Rice LIKE EARLY STARVATION1 cooperates with FLOURY ENDOSPERM 6 to modulate starch biosynthesis and endosperm development.

In cereal grains, starch is synthesized by the concerted actions of multiple enzymes on the surface of starch granules within the amyloplast. However, little is known about how starch-synthesizing enzymes access starch granules, especially for amylopectin biosynthesis. Here, we show that the rice (Oryza sativa) floury endosperm9 (flo9) mutant is defective in amylopectin biosynthesis, leading to grains exhibiting a floury endosperm with a hollow core. Molecular cloning revealed that FLO9 encodes a plant-specific protein homologous to Arabidopsis (Arabidopsis thaliana) LIKE EARLY STARVATION1 (LESV). Unlike Arabidopsis LESV, which is involved in starch metabolism in leaves, OsLESV is required for starch granule initiation in the endosperm. OsLESV can directly bind to starch by its C-terminal tryptophan (Trp)-rich region. Cellular and biochemical evidence suggests that OsLESV interacts with the starch-binding protein FLO6, and loss-of-function mutations of either gene impair ISOAMYLASE 1 (ISA1) targeting to starch granules. Genetically, OsLESV acts synergistically with FLO6 to regulate starch biosynthesis and endosperm development. Together, our results identify OsLESV-FLO6 as a non-enzymatic molecular module responsible for ISA1 localization on starch granules, and present a target gene for use in biotechnology to control starch content and composition in rice endosperm.

The rice peroxisomal receptor PEX5 negatively regulates resistance to rice blast fungus Magnaporthe oryzae.

The receptor protein PEX5, an important component of peroxisomes, regulates growth, development, and immunity in yeast and mammals. PEX5 also influences growth and development in plants, but whether it participates in plant immunity has remained unclear. Here, we report that knockdown of OsPEX5 enhances resistance to the rice blast fungus Magnaporthe oryzae. We demonstrate that OsPEX5 interacts with the E3 ubiquitin ligase APIP6, a positive regulator of plant immunity. APIP6 ubiquitinates OsPEX5 in vitro and promotes its degradation in vivo via the 26S proteasome pathway. In addition, OsPEX5 interacts with the aldehyde dehydrogenase OsALDH2B1, which functions in growth-defense trade-offs in rice. OsPEX5 stabilizes OsALDH2B1 to enhance its repression of the defense-related gene OsAOS2. Our study thus uncovers a previously unrecognized hierarchical regulatory mechanism in which an E3 ubiquitin ligase targets a peroxisome receptor protein that negatively regulates immunity in rice by stabilizing an aldehyde dehydrogenase that suppresses defense gene expression.

Editing a rice CDP-DAG synthase confers broad-spectrum resistance.

Lesion mimic mutations (LMMs) often confer broad-spectrum resistance (BSR) in plants, but with significant yield penalties. Sha et al. recently demonstrated that genome editing of the rice BSR gene RESISTANCE TO BLAST1 (RBL1), encoding a cytidine diphosphate diacylglycerol (CDP-DAG) synthase involved in phospholipid biosynthesis, confers multipathogen resistance without an obvious trade-off in yield.

A natural allele of proteasome maturation factor improves rice resistance to multiple pathogens.

Crops with broad-spectrum resistance loci are highly desirable in agricultural production because these loci often confer resistance to most races of a pathogen or multiple pathogen species. Here we discover a natural allele of proteasome maturation factor in rice, UMP1R2115, that confers broad-spectrum resistance to Magnaporthe oryzae, Rhizoctonia solani, Ustilaginoidea virens and Xanthomonas oryzae pv. oryzae. Mechanistically, this allele increases proteasome abundance and activity to promote the degradation of reactive oxygen species-scavenging enzymes including peroxidase and catalase upon pathogen infection, leading to elevation of H2O2 accumulation for defence. In contrast, inhibition of proteasome function or overexpression of peroxidase/catalase-encoding genes compromises UMP1R2115-mediated resistance. More importantly, introduction of UMP1R2115 into a disease-susceptible rice variety does not penalize grain yield while promoting disease resistance. Our work thus uncovers a broad-spectrum resistance pathway integrating de-repression of plant immunity and provides a valuable genetic resource for breeding high-yield rice with multi-disease resistance.

A VQ-motif-containing protein fine-tunes rice immunity and growth by a hierarchical regulatory mechanism.

