OsEIL2 balances rice immune responses against (hemi)biotrophic and necrotrophic pathogens via the salicylic acid and jasmonic acid synergism.

Plants typically activate distinct defense pathways against various pathogens. Heightened resistance to one pathogen often coincides with increased susceptibility to another pathogen. However, the underlying molecular basis of this antagonistic response remains unclear. Here, we demonstrate that mutants defective in the transcription factor ETHYLENE-INSENSITIVE 3-LIKE 2 (OsEIL2) exhibited enhanced resistance to the biotrophic bacterial pathogen Xanthomonas oryzae pv oryzae and to the hemibiotrophic fungal pathogen Magnaporthe oryzae, but enhanced susceptibility to the necrotrophic fungal pathogen Rhizoctonia solani. Furthermore, necrotroph-induced OsEIL2 binds to the promoter of OsWRKY67 with high affinity, leading to the upregulation of salicylic acid (SA)/jasmonic acid (JA) pathway genes and increased SA/JA levels, ultimately resulting in enhanced resistance. However, biotroph- and hemibiotroph-induced OsEIL2 targets OsERF083, resulting in the inhibition of SA/JA pathway genes and decreased SA/JA levels, ultimately leading to reduced resistance. Our findings unveil a previously uncharacterized defense mechanism wherein two distinct transcriptional regulatory modules differentially mediate immunity against pathogens with different lifestyles through the transcriptional reprogramming of phytohormone pathway genes.

Identification of two novel rice S genes through combination of association and transcription analyses with gene-editing technology.

Traditional rice blast resistance breeding largely depends on utilizing typical resistance (R) genes. However, the lack of durable R genes has prompted rice breeders to find new resistance resources. Susceptibility (S) genes are potential new targets for resistance genetic engineering using genome-editing technologies, but identifying them is still challenging. Here, through the integration of genome-wide association study (GWAS) and transcriptional analysis, we identified two genes, RNG1 and RNG3, whose polymorphisms in 3'-untranslated regions (3'-UTR) affected their expression variations. These polymorphisms could serve as molecular markers to identify rice blast-resistant accessions. Editing the 3'-UTRs using CRISPR/Cas9 technology affected the expression levels of two genes, which were positively associated with rice blast susceptibility. Knocking out either RNG1 or RNG3 in rice enhanced the rice blast and bacterial blight resistance, without impacting critical agronomic traits. RNG1 and RNG3 have two major genotypes in diverse rice germplasms. The frequency of the resistance genotype of these two genes significantly increased from landrace rice to modern cultivars. The obvious selective sweep flanking RNG3 suggested it has been artificially selected in modern rice breeding. These results provide new targets for S gene identification and open avenues for developing novel rice blast-resistant materials.

Identification of a Rice Leaf Width Gene Narrow Leaf 22 (NAL22) through Genome-Wide Association Study and Gene Editing Technology.

Rice leaf width (RLW) is a crucial determinant of photosynthetic area. Despite the discovery of several genes controlling RLW, the underlying genetic architecture remains unclear. In order to better understand RLW, this study conducted a genome-wide association study (GWAS) on 351 accessions from the rice diversity population II (RDP-II). The results revealed 12 loci associated with leaf width (LALW). In LALW4, we identified one gene, Narrow Leaf 22 (NAL22), whose polymorphisms and expression levels were associated with RLW variation. Knocking out this gene in Zhonghua11, using CRISPR/Cas9 gene editing technology, resulted in a short and narrow leaf phenotype. However, seed width remained unchanged. Additionally, we discovered that the vein width and expression levels of genes associated with cell division were suppressed in nal22 mutants. Gibberellin (GA) was also found to negatively regulate NAL22 expression and impact RLW. In summary, we dissected the genetic architecture of RLW and identified a gene, NAL22, which provides new loci for further RLW studies and a target gene for leaf shape design in modern rice breeding.

