MG1 interacts with a protease inhibitor and confers resistance to rice root-knot nematode.

The rice root-knot nematode (Meloidogyne graminicola) is one of the most destructive pests threatening rice (Oryza sativa L.) production in Asia; however, no rice resistance genes have been cloned. Here, we demonstrate that M. GRAMINICOLA-RESISTANCE GENE 1 (MG1), an R gene highly expressed at the site of nematode invasion, determines resistance against the nematode in several rice varieties. Introgressing MG1 into susceptible varieties increases resistance comparable to resistant varieties, for which the leucine-rich repeat domain is critical for recognizing root-knot nematode invasion. We also report transcriptome and cytological changes that are correlated with a rapid and robust response during the incompatible interaction that occurs in resistant rice upon nematode invasion. Furthermore, we identified a putative protease inhibitor that directly interacts with MG1 during MG1-mediated resistance. Our findings provide insight into the molecular basis of nematode resistance as well as valuable resources for developing rice varieties with improved nematode resistance.

Genomic decoding of breeding history to guide breeding-by-design in rice.

Deciphering the intrinsic molecular logic of empirical crop breeding from a genomic perspective is a decisive prerequisite for breeding-by-design (BbD), but remains not well established. Here, we decoded the historical features of past rice breeding by phenotyping and haplotyping 546 accessions covering the majority of cultivars bred in the history of Northeast China (NEC). We revealed that three groups founded the genetic diversities in NEC rice with distinct evolution patterns and traced and verified the breeding footprints to known or genome-wide association study (GWAS)-detected quantitative trait loci (QTLs), or introgressions from indica sub-species with chronological changes in allele frequencies. Then we summarized a rice breeding trend/principle in NEC, and combined with the successful example in breeding and application of Zhongkefa5 to demonstrate the guiding value of our conclusion for BbD in practice. Our study provides a paradigm for decoding the breeding history of a specific crop to guide BbD, which may have implications in different crop breeding.

An activated form of NB-ARC protein RLS1 functions with cysteine-rich receptor-like protein RMC to trigger cell death in rice.

A key event that follows pathogen recognition by a resistance (R) protein containing an NB-ARC (nucleotide-binding adaptor shared by Apaf-1, R proteins, and Ced-4) domain is hypersensitive response (HR)-type cell death accompanied by accumulation of reactive oxygen species and nitric oxide. However, the integral mechanisms that underlie this process remain relatively opaque. Here, we show that a gain-of-function mutation in the NB-ARC protein RLS1 (Rapid Leaf Senescence 1) triggers high-light-dependent HR-like cell death in rice. The RLS1-mediated defense response is largely independent of salicylic acid accumulation, NPR1 (Nonexpressor of Pathogenesis-Related Gene 1) activity, and RAR1 (Required for Mla12 Resistance 1) function. A screen for suppressors of RLS1 activation identified RMC (Root Meander Curling) as essential for the RLS1-activated defense response. RMC encodes a cysteine-rich receptor-like secreted protein (CRRSP) and functions as an RLS1-binding partner. Intriguingly, their co-expression resulted in a change in the pattern of subcellular localization and was sufficient to trigger cell death accompanied by a decrease in the activity of the antioxidant enzyme APX1. Collectively, our findings reveal an NB-ARC-CRRSP signaling module that modulates oxidative state, the cell death process, and associated immunity responses in rice.

Antagonistic control of seed dormancy in rice by two bHLH transcription factors.