Rice blast and bacterial blight, caused by the fungus Magnaporthe oryzae and the bacterium Xanthomonas oryzae pv. oryzae (Xoo), respectively, are devastating diseases affecting rice. Here, we report that a rice valine-glutamine (VQ) motif-containing protein, OsVQ25, balances broad-spectrum disease resistance and plant growth by interacting with a U-Box E3 ligase, OsPUB73, and a transcription factor, OsWRKY53. We show that OsPUB73 positively regulates rice resistance against M. oryzae and Xoo by interacting with and promoting OsVQ25 degradation via the 26S proteasome pathway. Knockout mutants of OsVQ25 exhibit enhanced resistance to both pathogens without a growth penalty. Furthermore, OsVQ25 interacts with and suppresses the transcriptional activity of OsWRKY53, a positive regulator of plant immunity. OsWRKY53 downstream defense-related genes and brassinosteroid signaling genes are upregulated in osvq25 mutants. Our findings reveal a ubiquitin E3 ligase-VQ protein-transcription factor module that fine-tunes plant immunity and growth at the transcriptional and posttranslational levels.

An ORFeome of rice E3 ubiquitin ligases for global analysis of the ubiquitination interactome.

BACKGROUND: Ubiquitination is essential for many cellular processes in eukaryotes, including 26S proteasome-dependent protein degradation, cell cycle progression, transcriptional regulation, and signal transduction. Although numerous ubiquitinated proteins have been empirically identified, their cognate ubiquitin E3 ligases remain largely unknown. RESULTS: Here, we generate a complete ubiquitin E3 ligase-encoding open reading frames (UbE3-ORFeome) library containing 98.94% of the 1515 E3 ligase genes in the rice (Oryza sativa L.) genome. In the test screens with four known ubiquitinated proteins, we identify both known and new E3s. The interaction and degradation between several E3s and their substrates are confirmed in vitro and in vivo. In addition, we identify the F-box E3 ligase OsFBK16 as a hub-interacting protein of the phenylalanine ammonia lyase family OsPAL1-OsPAL7. We demonstrate that OsFBK16 promotes the degradation of OsPAL1, OsPAL5, and OsPAL6. Remarkably, we find that overexpression of OsPAL1 or OsPAL6 as well as loss-of-function of OsFBK16 in rice displayed enhanced blast resistance, indicating that OsFBK16 degrades OsPALs to negatively regulate rice immunity. CONCLUSIONS: The rice UbE3-ORFeome is the first complete E3 ligase library in plants and represents a powerful proteomic resource for rapid identification of the cognate E3 ligases of ubiquitinated proteins and establishment of functional E3-substrate interactome in plants.

Du13 encodes a C2 H2 zinc-finger protein that regulates Wxb pre-mRNA splicing and microRNA biogenesis in rice endosperm.

Amylose content is a crucial physicochemical property responsible for the eating and cooking quality of rice (Oryza sativa L.) grain and is mainly controlled by the Waxy (Wx) gene. Previous studies have identified several Dull genes that modulate the expression of the Wxb allele in japonica rice by affecting the splicing efficiency of the Wxb pre-mRNA. Here, we uncover dual roles for a novel Dull gene in pre-mRNA splicing and microRNA processing. We isolated the dull mutant, du13, with a dull endosperm and low amylose content. Map-based cloning showed that Du13 encodes a C2 H2 zinc-finger protein. Du13 coordinates with the nuclear cap-binding complex to regulate the splicing of Wxb transcripts in rice endosperm. Moreover, Du13 also regulates alternative splicing of other protein-coding transcripts and affects the biogenesis of a subset of microRNAs. Our results reveal an evolutionarily conserved link between pre-mRNA splicing and microRNA biogenesis in rice endosperm. Our findings also provide new insights into the functions of Dull genes in rice and expand our knowledge of microRNA biogenesis in monocots.

Two VOZ transcription factors link an E3 ligase and an NLR immune receptor to modulate immunity in rice.

Nucleotide-binding leucine-rich repeat (NLR) proteins play critical roles in plant immunity. However, how NLRs are regulated and activate defense signaling is not fully understood. The rice (Oryza sativa) NLR receptor Piz-t confers broad-spectrum resistance to the fungal pathogen Magnaporthe oryzae and the RING-type E3 ligase AVRPIZ-T INTERACTING PROTEIN 10 (APIP10) negatively regulates Piz-t accumulation. In this study, we found that APIP10 interacts with two rice transcription factors, VASCULAR PLANT ONE-ZINC FINGER 1 (OsVOZ1) and OsVOZ2, and promotes their degradation through the 26S proteasome pathway. OsVOZ1 displays transcriptional repression activity while OsVOZ2 confers transcriptional activation activity in planta. The osvoz1 and osvoz2 single mutants display modest but opposite M. oryzae resistance in the non-Piz-t background. However, the osvoz1 osvoz2 double mutant exhibits strong dwarfism and cell death, and silencing of both genes via RNA interference also leads to dwarfism, mild cell death, and enhanced resistance to M. oryzae in the non-Piz-t background. Both OsVOZ1 and OsVOZ2 interact with Piz-t. Double silencing of OsVOZ1 and OsVOZ2 in the Piz-t background decreases Piz-t protein accumulation and transcription, reactive oxygen species-dependent cell death, and resistance to M. oryzae containing AvrPiz-t. Taken together, these results indicate that OsVOZ1 and OsVOZ2 negatively regulate basal defense but contribute positively to Piz-t-mediated immunity.