Genome-Wide Association Study Identifies a Plant-Height-Associated Gene OsPG3 in a Population of Commercial Rice Varieties.

Plant height is one of the most crucial components of plant structure. However, due to its complexity, the genetic architecture of rice plant height has not been fully elucidated. In this study, we performed a genome-wide association study (GWAS) to determine rice plant height using 178 commercial rice varieties and identified 37 loci associated with rice plant height (LAPH). Among these loci, in LAPH2, we identified a polygalacturonase gene, OsPG3, which was genetically and functionally associated with rice plant height. The rice plant exhibits a super dwarf phenotype when the knockout of the OsPG3 gene occurs via CRISPR-Cas9 gene-editing technology. RNA-Seq analysis indicated that OsPG3 modulates the expression of genes involved in phytohormone metabolism and cell-wall-biosynthesis pathways. Our findings suggest that OsPG3 plays a vital role in controlling rice plant height by regulating cell wall biosynthesis. Given that rice architecture is one of the most critical phenotypes in rice breeding, OsPG3 has potential in rice's molecular design breeding toward an ideal plant height.

Cloning and functional analysis of the novel rice blast resistance gene Pi65 in japonica rice.

KEY MESSAGE: Pi65, a leucine-rich repeat receptor-like kinase (LRR-RLK) domain cloned from Oryza sativa japonica, is a novel rice blast disease resistance gene. Rice blast seriously threatens rice production worldwide. Utilizing the rice blast resistance gene to breed rice blast-resistant varieties is one of the best ways to control rice blast disease. Using a map-based cloning strategy, we cloned a novel rice blast resistance gene, Pi65, from the resistant variety GangYu129 (abbreviated GY129, Oryza sativa japonica). Overexpression of Pi65 in the susceptible variety LiaoXing1 (abbreviated LX1, Oryza sativa japonica) enhanced rice blast resistance, while knockout of Pi65 in GY129 resulted in susceptibility to rice blast disease. Pi65 encodes two transmembrane domains, with 15 LRR domains and one serine/threonine protein kinase catalytic domain, conferring resistance to isolates of Magnaporthe oryzae (abbreviated M. oryzae) collected from Northeast China. There were sixteen amino acid differences between the Pi65 resistance and susceptible alleles. Compared with the Pi65-resistant allele, the susceptible allele exhibited one LRR domain deletion. Pi65 was constitutively expressed in whole plants, and it could be induced in the early stage of M. oryzae infection. Transcriptome analysis revealed that numerous genes associated with disease resistance were specifically upregulated in GY129 24 h post inoculation (HPI); in contrast, photosynthesis and carbohydrate metabolism-related genes were particularly downregulated at 24 HPI, demonstrating that disease resistance-associated genes were activated in GY129 (carrying Pi65) after rice blast fungal infection and that cellular basal metabolism and energy metabolism were inhibited simultaneously. Our study provides genetic resources for improving rice blast resistance and enriches the study of rice blast resistance mechanisms.

The rice RNase P protein subunit Rpp30 confers broad-spectrum resistance to fungal and bacterial pathogens.

RNase P functions either as a catalytic ribonucleoprotein (RNP) or as an RNA-free polypeptide to catalyse RNA processing, primarily tRNA 5' maturation. To the growing evidence of non-canonical roles for RNase P RNP subunits including regulation of chromatin structure and function, we add here a role for the rice RNase P Rpp30 in innate immunity. This protein (encoded by LOC_Os11g01074) was uncovered as the top hit in yeast two-hybrid assays performed with the rice histone deacetylase HDT701 as bait. We showed that HDT701 and OsRpp30 are localized to the rice nucleus, OsRpp30 expression increased post-infection by Pyricularia oryzae (syn. Magnaporthe oryzae), and OsRpp30 deacetylation coincided with HDT701 overexpression in vivo. Overexpression of OsRpp30 in transgenic rice increased expression of defence genes and generation of reactive oxygen species after pathogen-associated molecular pattern elicitor treatment, outcomes that culminated in resistance to a fungal (P. oryzae) and a bacterial (Xanthomonas oryzae pv. oryzae) pathogen. Knockout of OsRpp30 yielded the opposite phenotypes. Moreover, HA-tagged OsRpp30 co-purified with RNase P pre-tRNA cleavage activity. Interestingly, OsRpp30 is conserved in grass crops, including a near-identical C-terminal tail that is essential for HDT701 binding and defence regulation. Overall, our results suggest that OsRpp30 plays an important role in rice immune response to pathogens and provides a new approach to generate broad-spectrum disease-resistant rice cultivars.

