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The rice immune receptor XA21 confers resistance to Xanthomonas oryzae pv. oryzae (Xoo), and upon recognition of the RaxX21-sY peptide produced by Xoo, XA21 activates the plant immune response. Here we screened 21 000 mutant plants expressing XA21 to identify components involved in this response, and reported here the identification of a rice mutant, sxi4, which is susceptible to Xoo. The sxi4 mutant carries a 32-kb translocation from chromosome 3 onto chromosome 7 and displays an elevated level of DCL2a transcript, encoding a Dicer-like protein. Silencing of DCL2a in the sxi4 genetic background restores resistance to Xoo. RaxX21-sY peptide-treated leaves of sxi4 retain the hallmarks of XA21-mediated immune response. However, WRKY45-1, a known negative regulator of rice resistance to Xoo, is induced in the sxi4 mutant in response to RaxX21-sY peptide treatment. A CRISPR knockout of a short interfering RNA (TE-siRNA815) in the intron of WRKY45-1 restores the resistance phenotype in sxi4. These results suggest a model where DCL2a accumulation negatively regulates XA21-mediated immunity by altering the processing of TE-siRNA815.

Post-harvest processing efficiency and in-field disease resistance are two major traits for crop improvement. Higher grain-processing efficiency saves energy while stronger disease resistance reduces pesticide usage contributing to human health and environmental safety. However, simultaneous improvement on these two traits is exceptionally difficult due to the lack of suitable germplasm/gene resources and technologies. To address this problem in rice, we screened a rice mutant population and identified a rice mutant, improved brown rice processing efficiency 1 (ipe1), which has OsCesA7 mutated leading to improved brown rice processing efficiency. However, ipe1 shows reduced disease resistance. To restore disease resistance in ipe1 but keep its improved grain processing efficiency, we identified a rice intrinsic histone deacetylase 705 (HDA705) gene promoter, which has a similar expression pattern as the OsCesA7 promoter except with little expression in rice glumes, and expressed OsCesA7 with this promoter (proHDA705:OsCesA7). The proHDA705:OsCesA7 lines carry enhanced disease resistance and elevated brown rice processing efficiency with no compromises in other agronomic traits. This engineering strategy greatly saves energy used for brown rice processing and reduces pesticide usage, contributing not only to grain yield but also to human health, environmental safety, and energy saving.

MicroRNA168 (miR168) is a key miRNA that targets Argonaute1 (AGO1), a major component of the RNA-induced silencing complex1,2. Previously, we reported that miR168 expression was responsive to infection by Magnaporthe oryzae, the causal agent of rice blast disease3. However, how miR168 regulates immunity to rice blast and whether it affects rice development remains unclear. Here, we report our discovery that the suppression of miR168 by a target mimic (MIM168) not only improves grain yield and shortens flowering time in rice but also enhances immunity to M. oryzae. These results were validated through repeated tests in rice fields in the absence and presence of rice blast pressure. We found that the miR168–AGO1 module regulates miR535 to improve yield by increasing panicle number, miR164 to reduce flowering time, and miR1320 and miR164 to enhance immunity. Our discovery demonstrates that changes in a single miRNA enhance the expression of multiple agronomically important traits. In rice, it is known that miR168 regulates AGO1. Using a target mimic approach to suppress the microRNA enhances resistance against rice blast disease, increases grain yield and shortens growth period, including in field trials.

Chlorophylls function in photosynthesis, and are critical to plant developmental processes and responses to environmental stimuli. Chlorophyll b is synthesized from chlorophyll a by chlorophyll a oxygenase (CAO). Here, we characterize a yellow-green leaf (ygl) mutant and identify the causal gene which encodes a chlorophyll a oxygenase in maize (ZmCAO1). A 51 bp Popin transposon insertion in ZmCAO1 strongly disrupts its transcription. Low enzyme activity of ZmCAO1 leads to reduced concentrations of chlorophyll a and chlorophyll b, resulting in the yellow-green leaf phenotype of the ygl mutant. The net photosynthetic rate, stomatal conductance, and transpiration rate are decreased in the ygl mutant, while concentrations of δ-aminolevulinic acid (ALA), porphobilinogen (PBG) and protochlorophyllide (Pchlide) are increased. In addition, a ZmCAO1 mutation results in down-regulation of key photosynthetic genes, limits photosynthetic assimilation, and reduces plant height, ear size, kernel weight, and grain yield. Furthermore, the zmcao1 mutant shows enhanced reactive oxygen species production leading to sensitivity to waterlogging. These results demonstrate the pleiotropy of ZmCAO1 function in photosynthesis, grain yield, and waterlogging tolerance in maize.