Preharvest sprouting (PHS) due to lack of seed dormancy seriously threatens crop production worldwide. As a complex quantitative trait, breeding of crop cultivars with suitable seed dormancy is hindered by limited useful regulatory genes. Here by repeatable phenotypic characterization of fixed recombinant individuals, we report a quantitative genetic locus, Seed Dormancy 6 (SD6), from aus-type rice, encoding a basic helix-loop-helix (bHLH) transcription factor, which underlies the natural variation of seed dormancy. SD6 and another bHLH factor inducer of C-repeat binding factors expression 2 (ICE2) function antagonistically in controlling seed dormancy by directly regulating the ABA catabolism gene ABA8OX3, and indirectly regulating the ABA biosynthesis gene NCED2 via OsbHLH048, in a temperature-dependent manner. The weak-dormancy allele of SD6 is common in cultivated rice but undergoes negative selection in wild rice. Notably, by genome editing SD6 and its wheat homologs, we demonstrated that SD6 is a useful breeding target for alleviating PHS in cereals under field conditions.

Synergistic interplay of ABA and BR signal in regulating plant growth and adaptation.

Complex antagonistic interactions between abscisic acid (ABA) and brassinosteroid (BR) signalling pathways have been widely documented. However, whether or how ABA interacts synergistically with BR in plants remains to be elucidated. Here, we report that low, but not high, concentration of ABA increases lamina joint inclination of rice seedling, which requires functional BR biosynthesis and signalling. Transcriptome analyses confirm that about 60% of low-concentration ABA early response genes can be regulated by BR in the same directions. ABA activates BR signal in a fast, limited and short-term manner and the BR-biosynthesis regulatory gene, OsGSR1, plays a key role during this process, whose expression is induced slightly by ABA through transcriptional factor ABI3. Moreover, the early short-term BR signal activation is also important for ABA-mediated salt stress tolerance. Intriguingly, the process and effect of short-term BR signal activation were covered by high concentration of ABA, implying adaptive mechanisms existed in plants to cope with varying degrees of stress.

Strigolactone Signaling: Repressor Proteins Are Transcription Factors.

A recent landmark study by Wang et al. provides new insight into transcriptional regulation in strigolactone (SL) signaling. The finding that SUPPRESSOR OF MAX2 LIKE 6 (SMXL6) also functions as an autoregulated transcription factor (TF) causes a paradigm shift in the current view of transcriptional repressors in phytohormone signaling.

Mutation of a Nucleotide-Binding Leucine-Rich Repeat Immune Receptor-Type Protein Disrupts Immunity to Bacterial Blight.

Most characterized plant resistance proteins belong to the nucleotide-binding domain and Leu-rich repeat-containing (NLR) family. NLRs are present in an auto-inhibited state in the absence of specific pathogens, while gain-of-function mutations in NLRs usually cause autoimmunity. Here, we show that a gain-of-function mutation, weaker defense (wed), which caused a Phe-to-Leu substitution in the nucleotide-binding domain of a typical NLR in rice (Oryza sativa), led to enhanced susceptibility to Xanthomonas oryzae pv. Oryzae The unexpected accumulation of salicylic acid (SA), along with downregulation of NONEXPRESSOR OF PR1 (NPR1), in wed indicates the potential presence of a feedback regulation loop of SA biosynthesis in rice. Epistasis analyses illustrated that SA accumulation and the NLR-associated components RAR1, OsRac1, and PhyB are dispensable for the wed phenotypes. Intriguingly, besides pattern-triggered immunity, effector-triggered immunity conferred by different resistance proteins, including Xa3/Xa26, Xa4, and Xa21, was also disturbed by wed to a certain extent, indicating the existence of shared regulatory mechanisms for various defense systems. The identification of wed therefore provides a unique system for genetic dissection of shared immune signaling pathways activated by different types of immune receptors.

Control of rice pre-harvest sprouting by glutaredoxin-mediated abscisic acid signaling.