Rice stripe virus suppresses jasmonic acid-mediated resistance by hijacking brassinosteroid signaling pathway in rice.

Rice stripe virus (RSV) is one of the most destructive viral diseases affecting rice production. However, so far, only one RSV resistance gene has been cloned, the molecular mechanisms underlying host-RSV interaction are still poorly understood. Here, we show that increasing levels or signaling of brassinosteroids (BR) and jasmonic acid (JA) can significantly enhance the resistance against RSV. On the contrary, plants impaired in BR or JA signaling are more susceptible to RSV. Moreover, the enhancement of RSV resistance conferred by BR is impaired in OsMYC2 (a key positive regulator of JA response) knockout plants, suggesting that BR-mediated RSV resistance requires active JA pathway. In addition, we found that RSV infection suppresses the endogenous BR levels to increase the accumulation of OsGSK2, a key negative regulator of BR signaling. OsGSK2 physically interacts with OsMYC2, resulting in the degradation of OsMYC2 by phosphorylation and reduces JA-mediated defense to facilitate virus infection. These findings not only reveal a novel molecular mechanism mediating the crosstalk between BR and JA in response to virus infection and deepen our understanding about the interaction of virus and plants, but also suggest new effective means of breeding RSV resistant crops using genetic engineering.

Small Grain and Dwarf 2, encoding an HD-Zip II family transcription factor, regulates plant development by modulating gibberellin biosynthesis in rice.

Homeodomain leucine zipper (HD-Zip) proteins are transcription factors that regulate plant development. Bioactive gibberellin (GA) is a key endogenous hormone that participates in plant growth. However, the relationship between HD-Zip genes and modulation of GA biosynthesis in rice remains elusive. Here, we identified a rice mutant, designated as small grain and dwarf 2 (sgd2), which had reduced height and grain size compared with the wild type. Cytological observations indicated that the defective phenotype was mainly due to decreased cell length. Map-based cloning and complementation tests demonstrated that a 9 bp deletion in a homeodomain leucine zipper (HD-Zip) II family transcription factor was responsible for the sgd2 mutant phenotype. Expression of SGD2 was pronounced in developing panicles, and its protein was localized in nucleus. Luciferase reporter system and transactivation assays in yeast suggested that SGD2 functioned as a transcriptional repressor. High performance liquid chromatography assays showed that the endogenous GA1 level in the sgd2 mutant was dramatically decreased, and exogenous GA3 recovered the second leaf sheath to normal length. Results of qRT-PCR showed that the expression levels of genes positively regulating GA-biosynthesis were mostly down-regulated in the mutant. Our data identified the role of an HD-Zip transcription factor that affects rice plant development by modulating gibberellin biosynthesis.

OsPEX5 regulates rice spikelet development through modulating jasmonic acid biosynthesis.

Spikelet is the primary reproductive structure and a critical determinant of grain yield in rice. The molecular mechanisms regulating rice spikelet development still remain largely unclear. Here, we report that mutations in OsPEX5, which encodes a peroxisomal targeting sequence 1 (PTS1) receptor protein, cause abnormal spikelet morphology. We show that OsPEX5 can physically interact with OsOPR7, an enzyme involved in jasmonic acid (JA) biosynthesis and is required for its import into peroxisome. Similar to Ospex5 mutant, the knockout mutant of OsOPR7 generated via CRISPR-Cas9 technology has reduced levels of endogenous JA and also displays an abnormal spikelet phenotype. Application of exogenous JA can partially rescue the abnormal spikelet phenotype of Ospex5 and Osopr7. Furthermore, we show that OsMYC2 directly binds to the promoters of OsMADS1, OsMADS7 and OsMADS14 to activate their expression, and subsequently regulate spikelet development. Our results suggest that OsPEX5 plays a critical role in regulating spikelet development through mediating peroxisomal import of OsOPR7, therefore providing new insights into regulation of JA biosynthesis in plants and expanding our understanding of the biological role of JA in regulating rice reproduction.

FLOURY ENDOSPERM15 encodes a glyoxalase I involved in compound granule formation and starch synthesis in rice endosperm.