Trade-offs between plant growth and immunity are a well-known phenomenon in plants that are meant to ensure the best use of limited resources. Recently, many advances have been achieved on molecular regulations of the trade-offs between plant growth and immunity. Here, we provide an overview on molecular understanding of these trade-offs including those regulated at the transcriptional level or post-transcriptional level by transcriptional factors, microRNAs, and post-translational modifications of proteins, respectively The understanding on the molecular regulation of these trade-offs will provide new strategies to breed crops with high yield and enhanced resistance to disease.

bsr‐d1, an allele encoding a transcription factor identified from the rice cultivar Digu, confers durable, broad‐spectrum resistance to infections by strains of Magnaporthe oryzae. bsr‐d1 was predicted to inhibit M. oryzae‐induced expression of Bsr‐d1 RNA and degradation of hydrogen peroxide to achieve resistance to M. oryzae. However, the global effect of biological process and molecular function on blast resistance mediated by Bsr‐d1 remains unknown. In this study, we compared transcriptomic profiling between Bsr‐d1 knockout (Bsr‐d1KO) lines and the wild type, TP309. Our study revealed that bsr‐d1 mainly regulates the redox state of plant cells, but also affects amino acid and unsaturated fatty acid metabolism. We further found that BSR‐D1 indirectly regulates salicylic acid biosynthesis, metabolism, and signal transduction downstream of the activation of H2O2 signalling in the bsr‐d1‐mediated immune response. Furthermore, we identified a novel peroxidase‐encoding gene, Perox3, as a new BSR‐D1 target gene that reduces resistance to M. oryzae when overexpressed in TP309. These results provide new insights into the bsr‐d1‐mediated blast resistance., BSR‐D1 mainly regulates the redox state of plant cells by directly targeting another new gene, Perox3, but indirectly regulates salicylic acid biosynthesis and signal transduction after H2O2 signalling in the bsr‐d1‐mediated immune response.

In this article, we describe the development of the plant immunity field, starting with efforts to understand the genetic basis for disease resistance, which ∼30 y ago led to the discovery of diverse classes of immune receptors that recognize and respond to infectious microbes. We focus on knowledge gained from studies of the rice XA21 immune receptor that recognizes RaxX (required for activation of XA21 mediated immunity X), a sulfated microbial peptide secreted by the gram-negative bacterium Xanthomonas oryzae pv. oryzae. XA21 is representative of a large class of plant and animal immune receptors that recognize and respond to conserved microbial molecules. We highlight the complexity of this large class of receptors in plants, discuss a possible role for RaxX in Xanthomonas biology, and draw attention to the important role of sulfotyrosine in mediating receptor-ligand interactions.

Plant transcription factors (TFs), such as basic helix-loop-helix (bHLH) and AT-rich zinc-binding proteins (PLATZ), play critical roles in regulating the expression of developmental genes in cereals. We identified the bHLH protein TaPGS1 (T. aestivum Positive Regulator of Grain Size 1) specifically expressed in the seeds at 5-20 days post-anthesis in wheat. TaPGS1 was ectopically overexpressed (OE) in wheat and rice, leading to increased grain weight (up to 13.81% in wheat and 18.55% in rice lines) and grain size. Carbohydrate and total protein levels also increased. Scanning electron microscopy results indicated that the starch granules in the endosperm of TaPGS1 OE wheat and rice lines were smaller and tightly embedded in a proteinaceous matrix. Furthermore, TaPGS1 was bound directly to the E-box motif at the promoter of the PLATZ TF genes TaFl3 and OsFl3 and positively regulated their expression in wheat and rice. In rice, the OsFl3 CRISPR/Cas9 knockout lines showed reduced average thousand-grain weight, grain width, and grain length in rice. Our results reveal that TaPGS1 functions as a valuable trait-associated gene for improving cereal grain yield.

Many pathogenic fungi depend on the development of specialized infection structures called appressoria to invade their hosts and cause disease. Impairing the function of fungal infection structures therefore provides a potential means by which diseases could be prevented. In spite of this extraordinary potential, however, relatively few anti-penetrant drugs have been developed to control fungal diseases, of either plants or animals. In the present study, we report the identification of compounds that act specifically to prevent fungal infection. We found that the organization of septin GTPases, which are essential for appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae, requires very-long-chain fatty acids (VLCFAs), which act as mediators of septin organization at membrane interfaces. VLCFAs promote septin recruitment to curved plasma membranes and depletion of VLCFAs prevents septin assembly and host penetration by M. oryzae. We observed that VLCFA biosynthesis inhibitors not only prevent rice blast disease, but also show effective, broad-spectrum fungicidal activity against a wide range of fungal pathogens of maize, wheat and locusts, without affecting their respective hosts. Our findings reveal a mechanism underlying septin-mediated infection structure formation in fungi and provide a class of fungicides to control diverse diseases of plants and animals.

miRNAs contribute to plant resistance against pathogens. Previously, we found that the function of miR398b in immunity in rice differs from that in Arabidopsis. However, the underlying mechanisms are unclear. In this study, we characterized the mutants of miR398b target genes and demonstrated that multiple superoxide dismutase genes contribute to miR398b‐regulated rice immunity against the blast fungus Magnaporthe oryzae. Out of the four target genes of miR398b, mutations in Cu/Zn‐Superoxidase Dismutase1 (CSD1), CSD2 and Os11g09780 (Superoxide DismutaseX,SODX) led to enhanced resistance to M. oryzae and increased hydrogen peroxide (H2O2) accumulation. By contrast, mutations in Copper Chaperone for Superoxide Dismutase (CCSD) resulted in enhanced susceptibility. Biochemical studies revealed that csd1, csd2 and sodx displayed altered expression of CSDs and other superoxide dismutase (SOD) family members, leading to increased total SOD enzyme activity that positively contributed to higher H2O2 production. By contrast, the ccsd mutant showed CSD protein deletion, resulting in decreased CSD and total SOD enzyme activity. Our results demonstrate the roles of different SODs in miR398b‐regulated resistance to rice blast disease, and uncover an integrative regulatory network in which miR398b boosts total SOD activity to upregulate H2O2 concentration and thereby improve disease resistance.