Pre-harvest sprouting (PHS) is one of the major problems in cereal production worldwide, which causes significant losses of both yield and quality; however, the molecular mechanism underlying PHS remains largely unknown. Here, we identified a dominant PHS mutant phs9-D. The corresponding gene PHS9 encodes a higher plant unique CC-type glutaredoxin and is specifically expressed in the embryo at the late embryogenesis stage, implying that PHS9 plays some roles in the late stage of seed development. Yeast two-hybrid screening showed that PHS9 could interact with OsGAP, which is an interaction partner of the abscicic acid (ABA) receptor OsRCAR1. PHS9- or OsGAP overexpression plants showed reduced ABA sensitivity in seed germination, whereas PHS9 or OsGAP knock-out mutant plants showed increased ABA sensitivity in seed germination, suggesting that PHS9 and OsGAP acted as negative regulators in ABA signaling during seed germination. Interestingly, the germination of PHS9 and OsGAP overexpression or knock-out plant seeds was weakly promoted by H2 O2 , implying that PHS9 and OsGAP could affect reactive oxygen species (ROS) signaling during seed germination. These results indicate that PHS9 plays an important role in the regulation of rice PHS through the integration of ROS signaling and ABA signaling.

Parallel selection on a dormancy gene during domestication of crops from multiple families.

Domesticated species often exhibit convergent phenotypic evolution, termed the domestication syndrome, of which loss of seed dormancy is a component. To date, dormancy genes that contribute to parallel domestication across different families have not been reported. Here, we cloned the classical stay-green G gene from soybean and found that it controls seed dormancy and showed evidence of selection during soybean domestication. Moreover, orthologs in rice and tomato also showed evidence of selection during domestication. Analysis of transgenic plants confirmed that orthologs of G had conserved functions in controlling seed dormancy in soybean, rice, and Arabidopsis. Functional investigation demonstrated that G affected seed dormancy through interactions with NCED3 and PSY and in turn modulated abscisic acid synthesis. Therefore, we identified a gene responsible for seed dormancy that has been subject to parallel selection in multiple crop families. This may help facilitate the domestication of new crops.

Early selection of bZIP73 facilitated adaptation of japonica rice to cold climates.

Cold stress is a major factor limiting production and geographic distribution of rice (Oryza sativa). Although the growth range of japonica subspecies has expanded northward compared to modern wild rice (O. rufipogon), the molecular basis of the adaptation remains unclear. Here we report bZIP73, a bZIP transcription factor-coding gene with only one functional polymorphism (+511 G>A) between the two subspecies japonica and indica, may have facilitated japonica adaptation to cold climates. We show the japonica version of bZIP73 (bZIP73Jap) interacts with bZIP71 and modulates ABA levels and ROS homeostasis. Evolutionary and population genetic analyses suggest bZIP73 has undergone balancing selection; the bZIP73Jap allele has firstly selected from standing variations in wild rice and likely facilitated cold climate adaptation during initial japonica domestication, while the indica allele bZIP73Ind was subsequently selected for reasons that remain unclear. Our findings reveal early selection of bZIP73Jap may have facilitated climate adaptation of primitive rice germplasms.

Endosperm sugar accumulation caused by mutation of PHS8/ISA1 leads to pre-harvest sprouting in rice.

Pre-harvest sprouting (PHS) is an unfavorable trait in cereal crops that could seriously decrease grain yield and quality. Although some PHS-associated quantitative trait loci or genes in cereals have been reported, the molecular mechanism underlying PHS remains largely elusive. Here, we characterized a rice mutant, phs8, which exhibits PHS phenotype accompanied by sugary endosperm. Map-based cloning revealed that PHS8 encodes a starch debranching enzyme named isoamylase1. Mutation in PHS8 resulted in the phytoglycogen breakdown and sugar accumulation in the endosperm. Intriguingly, with increase of sugar contents, decreased expression of OsABI3 and OsABI5 as well as reduced sensitivity to abscisic acid (ABA) were found in the phs8 mutant. Using rice suspension cell system, we confirmed that exogenous sugar is sufficient to suppress the expression of both OsABI3 and OsABI5. Furthermore, overexpression of OsABI3 or OsABI5 could partially rescue the PHS phenotype of phs8. Therefore, our study presents important evidence supporting that endosperm sugar not only acts as an essential energy source for seed germination but also determines seed dormancy and germination by affecting ABA signaling.

MicroRNAs in crop improvement: fine-tuners for complex traits.