Genome-wide association study identifies an NLR gene that confers partial resistance to Magnaporthe oryzae in rice.

Because of the frequent breakdown of major resistance (R) genes, identification of new partial R genes against rice blast disease is an important goal of rice breeding. In this study, we used a core collection of the Rice Diversity Panel II (C-RDP-II), which contains 584 rice accessions and are genotyped with 700 000 single-nucleotide polymorphism (SNP) markers. The C-RDP-II accessions were inoculated with three blast strains collected from different rice-growing regions in China. Genome-wide association study identified 27 loci associated with rice blast resistance (LABRs). Among them, 22 LABRs were not associated with any known blast R genes or QTLs. Interestingly, a nucleotide-binding site leucine-rich repeat (NLR) gene cluster exists in the LABR12 region on chromosome 4. One of the NLR genes is highly conserved in multiple partially resistant rice cultivars, and its expression is significantly up-regulated at the early stages of rice blast infection. Knockout of this gene via CRISPR-Cas9 in transgenic plants partially reduced blast resistance to four blast strains. The identification of this new non-strain specific partial R gene, tentatively named rice blast Partial Resistance gene 1 (PiPR1), provides genetic material that will be useful for understanding the partial resistance mechanism and for breeding durably resistant cultivars against blast disease of rice.

Proteome-Wide Analyses Provide New Insights into the Compatible Interaction of Rice with the Root-Knot Nematode Meloidogyne graminicola.

The root-knot nematode Meloidogyne graminicola is an important pathogen in rice, causing huge yield losses annually worldwide. Details of the interaction between rice and M. graminicola and the resistance genes in rice still remain unclear. In this study, proteome-wide analyses of the compatible interaction of the japonica rice cultivar "Nipponbare" (NPB) with M. graminicola were performed. In total, 6072 proteins were identified in NPB roots with and without infection of M. graminicola by label-free quantitative mass spectrometry. Of these, 513 specifically or significantly differentially expressed proteins were identified to be uniquely caused by nematode infection. Among these unique proteins, 99 proteins were enriched on seven Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. By comparison of protein expression and gene transcription, LOC_Os01g06600 (ACX, a glutaryl-CoA dehydrogenase), LOC_Os09g23560 (CAD, a cinnamyl-alcohol dehydrogenase), LOC_Os03g39850 (GST, a glutathione S-transferase) and LOC_Os11g11960 (RPM1, a disease resistance protein) on the alpha-linolenic acid metabolism, phenylpropanoid biosynthesis, glutathione metabolism and plant-pathogen interaction pathways, respectively, were all associated with disease defense and identified to be significantly down-regulated in the compatible interaction of NPB with nematodes, while the corresponding genes were remarkably up-regulated in the roots of a resistant rice accession "Khao Pahk Maw" with infection of nematodes. These four genes likely played important roles in the compatible interaction of rice with M. graminicola. Conversely, these disease defense-related genes were hypothesized to be likely involved in the resistance of resistant rice lines to this nematode. The proteome-wide analyses provided many new insights into the interaction of rice with M. graminicola.

Genotyping-by-Sequencing-Based Genetic Analysis of African Rice Cultivars and Association Mapping of Blast Resistance Genes Against Magnaporthe oryzae Populations in Africa.