Map-based cloning of plant disease resistance (R) genes is time-consuming. Here, we reported the isolation of blast R gene Pid4 using comparative transcriptomic profiling and genome-wide sequence analysis. Pid4 encodes a coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) protein and is constitutively expressed at diverse developmental stages in the rice variety Digu. The Pid4 protein is localized in both the nucleus and cytoplasm. Introduction of Pid4 into susceptible rice cultivars confers race-specific resistance to leaf and neck blast. Amino acid sequence comparison and blast resistance spectrum tests showed that Pid4 is a novel R gene, different from the previously reported R genes located in the same gene cluster. A Pid4 Indel marker was developed to facilitate the identification of Pid4 in different rice varieties. We demonstrated that a plant R gene can be quickly isolated using transcriptomic profiling coupled with genome-wide sequence analysis.

Significance Crops carrying broad-spectrum resistance loci provide an effective strategy for controlling infectious disease. Despite their importance, few broad-spectrum resistance loci have been reported, and the underlying mechanisms controlling the trait remain largely unknown. This report describes the identification of a gene, called “bsr-k1,” conferring broad-spectrum resistance and demonstrates that the encoded protein regulates immunity-related genes. Loss of function of BSR-K1 in rice leads to enhanced broad-spectrum resistance to two serious rice diseases with no major penalty on yield. This report provides insights into broad-spectrum resistance and offers an efficient strategy to breeding durably resistant rice., Crops carrying broad-spectrum resistance loci provide an effective strategy for controlling infectious disease because these loci typically confer resistance to diverse races of a pathogen or even multiple species of pathogens. Despite their importance, only a few crop broad-spectrum resistance loci have been reported. Here, we report the identification and characterization of the rice bsr-k1 (broad-spectrum resistance Kitaake-1) mutant, which confers broad-spectrum resistance against Magnaporthe oryzae and Xanthomonas oryzae pv oryzae with no major penalty on key agronomic traits. Map-based cloning reveals that Bsr-k1 encodes a tetratricopeptide repeats (TPRs)-containing protein, which binds to mRNAs of multiple OsPAL (OsPAL1–7) genes and promotes their turnover. Loss of function of the Bsr-k1 gene leads to accumulation of OsPAL1–7 mRNAs in the bsr-k1 mutant. Furthermore, overexpression of OsPAL1 in wild-type rice TP309 confers resistance to M. oryzae, supporting the role of OsPAL1. Our discovery of the bsr-k1 allele constitutes a significant conceptual advancement and provides a valuable tool for breeding broad-spectrum resistant rice.

Broad-spectrum resistance is highly preferred in crop breeding programmes. Previously, we have reported the identification of the broad-spectrum resistance-Digu 1 (bsr-d1) allele from rice Digu. The bsr-d1 allele prevents activation of Bsr-d1 expression by Magnaporthe oryzae infection and degradation of H2 O2 by peroxidases, leading to resistance to M. oryzae. However, it remains unknown whether defence pathways other than H2 O2 burst and peroxidases contribute to the bsr-d1-mediated immunity. Blast resistance was determined in rice leaves by spray and punch inoculations. Target genes of OsMYB30 were identified by one-hybrid assays in yeast and electrophoretic mobility shift assay. Lignin content was measured by phloroglucinol-HCl staining, and acetyl bromide and thioacidolysis methods. Here, we report the involvement of the OsMYB30 gene in bsr-d1-mediated blast resistance. Expression of OsMYB30 was induced during M. oryzae infection or when Bsr-d1 was knocked out or downregulated, as occurs in bsr-d1 plants upon infection. We further found that OsMYB30 bound to and activated the promoters of 4-coumarate:coenzyme A ligase genes (Os4CL3 and Os4CL5) resulting in accumulation of lignin subunits G and S. This action led to obvious thickening of sclerenchyma cells near the epidermis, inhibiting M. oryzae penetration at the early stage of infection. Our study revealed novel components required for bsr-d1-mediated resistance and penetration-dependent immunity, and advanced our understanding of broad-spectrum disease resistance.

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., Using a forward genetic screening approach, this study identifies the PALD gene encoding a protein with three putative tyrosine phosphatase domains as a positive regulator of XA21-mediated immunity. PALD positively regulates XA21 protein levels and interacts with XA21 in vivo. The catalytic cysteine of the putative tyrosine phosphatase domain in PALD plays a critical role in resistance to Xanthomonas oryzae pv. oryzae.

The rice XA21 receptor kinase confers robust resistance to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). A tyrosine-sulfated peptide from Xoo, called RaxX, triggers XA21-mediated immune responses, including the production of ethylene and reactive oxygen species and the induction of defence gene expression. It has not been tested previously whether these responses confer effective resistance to Xoo. Here, we describe a newly established post-inoculation treatment assay that facilitates investigations into the effect of the sulfated RaxX peptide in planta. In this assay, rice plants were inoculated with a virulent strain of Xoo and then treated with the RaxX peptide 2 days after inoculation. We found that post-inoculation treatment of XA21 plants with the sulfated RaxX peptide suppresses the development of Xoo infection in XA21 rice plants. The treated plants display restricted lesion development and reduced bacterial growth. Our findings demonstrate that exogenous application of sulfated RaxX activates XA21-mediated immunity in planta, and provides a potential strategy for the control of bacterial disease in the field.