One of the most common challenges for both conventional and modern crop improvement is that the appearance of one desirable trait in a new crop variety is always balanced by the impairment of one or more other beneficial characteristics. The best way to overcome this problem is the flexible utilization of regulatory genes, especially genes that provide more efficient and precise regulation in a targeted manner. MicroRNAs (miRNAs), a type of short non-coding RNA, are promising candidates in this area due to their role as master modulators of gene expression at the post-transcriptional level, targeting messenger RNAs for cleavage or directing translational inhibition in eukaryotes. We herein highlight the current understanding of the biological role of miRNAs in orchestrating distinct agriculturally important traits by summarizing recent functional analyses of 65 miRNAs in 9 major crops worldwide. The integration of current miRNA knowledge with conventional and modern crop improvement strategies is also discussed.

Rapid stomatal response to fluctuating light: an under-explored mechanism to improve drought tolerance in rice.

Light inside a canopy constantly fluctuates. Under fluctuating light (FL) conditions, stomatal conductance and photosynthetic rate constantly change. In this study, we explored whether this dynamics of stomata movements upon FL influenced the water use efficiency of rice in the field. We used a USDA-curated rice mini-core diversity panel consisting of 204 worldwide distributed accessions. A priori model on dynamic stomatal response to FL was utilised to identify kinetic parameters describing the stomatal delays during the closing (τcl) and the opening (τop) phase. Result showed that τcl had a larger variation than τop across the mini-core panel. τcl was negatively correlated with water use efficiency (WUE) related traits, stem diameter, grain weight per tiller and heading time, but positively correlated with maximum annual temperature, carbon assimilation related traits and biomass (P<0.05). We further showed a strong correlation of τcl with the relative decrease of biomass under drought in 14 accessions with different τcl. We discussed the adjustment of stomatal conductance under fluctuating light in light of the trade-off between optimising CO2 uptake and optimising water saving. This study suggests that stomatal dynamics under fluctuating light is closely related to drought resistance and hence detailed study is needed to enable its application in breeding drought tolerance in rice.

Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies.

Asian cultivated rice (Oryza sativa L.) consists of two main subspecies, indica and japonica. Indica has higher nitrate-absorption activity than japonica, but the molecular mechanisms underlying that activity remain elusive. Here we show that variation in a nitrate-transporter gene, NRT1.1B (OsNPF6.5), may contribute to this divergence in nitrate use. Phylogenetic analysis revealed that NRT1.1B diverges between indica and japonica. NRT1.1B-indica variation was associated with enhanced nitrate uptake and root-to-shoot transport and upregulated expression of nitrate-responsive genes. The selection signature of NRT1.1B-indica suggests that nitrate-use divergence occurred during rice domestication. Notably, field tests with near-isogenic and transgenic lines confirmed that the japonica variety carrying the NRT1.1B-indica allele had significantly improved grain yield and nitrogen-use efficiency (NUE) compared to the variety without that allele. Our results show that variation in NRT1.1B largely explains nitrate-use divergence between indica and japonica and that NRT1.1B-indica can potentially improve the NUE of japonica.

OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice.

It has long been established that premature leaf senescence negatively impacts the yield stability of rice, but the underlying molecular mechanism driving this relationship remains largely unknown. Here, we identified a dominant premature leaf senescence mutant, prematurely senile 1 (ps1-D). PS1 encodes a plant-specific NAC (no apical meristem, Arabidopsis ATAF1/2, and cup-shaped cotyledon2) transcriptional activator, Oryza sativa NAC-like, activated by apetala3/pistillata (OsNAP). Overexpression of OsNAP significantly promoted senescence, whereas knockdown of OsNAP produced a marked delay of senescence, confirming the role of this gene in the development of rice senescence. OsNAP expression was tightly linked with the onset of leaf senescence in an age-dependent manner. Similarly, ChIP-PCR and yeast one-hybrid assays demonstrated that OsNAP positively regulates leaf senescence by directly targeting genes related to chlorophyll degradation and nutrient transport and other genes associated with senescence, suggesting that OsNAP is an ideal marker of senescence onset in rice. Further analysis determined that OsNAP is induced specifically by abscisic acid (ABA), whereas its expression is repressed in both aba1 and aba2, two ABA biosynthetic mutants. Moreover, ABA content is reduced significantly in ps1-D mutants, indicating a feedback repression of OsNAP on ABA biosynthesis. Our data suggest that OsNAP serves as an important link between ABA and leaf senescence. Additionally, reduced OsNAP expression leads to delayed leaf senescence and an extended grain-filling period, resulting in a 6.3% and 10.3% increase in the grain yield of two independent representative RNAi lines, respectively. Thus, fine-tuning OsNAP expression should be a useful strategy for improving rice yield in the future.