Understanding the genetic diversity of rice germplasm is important for the sustainable use of genetic materials in rice breeding and production. Africa is rich in rice genetic resources that can be utilized to boost rice productivity on the continent. A major constraint to rice production in Africa is rice blast, caused by the hemibiotrophic fungal pathogen Magnaporthe oryzae. In this report, we present the results of a genotyping-by-sequencing (GBS)-based diversity analysis of 190 African rice cultivars and an association mapping of blast resistance (R) genes and quantitative trait loci (QTLs). The 190 African cultivars were clustered into three groups based on the 184K single nucleotide polymorphisms generated by GBS. We inoculated the rice cultivars with six African M. oryzae isolates. Association mapping identified 25 genomic regions associated with blast resistance (RABRs) in the rice genome. Moreover, PCR analysis indicated that RABR_23 is associated with the Pi-ta gene on chromosome 12. Our study demonstrates that the combination of GBS-based genetic diversity population analysis and association mapping is effective in identifying rice blast R genes/QTLs that contribute to resistance against African populations of M. oryzae. The identified markers linked to the RABRs and 14 highly resistant cultivars in this study will be useful for rice breeding in Africa.

Genome-Wide Association Mapping of Rice Resistance Genes Against Magnaporthe oryzae Isolates from Four African Countries.

Rice blast disease is emerging as a major constraint to rice production in Africa. Although a traditional gene-tagging strategy using biparental crosses can effectively identify resistance (R) genes or quantitative trait loci (QTL) against Magnaporthe oryzae, the mapping procedure required is time consuming and requires many populations to investigate the genetics of resistance. In this report, we conducted a genome-wide association study (GWAS) to rapidly map rice genes conferring resistance against eight M. oryzae isolates from four African countries. We inoculated 162 rice cultivars, which were part of the rice diversity panel 1 (RDP1) and were previously genotyped with the 44,000 single-nucleotide polymorphism (SNP) chip, with the eight isolates. The GWAS identified 31 genomic regions associated with blast resistance (RABR) in the rice genome. In addition, we used polymerase chain reaction analysis to confirm the association between the Pish gene and a major RABR on chromosome 1 that was associated with resistance to four M. oryzae isolates. Our study has demonstrated the power of GWAS for the rapid identification of rice blast R or QTL genes that are effective against African populations of M. oryzae. The identified SNP markers associated with RABR can be used in breeding for resistance against rice blast in Africa.

Comprehensive profiling of the rice ubiquitome reveals the significance of lysine ubiquitination in young leaves.

Protein ubiquitination is a major post-translational modification that regulates development, apoptosis, responses to environmental cues, and other processes in eukaryotes. Although several ubiquitinated proteins have been identified in rice, large-scale profiling of the rice ubiquitome has not been reported because of limitations in the current analytical methods. Here, we report the first rice ubiquitome, determined by combining highly sensitive immune affinity purification and high resolution LC-MS/MS. We identified 861 di-Gly-Lys-containing peptides in 464 proteins in rice leaf cells. Bioinformatic analyses of the ubiquitome identified a variety of cellular functions and diverse subcellular localizations for the ubiquitinated proteins, and also revealed seven putative ubiquitination motifs in rice. Proteins related to binding and catalytic activity were predicted to be the preferential targets of lysine ubiquitination. A protein interaction network and KEGG analysis indicated that a wide range of signaling and metabolic pathways are modulated by protein ubiquitination in rice. Our results demonstrate the usefulness of the significantly improved method for assaying proteome-wide ubiquitination in plants. The identification of the 464 ubiquitinated proteins in rice leaves provides a foundation for the analysis of the physiological roles of these ubiquitination-related proteins.

Microbiome homeostasis on rice leaves is regulated by a precursor molecule of lignin biosynthesis.