Additional file 1: Supplementary Methods: Sequencing, genome assembly and construction of pseudomolecule chromosomes, and BAC library construction. Table S1. Genomic libraries included in the Oryza sativa (KitaakeX) genome assembly and their respective assembled sequence coverage levels in the final release. Table S2. PACBIO library statistics for single pass yield of the 42 chips included in the Oryza sativa (KitaakeX) genome assembly and their respective assembled sequence coverage levels. Table S3. Summary statistics of the output of the SuperNova whole genome shotgun assembly prior to integration with the PACBIO assembly. Table S4. Summary statistics of the raw output of the MECAT whole genome shotgun assembly. Figure S1-S12. Syntenic Japonica sequence placements on the Oryza sativa (var. KitaakeX) chromosomes. Each figure shows one chromosome. Table S5. Final summary assembly statistics for chromosome scale assembly Figure S13. Dot plot of BAC clone 119,492 on a region of Chr_02. Figure S14. Dot plot of BAC clone 120,743 on a region of Chr_12. Figure S15. Dot plot of BAC clone 119,503 in a region of Chr_06. Table S6. KitaakeX BAC libraries used for genome assembly and construction of pseudomolecule chromosomes. For Figures S1-S12, plot of the marker placements for each chromosome is shown.

Autophagy is essential for appressorium-mediated plant infection by Magnaporthe oryzae, the causal agent of rice blast disease and a major threat to global food security. The regulatory mechanism of pathogenicity-associated autophagy, however, remains largely unknown. Here, we report the identification and functional characterization of a plausible ortholog of yeast SNT2 in M. oryzae, which we term MoSNT2. Deletion mutants of MoSNT2 are compromised in autophagy homeostasis and display severe defects in autophagy-dependent fungal cell death and pathogenicity. These mutants are also impaired in infection structure development, conidiation, oxidative stress tolerance and cell wall integrity. MoSnt2 recognizes histone H3 acetylation through its PHD1 domain and thereby recruits the histone deacetylase complex, resulting in deacetylation of H3. MoSnt2 binds to promoters of autophagy genes MoATG6, 15, 16, and 22 to regulate their expression. In addition, MoTor controls MoSNT2 expression to regulate MoTor signaling which leads to autophagy and rice infection. Our study provides evidence of a direct link between MoSnt2 and MoTor signaling and defines a novel epigenetic mechanism by which MoSNT2 regulates infection-associated autophagy and plant infection by the rice blast fungus. Abbreviations: M. oryzae: Magnaporthe oryzae; S. cerevisiae: Saccharomyces cerevisiae; F. oxysporum: Fusarium oxysporum; U. maydis: Ustilago maydis; Compl.: complemented strains of ΔMosnt2 expressing MoSNT2-GFP; ATG: autophagy-related; HDAC: histone deacetylase complex; Tor: target of rapamycin kinase; MTOR: mechanistic target of rapamycin kinase in mammals; MoSnt2: DNA binding SaNT domain protein in M. oryzae; MoTor: target of rapamycin kinase in M. oryzae; MoAtg8: autophagy-related protein 8 in M. oryzae; MoHos2: hda one similar protein in M. oryzae; MoeIf4G: eukaryotic translation initiation factor 4 G in M. oryzae; MoRs2: ribosomal protein S2 in M. oryzae; MoRs3: ribosomal protein S3 in M. oryzae; MoIcl1: isocitrate lyase in M. oryzae; MoSet1: histone H3K4 methyltransferase in M. oryzae; Asd4: ascus development 4; Abl1: AMP-activated protein kinase β subunit-like protein; Tig1: TBL1-like gene required for invasive growth; Rpd3: reduced potassium dependency; KAT8: lysine (K) acetyltransferase 8; PHD: plant homeodomain; ELM2: Egl-27 and MTA1 homology 2; GFP: green fluorescent protein; YFP: yellow fluorescent protein; YFP(CTF): C-terminal fragment of YFP; YFP(NTF): N-terminal fragment of YFP; GST: glutathione S-transferase; bp: base pairs; DEGs: differentially expressed genes; CM: complete medium; MM-N: minimum medium minus nitrogen; CFW: calcofluor white; CR: congo red; DAPI: 4ʹ, 6-diamidino-2-phenylindole; BiFC: bimolecular fluorescence complementation; RT: reverse transcription; PCR: polymerase chain reaction; qPCR: quantitative polymerase chain reaction; RNAi: RNA interference; ChIP: chromatin immunoprecipitation

Flexible growth and immune responses in rice Plants that are fighting microbial pathogens often divert resources that could be used for growth into the immune response. For crops, this translates into lower yield when plant immunity is activated. Wang et al. show that, in rice, reversible phosphorylation of a key transcription factor allows the plant to defend against fungal attack when needed but then, within days, reallocate resources back to growth (see the Perspective by Greene and Dong). Thus, both pathogen defense and crop yield can be sustained. Science , this issue p. 1026 ; see also p. 976

Receptor-like cytoplasmic kinases (RLCKs) represent a large family of proteins in plants. However, few RLCKs have been well characterized. Here, we report the functional characterization of four rice RLCKs - OsRLCK57, OsRLCK107, OsRLCK118 and OsRLCK176 from subfamily VII. These OsRLCKs interact with the rice brassinosteroid receptor, OsBRI1 in yeast cell, but not the XA21 immune receptor. Transgenic lines silenced for each of these genes have enlarged leaf angles and are hypersensitive to brassinolide treatment compared to wild type rice. Transgenic plants silenced for OsRLCK57 had significantly fewer tillers and reduced panicle secondary branching, and lines silenced for OsRLCK107 and OsRLCK118 produce fewer seeds. Silencing of these genes decreased Xa21 gene expression and compromised XA21-mediated immunity to Xanthomonas oryzae pv. oryzae. Our study demonstrates that these OsRLCKs negatively regulate BR signalling, while positively regulating immune responses by contributing to the expression of the immune receptor XA21.

