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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 underlying mechanisms of host-driven assembly of phyllosphere microbiota remain largely unknown. Here, 4-hydroxycinnamic acid synthesized by the rice plant’s PAL02 in the phenylpropanoid biosynthesis pathway is shown to be the main driver for enrichment of Pseudomonadales bacteria.

Background Balancing the yield, quality and resistance to disease is a daunting challenge in crop breeding due to the negative relationship among these traits. Large-scale genomic landscape analysis of germplasm resources is considered to be an efficient approach to dissect the genetic basis of the complex traits. Central China is one of the main regions where the japonica rice is produced. However, dozens of high-yield rice varieties in this region still exist with low quality or susceptibility to blast disease, severely limiting their application in rice production. Results Here, we re-sequence 200 japonica rice varieties grown in central China over the past 30 years and analyze the genetic structure of these cultivars using 2.4 million polymorphic SNP markers. Genome-wide association mapping and selection scans indicate that strong selection for high-yield and taste quality associated with low-amylose content may have led to the loss of resistance to the rice blast fungus Magnaporthe oryzae . By extensive bioinformatic analyses of yield components, resistance to rice blast, and taste quality, we identify several superior alleles for these traits in the population. Based on this information, we successfully introduce excellent taste quality and blast-resistant alleles into the background of two high-yield cultivars and develop two elite lines, XY99 and JXY1, with excellent taste, high yield, and broad-spectrum of blast resistance. Conclusions This is the first large-scale genomic landscape analysis of japonica rice varieties grown in central China and we demonstrate a balancing of multiple agronomic traits by genomic-based strategy.

Key messagePi65, 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.

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.

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.

The Cucurbitaceae includes important crops such as cucumber, melon, watermelon, squash and pumpkin. However, few genetic and genomic resources are available for plant improvement. Some cucurbit species such as cucumber have a narrow genetic base, which impedes construction of saturated molecular linkage maps. We report herein the development of highly polymorphic simple sequence repeat (SSR) markers originated from whole genome shotgun sequencing and the subsequent construction of a high-density genetic linkage map. This map includes 995 SSRs in seven linkage groups which spans in total 573 cM, and defines ~680 recombination breakpoints with an average of 0.58 cM between two markers. These linkage groups were then assigned to seven corresponding chromosomes using fluorescent in situ hybridization (FISH). FISH assays also revealed a chromosomal inversion between Cucumis subspecies [C. sativus var. sativus L. and var. hardwickii (R.) Alef], which resulted in marker clustering on the genetic map. A quarter of the mapped markers showed relatively high polymorphism levels among 11 inbred lines of cucumber. Among the 995 markers, 49%, 26% and 22% were conserved in melon, watermelon and pumpkin, respectively. This map will facilitate whole genome sequencing, positional cloning, and molecular breeding in cucumber, and enable the integration of knowledge of gene and trait in cucurbits.

Background Rice is a temperature-sensitive crop and its production is severely affected by low temperature in temperate and sub-tropical regions. To understand the genetic basis of cold tolerance in rice, we evaluated the cold tolerance at the seedling stage (CTS) of 295 rice cultivars in the rice diversity panel 1 (RDP1), these cultivars were collected from 82 countries. Results The evaluations revealed that both temperate and tropical japonica rice cultivars are more tolerant to cold stress than indica and AUS cultivars. Using the cold tolerance phenotypes and 44 K SNP chip dataset of RDP1, we performed genome-wide association mapping of quantitative trait loci (QTLs) for CTS. The analysis identified 67 QTLs for CTS that are located on 11 chromosomes. Fifty-six of these QTLs are located in regions without known cold tolerance-related QTLs. Conclusion Our study has provided new information on the genetic architecture of rice cold tolerance and has also identified highly cold tolerant cultivars and CTS-associated SNP markers that will be useful rice improvement.

BackgroundEffective management of rice blast, caused by the fungus Magnaporthe oryzae, requires an understanding of the genetic architecture of the resistance to the disease in rice. Rice resistance varies with M. oryzae strains, and many quantitative trait loci (QTLs) affecting rice blast resistance have been mapped using different strains of M. oryzae from different areas. However, little is known about the genetic architecture of rice resistance against the M. oryzae population in Hunan Province, which is a main rice production area in South China.ResultsIn this study, we used three isolates from Hunan Province and the rice diversity panel 1 to perform a genome-wide association study (GWAS) of blast resistance in rice. A total of 56 QTLs were identified. One of the QTLs is localized with the resistance gene Pik locus which confers resistance to all three isolates. Genomic sequence analysis of the resistant cultivars led to the identification of a new Pik allele, which we named Pikx. Yeast two-hybrid and co-immunoprecipitation assays between AvrPiks and Pikx confirmed that Pikx is a new allele at the Pik locus.ConclusionsOur GWAS has identified many new blast resistance QTLs. The identified new Pik allele Pikx will be useful for breeding cultivars with high resistance to blast in Hunan and other South China provinces. Further research on the relationship between AvrPiks and Pikx will provide new insights into the molecular mechanism of rice resistance to M. oryzae.

Cold tolerance at the bud burst stage (CTB) is a key trait for direct-seeded rice. Although quantitative trait loci (QTL) affecting CTB in rice have been mapped using traditional linkage mapping and genome-wide association study (GWAS) methods, the underlying genes remain unknown. In this study, we evaluated the CTB phenotype of 339 cultivars in the Rice Diversity Panel II (RDP II) collection. GWAS identified four QTLs associated with CTB (qCTBs), distributed on chromosomes 1–3. Among them, qCTB-1-1 overlaps with Osa-miR319b, a known cold tolerance micro RNA gene. The other three qCTBs have not been reported. In addition, we characterised the candidate gene OsRab11C1 for qCTB-1-2 that encodes a Rab protein belonging to the small GTP-binding protein family. Overexpression of OsRab11C1 significantly reduced CTB, while gene knockout elevated CTB as well as cold tolerance at the seedling stage, suggesting that OsRab11C1 negatively regulates rice cold tolerance. Molecular analysis revealed that OsRab11C1 modulates cold tolerance by suppressing the abscisic acid signalling pathway and proline biosynthesis. Using RDP II and GWAS, we identified four qCTBs that are involved in CTB and determined the function of the candidate gene OsRab11C1 in cold tolerance. Our results demonstrate that OsRab11C1 is a negative regulator of cold tolerance and knocking out of the gene by genome-editing may provide enhanced cold tolerance in rice.

BackgroundRice tiller number (TN) is one of the most important components associated with rice grain yield. Around one hundred rice TN genes have been identified, but dissecting the genetic architecture of rice TN variations remains difficult because of its complex trait and control by both major genes and quantitative trait loci (QTLs).ResultsIn this study, we used a subset of the rice diversity population II (S-RDP-II), genotyped with 700,000 single nucleotide polymorphisms (SNPs), to identify the loci associated with tiller number variations (LATNs) through a genome-wide association study (GWAS). The analysis revealed that 23 LATNs are significantly associated with TN variations. Among the 23 LATNs, eight are co-localized with previously cloned TN genes, and the remaining 15 LATNs are novel. DNA sequence analysis of the 15 novel LATNs led to the identification of five candidate genes using the accessions with extreme TN phenotypes. Genetic variations in two of the genes are mainly located in the promoter regions. qRT-PCR analysis showed that the expression levels of these two genes are also closely associated with TN variations.ConclusionsWe identified 15 novel LATNs that contribute significantly to the genetic variation of rice TN. Of these 15, the five identified TN-associated candidate genes will enhance our understanding of rice tillering and can be used as molecular markers for improving rice yield.