In terrestrial ecosystems, plant leaves provide the largest biological habitat for highly diverse microbial communities, known as the phyllosphere microbiota. However, the underlying mechanisms of host-driven assembly of these ubiquitous communities remain largely elusive. Here, we conduct a large-scale and in-depth assessment of the rice phyllosphere microbiome aimed at identifying specific host-microbe links. A genome-wide association study reveals a strong association between the plant genotype and members of four bacterial orders, Pseudomonadales, Burkholderiales, Enterobacterales and Xanthomonadales. Some of the associations are specific to a distinct host genomic locus, pathway or even gene. The compound 4-hydroxycinnamic acid (4-HCA) is identified as the main driver for enrichment of bacteria belonging to Pseudomonadales. 4-HCA can be synthesized by the host plant's OsPAL02 from the phenylpropanoid biosynthesis pathway. A knockout mutant of OsPAL02 results in reduced Pseudomonadales abundance, dysbiosis of the phyllosphere microbiota and consequently higher susceptibility of rice plants to disease. Our study provides a direct link between a specific plant metabolite and rice phyllosphere homeostasis opening possibilities for new breeding strategies.

The F-box protein OsFBX156 positively regulates rice defence against the blast fungus Magnaporthe oryzae by mediating ubiquitination-dependent degradation of OsHSP71.1.

F-box protein is a subunit of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which plays a critical role in regulating different pathways in plant immunity. In this study, we identified the rice (Oryza sativa) F-box protein OsFBX156, which targets the heat shock protein 70 (OsHSP71.1) to regulate resistance to the rice blast fungus Magnaporthe oryzae. Overexpression of OsFBX156 or knockout of OsHSP71.1 in rice resulted in the elevation of pathogenesis-related (PR) genes and an induction burst of reactive oxygen species (ROS) after flg22 and chitin treatments, thereby enhancing resistance to M. oryzae. Furthermore, OsFBX156 can promote the degradation of OsHSP71.1 through the 26S proteasome pathway. This study sheds lights on a novel mechanism wherein the F-box protein OsFBX156 targets OsHSP71.1 for degradation to promote ROS production and PR gene expression, thereby positively regulating rice innate immunity.

Antagonistic control of rice immunity against distinct pathogens by the two transcription modules via salicylic acid and jasmonic acid pathways.

Although the antagonistic effects of host resistance against biotrophic and necrotrophic pathogens have been documented in various plants, the underlying mechanisms are unknown. Here, we investigated the antagonistic resistance mediated by the transcription factor ETHYLENE-INSENSITIVE3-LIKE 3 (OsEIL3) in rice. The Oseil3 mutant confers enhanced resistance to the necrotroph Rhizoctonia solani but greater susceptibility to the hemibiotroph Magnaporthe oryzae and biotroph Xanthomonas oryzae pv. oryzae. OsEIL3 directly activates OsERF040 transcription while repressing OsWRKY28 transcription. The infection of R. solani and M. oryzae or Xoo influences the extent of binding of OsEIL3 to OsWRKY28 and OsERF040 promoters, resulting in the repression or activation of both salicylic acid (SA)- and jasmonic acid (JA)-dependent pathways and enhanced susceptibility or resistance, respectively. These results demonstrate that the distinct effects of plant immunity to different pathogen types are determined by two transcription factor modules that control transcriptional reprogramming and the SA and JA pathways.

ABCC Transporter Gene MoABC-R1 Is Associated with Pyraclostrobin Tolerance in Magnaporthe oryzae.