Summary Rice blast caused by the fungal pathogen Magnaporthe oryzae is one of the most destructive diseases worldwide. Although the rice–M. oryzae interaction has been studied extensively, the early molecular events that occur in rice before full maturation of the appressorium during M. oryzae invasion are unknown. Here, we report a comparative transcriptomics analysis of the durably resistant rice variety Digu and the susceptible rice variety Lijiangxintuanheigu (LTH) in response to infection by M. oryzae (5, 10 and 20 h post-inoculation, prior to full development of the appressorium). We found that the transcriptional responses differed significantly between these two rice varieties. Gene ontology and pathway analyses revealed that many biological processes, including extracellular recognition and biosynthesis of antioxidants, terpenes and hormones, were specifically activated in Digu shortly after infection. Forty-eight genes encoding receptor kinases (RKs) were significantly differentially regulated by M. oryzae infection in Digu. One of these genes, LOC_Os08g10300, encoding a leucine-rich repeat RK from the LRR VIII-2 subfamily, conferred enhanced resistance to M. oryzae when overexpressed in rice. Our study reveals that a multitude of molecular events occur in the durably resistant rice Digu before the full maturation of the appressorium after M. oryzae infection and that membrane-associated RKs play important roles in the early response.

© 2017 ASPB. The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a singlenucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa ssp japonica), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations.

Among the phosphofructokinase B-type carbohydrate kinase (PCK) family proteins, only few proteins, like the FRUCTOKINASE-LIKE 1 and 2, have been functionally characterized in regulation of chloroplast development. Here, we report the involvement of a PCK protein PFKB1 in chloroplast development by identification of a new rice mutant, revertible early yellowing Kitaake 2 [rey(k2)]. The mutant rey(k2) shows yellow leaf phenotype, reduced photosynthetic pigments, and retarded chloroplast development during early stages of seedlings, but gradually recovered at later stages. The phenotype of rey(k2) is resulted from the disruption of the PFKB1 protein. The Pfkb1 gene is ubiquitously expressed, and its protein is mainly targeted to the chloroplast and, in some cells, to the nucleus. In addition, the PFKB1 protein possesses phosphofructokinase activity in vitro. The rey(k2) mutant affects RNA levels of chloroplast-associated genes. In particular, the nuclear-encoded RNA polymerase (NEP)-dependent genes are expressed at a sustained high level in rey(k2) even after turning green, indicating that PFKB1 is essential for suppressing the expression of NEP-dependent genes. Taken together, our study suggests that PFKB1 functions as a novel regulator indispensable for early chloroplast development, at least partly by regulating chloroplast-associated genes.

Supplemental FiguresxDownload (18.78 MB ) Supplemental FiguresSupplementary Table 1Cell wall compositionof theOsirx10mutantand the wild -type NPBxDownload (.02 MB ) Supplementary Table 1Cell wall compositionof theOsirx10mutantand the wild -type NPB

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitates functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa L. ssp. japonica), which completes its life cycle in 9 weeks. We sequenced 1,504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. Using this mutant population, we identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line with a small segregating population. This result reveals the usefulness of the resource for efficient identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks, enabling rapid functional genomic studies of rice.One-sentence summaryWe have sequenced 1,504 mutant lines generated in the short life cycle rice variety Kitaake (9 weeks) and established a publicly available database, enabling rapid functional genomic studies of rice.

Non-expresser of pathogenesis-related genes 1 (NPR1) is the master regulator of salicylic acid-mediated systemic acquired resistance. Over-expression of Arabidopsis NPR1 and rice NH1 (NPR1 homolog1)/OsNPR1 in rice results in enhanced resistance. While there are four rice NPR1 paralogs in the rice genome, none have been demonstrated to function in disease resistance. To study rice NPR1 paralog 3, we introduced constructs into rice and tested for effects on resistance to infection by Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. While over-expression of NH3 using the maize ubiquitin-1 promoter failed to enhance resistance, introduction of an extra copy of NH3 driven by its own promoter (nNT-NH3) resulted in clear, enhanced resistance. Progeny analysis confirms that the enhanced resistance phenotype, measured by Xoo-induced lesion length, is associated with the NH3 transgene. Bacterial growth curve analysis indicates that bacterial population levels are reduced 10-fold in nNT-NH3 lines compared to control rice lines. The transgenic plants exhibit higher sensitivity to benzothiadiazole (BTH) and 2,6-dichloroisonicotinic acid (INA) treatment as measured by increased cell death. Expression analysis of pathogenesis-related (PR) genes showed that nNT-NH3 plants display greatly enhanced induction of PR genes only after treatment with BTH. Our study demonstrates an alternative method to employ a regulatory protein to enhance plant defence. This approach avoids using undesirable constitutive, high-level expression and may prove to be more practical for engineering resistance.

