rpa2c, chiasma frequency is reduced by ~78% compared with the wild type and is accompanied by loss of the obligate chiasma. The residual ~22% chiasmata fit a Poisson distribution, suggesting loss of crossover control. RPA2c colocalized with the meiotic cohesion subunit REC8 and the axis-associated protein PAIR2. Localization of REC8 was necessary for loading of RPA2c to the chromosomes. In addition, RPA2c partially colocalized with MER3 during late leptotene, thus indicating that RPA2c is required for class I crossover formation at a late stage of homologous recombination. Furthermore, we identified RPA1c, an RPA1 subunit with nearly overlapping distribution to RPA2c, required for ~79% of chiasmata formation. Our results demonstrate that an RPA complex comprising RPA2c and RPA1c is required to promote meiotic crossovers in rice.

High-level hemicellulosic arabinose predominately affects lignocellulose crystallinity for genetically enhancing both plant lodging resistance and biomass enzymatic digestibility in rice mutants.

Rice is a major food crop with enormous biomass residue for biofuels. As plant cell wall recalcitrance basically decides a costly biomass process, genetic modification of plant cell walls has been regarded as a promising solution. However, due to structural complexity and functional diversity of plant cell walls, it becomes essential to identify the key factors of cell wall modifications that could not much alter plant growth, but cause an enhancement in biomass enzymatic digestibility. To address this issue, we performed systems biology analyses of a total of 36 distinct cell wall mutants of rice. As a result, cellulose crystallinity (CrI) was examined to be the key factor that negatively determines either the biomass enzymatic saccharification upon various chemical pretreatments or the plant lodging resistance, an integrated agronomic trait in plant growth and grain production. Notably, hemicellulosic arabinose (Ara) was detected to be the major factor that negatively affects cellulose CrI probably through its interlinking with ß-1,4-glucans. In addition, lignin and G monomer also exhibited the positive impact on biomass digestion and lodging resistance. Further characterization of two elite mutants, Osfc17 and Osfc30, showing normal plant growth and high biomass enzymatic digestion in situ and in vitro, revealed the multiple GH9B candidate genes for reducing cellulose CrI and XAT genes for increasing hemicellulosic Ara level. Hence, the results have suggested the potential cell wall modifications for enhancing both biomass enzymatic digestibility and plant lodging resistance by synchronically overexpressing GH9B and XAT genes in rice.

Rice APOPTOSIS INHIBITOR5 coupled with two DEAD-box adenosine 5'-triphosphate-dependent RNA helicases regulates tapetum degeneration.

Programmed cell death (PCD) during tapetum degeneration in postmeiotic anthers is critical for the proper development of male gametophytes in flowering plants. Although several genes involved in this process have been identified recently, the molecular mechanism is still poorly understood. Here, we show that knockout of rice (Oryza sativa) APOPTOSIS INHIBITOR5 (API5), which encodes a putative homolog of antiapoptosis protein Api5 in animals, results in delayed degeneration of the tapetum due to inhibition of the tapetal PCD process leading to defects in formation of male gametophytes. Os API5 is a nuclear protein that interacts with two DEAD-box ATP-dependent RNA helicases, API5-INTERACTING PROTEIN1 (AIP1) and AIP2. AIP1 and AIP2 are homologs of yeast (Saccharomyces cerevisiae) Suppressor of Bad Response to Refrigeration1 protein 2 (SUB2p) that have critical roles in transcription elongation and pre-mRNA splicing. Os AIP1 and AIP2 can form dimers and interact directly with the promoter region of CP1, a rice cysteine protease gene. Suppression of Os AIP1/2 leads to down-regulation of CP1, resulting in sterility, which is highly similar to the effects of suppressed expression of Os CP1. Our results uncover a previously unknown pathway for regulating PCD during tapetum degeneration in rice, one that may be conserved among eukaryotic organisms.

DNA methylation remodeling and the functional implication during male gametogenesis in rice.

BACKGROUND: Epigenetic marks are reprogrammed during sexual reproduction. In flowering plants, DNA methylation is only partially remodeled in the gametes and the zygote. However, the timing and functional significance of the remodeling during plant gametogenesis remain obscure. RESULTS: Here we show that DNA methylation remodeling starts after male meiosis in rice, with non-CG methylation, particularly at CHG sites, being first enhanced in the microspore and subsequently decreased in sperm. Functional analysis of rice CHG methyltransferase genes CMT3a and CMT3b indicates that CMT3a functions as the major CHG methyltransferase in rice meiocyte, while CMT3b is responsible for the increase of CHG methylation in microspore. The function of the two histone demethylases JMJ706 and JMJ707 that remove H3K9me2 may contribute to the decreased CHG methylation in sperm. During male gametogenesis CMT3a mainly silences TE and TE-related genes while CMT3b is required for repression of genes encoding factors involved in transcriptional and translational activities. In addition, CMT3b functions to repress zygotic gene expression in egg and participates in establishing the zygotic epigenome upon fertilization. CONCLUSION: Collectively, the results indicate that DNA methylation is dynamically remodeled during male gametogenesis, distinguish the function of CMT3a and CMT3b in sex cells, and underpin the functional significance of DNA methylation remodeling during rice reproduction.