Activation of the jasmonic acid pathway by depletion of the hydroperoxide lyase OsHPL3 reveals crosstalk between the HPL and AOS branches of the oxylipin pathway in rice.

The allene oxide synthase (AOS) and hydroperoxide lyase (HPL) branches of the oxylipin pathway, which underlie the production of jasmonates and aldehydes, respectively, function in plant responses to a range of stresses. Regulatory crosstalk has been proposed to exist between these two signaling branches; however, there is no direct evidence of this. Here, we identified and characterized a jasmonic acid (JA) overproduction mutant, cea62, by screening a rice T-DNA insertion mutant library for lineages that constitutively express the AOS gene. Map-based cloning was used to identify the underlying gene as hydroperoxide lyase OsHPL3. HPL3 expression and the enzyme activity of its product, (E)-2-hexenal, were depleted in the cea62 mutant, which resulted in the dramatic overproduction of JA, the activation of JA signaling, and the emergence of the lesion mimic phenotype. A time-course analysis of lesion formation and of the induction of defense responsive genes in the cea62 mutant revealed that the activation of JA biosynthesis and signaling in cea62 was regulated in a developmental manner, as was OsHPL3 activity in the wild-type plant. Microarray analysis showed that the JA-governed defense response was greatly activated in cea62 and this plant exhibited enhanced resistance to the T1 strain of the bacterial blight pathogen Xanthomonasoryzaepvoryzae (Xoo). The wounding response was attenuated in cea62 plants during the early stages of development, but partially recovered when JA levels were elevated during the later stages. In contrast, the wounding response was not altered during the different developmental stages of wild-type plants. These findings suggest that these two branches of the oxylipin pathway exhibit crosstalk with regards to biosynthesis and signaling and cooperate with each other to function in diverse stress responses.

The histone methyltransferase SDG724 mediates H3K36me2/3 deposition at MADS50 and RFT1 and promotes flowering in rice.

Chromatin modifications affect flowering time in the long-day plant Arabidopsis thaliana, but the role of histone methylation in flowering time regulation of rice (Oryza sativa), a short-day plant, remains to be elucidated. We identified a late-flowering long vegetative phase1 (lvp1) mutant in rice and used map-based cloning to reveal that lvp1 affects the SET domain group protein 724 (SDG724). SDG724 functions as a histone methyltransferase in vitro and contributes to a major fraction of global histone H3 lysine 36 (H3K36) methylation in vivo. Expression analyses of flowering time genes in wild-type and lvp1 mutants revealed that Early heading date1, but not Heading date1, are misregulated in lvp1 mutants. In addition, the double mutant of lvp1 with photoperiod sensitivity5 (se5) flowered later than the se5 single mutant, indicating that lvp1 delays flowering time irrespective of photoperiod. Chromatin immunoprecipitation assays showed that lvp1 had reduced levels of H3K36me2/3 at MADS50 and RFT1. This suggests that the divergent functions of paralogs RFT1 and Hd3a, and of MADS50 and MADS51, are in part due to differential H3K36me2/3 deposition, which also correlates with higher expression levels of MADS50 and RFT1 in flowering promotion in rice.