The rice Xa21 gene, which confers resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), encodes a receptor-like kinase. Few components involved in transducing the Xa21-mediated defense response have yet been identified. Here, we report that XA21 binds to a WRKY transcription factor, called OsWRKY62. The OsWRKY62 gene encodes two splice variants (OsWRKY62.1 and OsWRKY62.2). OsWRKY62.1:smGFP2 and OsWRKY62.2:smGFP2 fusion proteins partially localize to the nucleus. Transgenic plants overexpressing OsWRKY62.1 are compromised in basal defense and Xa21-mediated resistance to Xoo. Furthermore, overexpression of OsWRKY62.1 suppresses the activation of defense-related genes. These results imply that OsWRKY62 functions as a negative regulator of innate immunity in rice, and serves as a critical mediator of both basal and race-specific defense responses.

A sulfated peptide activates a rice immune receptor., Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals.

Summary Rice blast caused by the fungal pathogen M agnaporthe oryzae is one of the most destructive diseases worldwide. Although the rice–M . oryzae interaction has been studied extensively, the early molecular events that occur in rice before full maturation of the appressorium during M . oryzae invasion are unknown. Here, we report a comparative transcriptomics analysis of the durably resistant rice variety Digu and the susceptible rice variety Lijiangxintuanheigu (LTH) in response to infection by M . oryzae (5, 10 and 20 h post‐inoculation, prior to full development of the appressorium). We found that the transcriptional responses differed significantly between these two rice varieties. Gene ontology and pathway analyses revealed that many biological processes, including extracellular recognition and biosynthesis of antioxidants, terpenes and hormones, were specifically activated in Digu shortly after infection. Forty‐eight genes encoding receptor kinases (RKs) were significantly differentially regulated by M . oryzae infection in Digu. One of these genes, LOC _Os08g10300, encoding a leucine‐rich repeat RK from the LRR VIII‐2 subfamily, conferred enhanced resistance to M . oryzae when overexpressed in rice. Our study reveals that a multitude of molecular events occur in the durably resistant rice Digu before the full maturation of the appressorium after M . oryzae infection and that membrane‐associated RKs play important roles in the early response.

Receptor-like cytoplasmic kinases (RLCKs) represent a large family of proteins in plants. However, few RLCKs have been well characterized. Here, we report the functional characterization of four rice RLCKs - OsRLCK57, OsRLCK107, OsRLCK118 and OsRLCK176 from subfamily VII. These OsRLCKs interact with the rice brassinosteroid receptor, OsBRI1 in yeast cell, but not the XA21 immune receptor. Transgenic lines silenced for each of these genes have enlarged leaf angles and are hypersensitive to brassinolide treatment compared to wild type rice. Transgenic plants silenced for OsRLCK57 had significantly fewer tillers and reduced panicle secondary branching, and lines silenced for OsRLCK107 and OsRLCK118 produce fewer seeds. Silencing of these genes decreased Xa21 gene expression and compromised XA21-mediated immunity to Xanthomonas oryzae pv. oryzae. Our study demonstrates that these OsRLCKs negatively regulate BR signalling, while positively regulating immune responses by contributing to the expression of the immune receptor XA21.

Arabidopsis NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance. Over-expression of Arabidopsis NPR1 or the NPR1 homolog 1 (NH1) in rice results in enhanced resistance to the pathogen Xanthomonasoryzae pv. oryzae (Xoo), suggesting the presence of a related defense pathway in rice. We investigated this pathway in rice by identifying proteins that interact with NH1. Here we report the isolation and characterization of a rice cDNA encoding a novel protein, named NRR (for negative regulator of resistance). NRR interacts with NPR1 in the NPR1-interacting domain (NI25) consisting of 25 amino acids. NRR also interacts with NH1; however, NI25 was not sufficient for a strong interaction, indicating a difference between the rice and the Arabidopsis proteins. Silencing of NRR in rice had little effect on resistance to Xoo. When constitutively over-expressed in rice, NRR affected basal resistance, age-related resistance and Xa21-mediated resistance, causing enhanced susceptibility to Xoo. This phenotype was correlated with elevated NRR mRNA and protein levels and increased Xoo growth. Over-expression of NRR suppressed the induction of defense-related genes. NRR:GFP (green fluorescent protein) protein was localized to the nucleus, indicating that NRR may act directly to suppress the activation of defense genes. The fact that NRR compromises Xa21-mediated resistance indicates cross-talk or overlap between NH1- and Xa21-mediated pathways.

*† Summary In dicotyledonous plants broad-spectrum resistance to pathogens is established after the induction of the systemic acquired resistance (SAR) response. In Arabidopsis the NPR1 protein can regulate SAR by interacting with members of the TGA class of basic, leucine-zipper transcription factors to alter pathogenesis-related (PR) gene expression. Overexpression of (At)NPR1 in Arabidopsis enhances resistance to multiple pathogens. Similarly, overexpression of (At)NPR1 in rice enhances resistance to the bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). These results suggest that components of the (At)NPR1-mediated SAR defense response may be conserved between monocots and dicots. To determine whether or not rice TGA factors are involved in disease resistance responses, the effect of altering the function of rice TGA2.1 was analyzed in transgenic plants. Transgenic rice overexpressing an rTGA2.1 mutant, that can no longer bind DNA, and transgenic rice that have the endogenous rTGA2.1 silenced by dsRNA-mediated silencing were generated. Both types of transgenic rice displayed increased tolerance to Xoo, were dwarfed, and had altered accumulation of PR genes. The results presented in this study suggest that wild-type rTGA2.1 has primarily a negative role in rice basal defense responses to bacterial pathogens.