Rice blast is a worldwide fungal disease that poses a threat to food security. Fungicide treatment is one of the most effective methods to control rice blast disease. However, the emergence of fungicide tolerance hampers the control efforts against rice blast. ATP-binding cassette (ABC) transporters have been found to be crucial in multidrug tolerance in various phytopathogenic fungi. This study investigated the association between polymorphisms in 50 ABC transporters and pyraclostrobin sensitivity in 90 strains of rice blast fungus. As a result, we identified MoABC-R1, a gene associated with fungicide tolerance. MoABC-R1 belongs to the ABCC-type transporter families. Deletion mutants of MoABC-R1, abc-r1, exhibited high sensitivity to pyraclostrobin at the concentration of 0.01 µg/mL. Furthermore, the pathogenicity of abc-r1 was significantly diminished. These findings indicate that MoABC-R1 not only plays a pivotal role in fungicide tolerance but also regulates the pathogenicity of rice blast. Interestingly, the combination of MoABC-R1 deletion with fungicide treatment resulted in a three-fold increase in control efficiency against rice blast. This discovery highlights MoABC-R1 as a potential target gene for the management of rice blast.

Molecular mapping of the blast resistance genes Pi2-1 and Pi51(t) in the durably resistant rice 'Tianjingyeshengdao'.

Tianjingyeshengdao' (TY) is a rice cultivar with durable resistance to populations of Magnaporthe oryzae (the causal agent of blast) in China. To understand the genetic basis of its resistance to blast, we developed a population of recombinant inbred lines from a cross between TY and the highly susceptible 'CO39' for gene mapping analysis. In total, 22 quantitative trait loci (QTLs) controlling rice blast resistance were identified on chromosomes 1, 3, 4, 5, 6, 9, 11, and 12 from the evaluation of four disease parameters in both greenhouse and blast nursery conditions. Among these QTLs, 19 were contributed by TY and three by CO39. Two QTL clusters on chromosome 6 and 12 were named Pi2-1 and Pi51(t), respectively. Pi2-1 was detected under both growth chamber and natural blast nursery conditions, and explained 31.24 to 59.73% of the phenotypic variation. Pi51(t) was only detected in the natural blast nursery and explained 3.67 to 10.37% of the phenotypic variation. Our results demonstrate that the durable resistance in TY is controlled by two major and seven minor genes. Identification of the markers linked to both Pi2-1 and Pi51(t) in this study should be useful for marker-aided selection in rice breeding programs as well as for molecular cloning of the identified resistance genes.

Genome-wide association study of rice leaf metabolites and volatiles.

Metabolites and volatiles in rice (Oryza sativa) are closely related to their development, biotic and abiotic stresses. To further analyze the biosynthetic pathways and regulatory networks of metabolites and volatiles in O. sativa, a genome-wide association study was conducted using 207 diverse accessions with single-nucleotides polymorphisms. In this study, 343 metabolites and 24 volatiles were detected, and strong genetic associations were identified for some important compounds, especially stigmasterol, α-Tocopherol, isoleucine, methyl palmitoleate, naringin and cymene. Additionally, some compounds were significantly associated with important phenotypes, such as small brown planthopper repellent, biomass, and plant height. This research can promote the understanding of biosynthetic pathways and regulatory networks of metabolites and volatiles in rice.

Recovery of metagenome-assembled genomes from the phyllosphere of 110 rice genotypes.

The plant microbiota plays crucial roles in sustaining plant health and productivity. Advancing plant microbiome research and designing sustainable practices for agriculture requires in-depth assessments of microorganisms associated with different host plants; however, there is little information on functional aspects of many microorganisms of interest. Therefore, we enriched microorganisms from the phyllosphere of 110 rice genotypes and subjected them to shotgun metagenomic sequencing to reconstruct bacterial genomes from the obtained datasets. The approach yielded a total of 1.34 terabases of shotgun-sequenced metagenomic data. By separately recovering bacterial genomes from each of the 110 rice genotypes, we recovered 569 non-redundant metagenome-assembled genomes (MAGs) with a completeness higher than 50% and contaminations less than 10%. The MAGs were primarily assigned to Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia. The presented data provides an extended basis for microbiome analyses of plant-associated microorganisms. It is complemented by detailed metadata to facilitate implementations in ecological studies, biotechnological mining approaches, and comparative assessments with genomes or MAGs from other studies.

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.