KEY MESSAGE: FLO15encodes a plastidic glyoxalase I protein, OsGLYI7, which affects compound starch granule formation and starch synthesis in rice endosperm. Starch synthesis in rice (Oryza sativa) endosperm is a sophisticated process, and its underlying molecular machinery still remains to be elucidated. Here, we identified and characterized two allelic rice floury endosperm 15 (flo15) mutants, both with a white-core endosperm. The flo15 grains were characterized by defects in compound starch granule development, along with decreased starch content. Map-based cloning of the flo15 mutants identified mutations in OsGLYI7, which encodes a glyoxalase I (GLYI) involved in methylglyoxal (MG) detoxification. The mutations of FLO15/OsGLYI7 resulted in increased MG content in flo15 developing endosperms. FLO15/OsGLYI7 localizes to the plastids, and the in vitro GLYI activity derived from flo15 was significantly decreased relative to the wild type. Moreover, the expression of starch synthesis-related genes was obviously altered in the flo15 mutants. These findings suggest that FLO15 plays an important role in compound starch granule formation and starch synthesis in rice endosperm.

OsPKpα1 encodes a plastidic pyruvate kinase that affects starch biosynthesis in the rice endosperm.

Pyruvate kinase (PK) is a key enzyme in glycolysis and carbon metabolism. Here, we isolated a rice (Oryza sativa) mutant, w59, with a white-core floury endosperm. Map-based cloning of w59 identified a mutation in OsPKpα1, which encodes a plastidic isoform of PK (PKp). OsPKpα1 localizes to the amyloplast stroma in the developing endosperm, and the mutation of OsPKpα1 in w59 decreases the plastidic PK activity, resulting in dramatic changes to the lipid biosynthesis in seeds. The w59 grains were also characterized by a marked decrease in starch content. Consistent with a decrease in number and size of the w59 amyloplasts, large empty spaces were observed in the central region of the w59 endosperm, at the early grain-filling stage. Moreover, a phylogenetic analysis revealed four potential rice isoforms of OsPKp. We validated the in vitro PK activity of these OsPKps through reconstituting active PKp complexes derived from inactive individual OsPKps, revealing the heteromeric structure of rice PKps, which was further confirmed using a protein-protein interaction analysis. These findings suggest a functional connection between lipid and starch synthesis in rice endosperm amyloplasts.

ADP-glucose pyrophosphorylase large subunit 2 is essential for storage substance accumulation and subunit interactions in rice endosperm.

ADP-glucose pyrophosphorylase (AGPase) controls a rate-limiting step in the starch biosynthetic pathway in higher plants. Here we isolated a shrunken rice mutant w24. Map-based cloning identified OsAGPL2, a large subunit of the cytosolic AGPase in rice endosperm, as the gene responsible for the w24 mutation. In addition to severe inhibition of starch synthesis and significant accumulation of sugar, the w24 endosperm showed obvious defects in compound granule formation and storage protein synthesis. The defect in OsAGPL2 enhanced the expression levels of the AGPase family. Meanwhile, the elevated activities of starch phosphorylase 1 and sucrose synthase in the w24 endosperm might possibly partly account for the residual starch content in the mutant seeds. Moreover, the expression of OsAGPL2 and its counterpart, OsAGPS2b, was highly coordinated in rice endosperm. Yeast two-hybrid and BiFC assays verified direct interactions between OsAGPL2 and OsAGPS2b as well as OsAGPL1 and OsAGPS1, supporting the model for spatiotemporal complex formation of AGPase isoforms in rice endosperm. Besides, our data provided no evidence for the self-binding of OsAGPS2b, implying that OsAGPS2b might not interact to form higher molecular mass aggregates in the absence of OsAGPL2. Therefore, the molecular mechanism of rice AGPase assembly might differ from that of Arabidopsis.

SLG controls grain size and leaf angle by modulating brassinosteroid homeostasis in rice.

Grain size and leaf angle are two important traits determining grain yield in rice. However, the mechanisms regulating the two traits remain largely unknown. Here, we characterized a rice gain-of-function mutant, slender grain Dominant (slg-D), which exhibited longer and narrower grains and larger leaf angles, similar to plants with elevated brassinosteroid (BR) levels or strengthened BR signaling. The increased cell length is responsible for the mutant phenotypes in slg-D We demonstrated that the phenotype of slg-D is caused by enhanced expression of SLG, a BAHD acyltransferase-like protein gene. SLG is preferentially expressed in young panicles and lamina joints, implying its role in controlling cell growth in those two tissues. slg-D was restored to wild type by treatment with brassinazole, an inhibitor of BR biosynthesis. Overexpression of SLG in d11-2 (deficient in BR synthesis) and d61-1 (deficient in BR signaling) did not change the existing phenotypes. The slg-D plants had elevated BR contents and, accordingly, expression of BR-related genes was changed in a manner similar to BR treatment. Moreover, SLG RNAi plants displayed mild BR-deficient phenotypes including shorter grains, smaller leaf angles, and compact semi-dwarf plant types. The in vitro biochemical assays and transgenic approaches collectively demonstrated that SLG functions as homomers. Taken together, we conclude that SLG is an important regulator in BR homeostasis and that manipulation of SLG expression to an optimal level may provide a way to develop an ideal plant type.