The tolerance of crops to a shortage of boron (B) in the soil varies markedly among species. This variation in tolerance is due, in part, to a species ability to form phloem mobile B-sugar-alcohol complexes (such as B-mannitol or B-sorbitol) which enhance the remobilization of B within the plant. Species lacking the capacity to form B-sugar alcohol complexes are intolerant of even short-term deficits in soil B supply. Here we have genetically engineered rice (Oryza sativa L.) cultivar Taipei 309 (TP309) with the sorbitol-6-phosphate dehydrogenase (S6PDH) gene, a key enzyme for sorbitol production, and determined the effect of this transformation on the physiology of B remobilization. Sorbitol was detected in the S6PDH transgenic plants as well as in vector-transformed plants and wild-type (TP 309) plants, although the concentration of sorbitol in the S6PDH transgenic plants was significantly enhanced. Remobilization of B from mature leaves to flag leaves correlated with increased levels of sorbitol. The presence of sorbitol and detection of B remobilization in the wild-type and vector-transformed plants suggests that rice utilizes an unknown pathway for sorbitol synthesis and may partly explain the relative insensitivity of rice to B deficits when compared to other graminaceous crops.

Summary Rice feeds half the world's population, and rice blast is often a destructive disease that results in significant crop loss. Non-race-specific resistance has been more effective in controlling crop diseases than race-specific resistance because of its broad spectrum and durability. Through a genome-wide association study, we report the identification of a natural allele of a C 2 H 2 -type transcription factor in rice that confers non-race-specific resistance to blast. A survey of 3,000 sequenced rice genomes reveals that this allele exists in 10% of rice, suggesting that this favorable trait has been selected through breeding. This allele causes a single nucleotide change in the promoter of the bsr-d1 gene, which results in reduced expression of the gene through the binding of the repressive MYB transcription factor and, consequently, an inhibition of H 2 O 2 degradation and enhanced disease resistance. Our discovery highlights this novel allele as a strategy for breeding durable resistance in rice.

Background The nonexpressor of pathogenesis-related genes 1, NPR1 (also known as NIM1 and SAI1), is a key regulator of SA-mediated systemic acquired resistance (SAR) in Arabidopsis. In rice, the NPR1 homolog 1 (NH1) interacts with TGA transcriptional regulators and the Negative Regulator of Resistance (NRR) protein to modulate the SAR response. Though five NPR1 homologs (NHs) have been identified in rice, only NH1 and NH3 enhance immunity when overexpressed. To understand why NH1 and NH3, but not NH2, NH4, or NH5, contribute to the rice immune response, we screened TGA transcription factors and NRR-like proteins for interactions specific to NH1 and NH3. We also examined their co-expression patterns using publicly available microarray data. Results We tested five NHs, four NRR homologs (RHs), and 13 rice TGA proteins for pair-wise protein interactions using yeast two-hybrid (Y2H) and split YFP assays. A survey of 331 inter-family interactions revealed a broad, complex protein interaction network. To investigate preferred interaction partners when all three families of proteins were present, we performed a bridged split YFP assay employing YFPN-fused TGA, YFPC-fused RH, and NH proteins without YFP fusions. We found 64 tertiary interactions mediated by NH family members among the 120 sets we examined. In the yeast two-hybrid assay, each NH protein was capable of interacting with most TGA and RH proteins. In the split YFP assay, NH1 was the most prevalent interactor of TGA and RH proteins, NH3 ranked the second, and NH4 ranked the third. Based on their interaction with TGA proteins, NH proteins can be divided into two subfamilies: NH1, NH2, and NH3 in one family and NH4 and NH5 in the other. In addition to evidence of overlap in interaction partners, co-expression analyses of microarray data suggest a correlation between NH1 and NH3 expression patterns, supporting their common role in rice immunity. However, NH3 is very tightly co-expressed with RH1 and RH2, while NH1 is strongly, inversely co-expressed with RH proteins, representing a difference between NH1 and NH3 expression patterns. Conclusions Our genome-wide surveys reveal that each rice NH protein can partner with many rice TGA and RH proteins and that each NH protein prefers specific interaction partners. NH1 and NH3 are capable of interacting strongly with most rice TGA and RH proteins, whereas NH2, NH4, and NH5 have weaker, limited interaction with TGA and RH proteins in rice cells. We have identified rTGA2.1, rTGA2.2, rTGA2.3, rLG2, TGAL2 and TGAL4 proteins as the preferred partners of NH1 and NH3, but not NH2, NH4, or NH5. These TGA proteins may play an important role in NH1- and NH3-mediated immune responses. In contrast, NH4 and NH5 preferentially interact with TGAL5, TGAL7, TGAL8 and TGAL9, which are predicted to be involved in plant development. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-461) contains supplementary material, which is available to authorized users.