Transcriptome-wide association analyses reveal the impact of regulatory variants on rice panicle architecture and causal gene regulatory networks.

Panicle architecture is a key determinant of rice grain yield and is mainly determined at the 1-2 mm young panicle stage. Here, we investigated the transcriptome of the 1-2 mm young panicles from 275 rice varieties and identified thousands of genes whose expression levels were associated with panicle traits. Multimodel association studies suggested that many small-effect genetic loci determine spikelet per panicle (SPP) by regulating the expression of genes associated with panicle traits. We found that alleles at cis-expression quantitative trait loci of SPP-associated genes underwent positive selection, with a strong preference for alleles increasing SPP. We further developed a method that integrates the associations of cis- and trans-expression components of genes with traits to identify causal genes at even small-effect loci and construct regulatory networks. We identified 36 putative causal genes of SPP, including SDT (MIR156j) and OsMADS17, and inferred that OsMADS17 regulates SDT expression, which was experimentally validated. Our study reveals the impact of regulatory variants on rice panicle architecture and provides new insights into the gene regulatory networks of panicle traits.

OsLIS-L1 encoding a lissencephaly type-1-like protein with WD40 repeats is required for plant height and male gametophyte formation in rice.

Although a large number of genes encoding the WD40 motif have been identified as being involved in various developmental processes in Arabidopsis, little is known about the function of these genes in rice (Oryza sativa). Here, we report the cloning and functional characterization of a novel rice gene OsLIS-L1 (Lissencephaly type-1-like 1), which is required for normal fertility and the first internode elongation. OsLIS-L1 encodes a lissencephaly type-1-like protein containing the WD40 motif that is required for brain development in human. SMART algorithm analysis indicated that OsLIS-L1 contains a LIS1 homology (LisH) domain, a C terminus to LisH (CTLH) domain, a five WD40-repeat domain in the middle, and a domain with four WD40 repeats which is homologous to the ß subunit of trimeric G-proteins (G(ß)). OsLIS-L1 transcript is relatively highly abundant in stem and panicle and has a dynamic expression pattern at different panicle developmental stages. Two independent alleles, designated oslis-l1-1 and oslis-l1-2, exhibited similar abnormal developmental phenotypes, including semi-dwarf, shorter panicle length, and reduced male fertility. Cytological examination confirmed that OsLIS-L1 does not affect the meiosis in pollen mother cells. Compared with wild type, the oslis-l1 mutant had abnormal male gametophyte formation, but anther cell wall and pollen wall development were not affected. Histological analysis revealed that OsLIS-L1 regulates the cell proliferation in the first internode under the panicle. Our results indicate that OsLIS-L1 plays an important role in male gametophyte formation and the first internode elongation in rice.

Effort and contribution of T-DNA Insertion mutant library for rice functional genomics research in China: review and perspective.

With the completion of the rice (Oryza sativa L.) genome-sequencing project, the rice research community proposed to characterize the function of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.

miR395-regulated sulfate metabolism exploits pathogen sensitivity to sulfate to boost immunity in rice.

MicroRNAs (miRNAs) play important roles in plant physiological activities. However, their roles and molecular mechanisms in boosting plant immunity, especially through the modulation of macronutrient metabolism in response to pathogens, are largely unknown. Here, we report that an evolutionarily conserved miRNA, miR395, promotes resistance to Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc), two destructive bacterial pathogens, by regulating sulfate accumulation and distribution in rice. Specifically, miR395 targets and suppresses the expression of the ATP sulfurylase gene OsAPS1, which functions in sulfate assimilation, and two sulfate transporter genes, OsSULTR2;1 and OsSULTR2;2, which function in sulfate translocation, to promote sulfate accumulation, resulting in broad-spectrum resistance to bacterial pathogens in miR395-overexpressing plants. Genetic analysis revealed that miR395-triggered resistance is involved in both pathogen-associated molecular pattern-triggered immunity and R gene-mediated resistance. Moreover, we found that accumulated sulfate but not S-metabolites inhibits proliferation of pathogenic bacteria, revealing a sulfate-mediated antibacterial defense mechanism that differs from sulfur-induced resistance. Furthermore, compared with other bacteria, Xoo and Xoc, which lack the sulfate transporter CysZ, are sensitive to high levels of extracellular sulfate. Accordingly, miR395-regulated sulfate accumulation impaired the virulence of Xoo and Xoc by decreasing extracellular polysaccharide production and biofilm formation. Taken together, these results suggest that rice miR395 modulates sulfate metabolism to exploit pathogen sensitivity to sulfate and thereby promotes broad-spectrum resistance.