*† Summary The Arabidopsis NPR1/NIM1 gene is a key regulator of systemic acquired resistance (SAR). Overexpression of NPR1 leads to enhanced resistance in Arabidopsis. To investigate the role of NPR1 in monocots, we over-expressed the Arabidopsis NPR1 in rice and challenged the transgenic plants with Xanthomonas oryzae pv. oryzae (Xoo), the rice bacterial blight pathogen. The transgenic plants displayed enhanced resistance to Xoo. RNA blot hybridization indicates that enhanced resistance requires expression of NPR1 mRNA above a threshold level in rice. To identify components mediating the resistance controlled by NPR1, we used NPR1 as bait in a yeast two-hybrid screen. We isolated four cDNA clones encoding rice NPR1 interactors (named rTGA2.1, rTGA2.2, rTGA2.3 and rLG2) belonging to the bZIP family. rTGA2.1, rTGA2.2 and rTGA2.3 share 75, 76 and 78% identity with Arabidopsis TGA2, respectively. In contrast, rLG2 shares highest identity (81%) to the maize liguleless (LG2) gene product, which is involved in establishing the leaf blade‐sheath boundary. The interaction of NPR1 with the rice bZIP proteins in yeast was impaired by the npr1-1 and npr1-2 mutations, but not by the nim1-4 mutation. The NPR1‐rTGA2.1 interaction was confirmed by an in vitro pull-down experiment. In gel mobility shift assays, rTGA2.1 binds to the rice RCH10 promoter and to a cis-element required sequencespecifically for salicylic acid responsiveness. This is the first demonstration that the Arabidopsis NPR1 gene can enhance disease resistance in a monocot plant. These results also suggest that monocot and dicot plants share a conserved signal transduction pathway controlling NPR1-mediated resistance.

Over-expression of rice NH1 (NH1ox), the ortholog of Arabidopsis NPR1, confers immunity to bacterial and fungal pathogens and induces the appearance of necrotic lesions due to activation of defense genes at the pre-flowering stage. This lesion-mimic phenotype can be enhanced by the application of benzothiadiazole (BTH). To identify genes regulating these responses, we screened a fast neutron-irradiated NH1ox rice population. We identified one mutant, called snl1 (suppressor of NH1-mediated lesion-mimic 1), which is impaired both in BTH-induced necrotic lesion formation and in the immune response. Using a comparative genome hybridization approach employing rice whole genome tiling array, we identified 11 genes associated with the snl1 phenotype. Transgenic analysis revealed that RNA interference of two of the genes, encoding previously uncharacterized cysteine-rich receptor-like kinases (CRK6 and CRK10), re-created the snl1 phenotype. Elevated expression of CRK10 using an inducible expression system resulted in enhanced immunity. Quantitative PCR revealed that BTH treatment and elevated levels of rice NH1 and its paralog NH3 induced expression of CRK10 and CRK6 RNA. These results indicate that CRK6 and CRK10 are required for the BTH-activated immune response mediated by NH1.

SUMMARY We show that OsSERK2 is a regulator of innate immune signaling mediated by multiple non-RD receptor kinases (RKs) including XA21, XA3, and OsFLS2. OsSerk2-silenced rice lines are impaired in XA21-mediated immunity to Xoo PXO99, XA3-mediated immunity to Xoo PXO86, and OsFLS2-mediated defense responses. Thus, OsSERK2 is broadly involved in PRR-mediated immunity in rice., The rice XA21 immune receptor kinase and the structurally related XA3 receptor confer immunity to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 (rice somatic embryogenesis receptor kinase 2) and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 (OsFLS2). Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast two-hybrid system in a kinase activity-dependent manner. OsSERK2 undergoes bidirectional transphosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. These results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function. Taken together, our findings suggest that the mechanism of OsSERK2-meditated regulation of rice XA21, XA3, and FLS2 differs from that of AtSERK3/BAK1-mediated regulation of Arabidopsis FLS2 and EFR.

Background Despite the importance of wheat as a major staple crop and the negative impact of diseases on its production worldwide, the genetic mechanisms and gene interactions involved in the resistance response in wheat are still poorly understood. The complete sequence of the rice genome has provided an extremely useful parallel road map for genetic and genomics studies in wheat. The recent construction of a defense response interactome in rice has the potential to further enhance the translation of advances in rice to wheat and other grasses. The objective of this study was to determine the degree of conservation in the protein-protein interactions in the rice and wheat defense response interactomes. As entry points we selected proteins that serve as key regulators of the rice defense response: the RAR1/SGT1/HSP90 protein complex, NPR1, XA21, and XB12 (XA21 interacting protein 12). Results Using available wheat sequence databases and phylogenetic analyses we identified and cloned the wheat orthologs of these four rice proteins, including recently duplicated paralogs, and their known direct interactors and tested 86 binary protein interactions using yeast-two-hybrid (Y2H) assays. All interactions between wheat proteins were further tested using in planta bimolecular fluorescence complementation (BiFC). Eighty three percent of the known rice interactions were confirmed when wheat proteins were tested with rice interactors and 76% were confirmed using wheat protein pairs. All interactions in the RAR1/SGT1/ HSP90, NPR1 and XB12 nodes were confirmed for the identified orthologous wheat proteins, whereas only forty four percent of the interactions were confirmed in the interactome node centered on XA21. We hypothesize that this reduction may be associated with a different sub-functionalization history of the multiple duplications that occurred in this gene family after the divergence of the wheat and rice lineages. Conclusions The observed high conservation of interactions between proteins that serve as key regulators of the rice defense response suggests that the existing rice interactome can be used to predict interactions in wheat. Such predictions are less reliable for nodes that have undergone a different history of duplications and sub-functionalization in the two lineages.