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.

Genetic mapping and molecular marker development for Pi65(t), a novel broad-spectrum resistance gene to rice blast using next-generation sequencing.

KEY MESSAGE: A novel R gene was mapped to a locus on chromosome 11 from 30.42 to 30.85 Mb, which was proven to be efficient in the improvement of rice blast resistance. Rice blast is a devastating fungal disease worldwide. The use of blast resistance (R) genes is the most important approach to control the disease in rice breeding. In the present study, we finely mapped a novel resistance gene Pi65(t), conferring a broad-spectrum resistance to the fungus Magnaporthe oryzae, using bulked segregant analysis in combination with next-generation sequencing technology. Segregation in a doubled haploid (DH) population and a BC1F2 population suggested that resistance to blast in Gangyu129 was likely conferred by a single dominant gene, designated Pi65(t); it was located on chromosome 11 from 30.20 to 31.20 Mb using next-generation sequencing. After screening recombinants with newly developed molecular markers, the region was narrowed down to 0.43 Mb, flanked by SNP-2 and SNP-8 at the physical location from 30.42 to 30.85 Mb based on the Nipponbare reference database in build 5. Using the software QTL IciMapping, Pi65(t) was further mapped to a locus between InDel-1 and SNP-4 with genetic distances of 0.11 and 0.98 cM, respectively. Within this region, 4 predicted R genes were found with nucleotide binding site and leucine-rich repeat (NBS-LRR) domains. We developed molecular markers to genotype 305 DH lines and found that InDel-1 was closely linked with Pi65(t). Using InDel-1, a new rice variety Chuangxin1 containing Pi65(t) was developed, and it is highly resistant to rice blast and produces a high yield in Liaoning province of China. This indicated that Pi65(t) could play a key role in the improvement of rice blast resistance.

Influence of planting methods and organic amendments on rice yield and bacterial communities in the rhizosphere soil.

A comprehensive understanding of rice cultivation techniques and organic amendments affecting soil quality, enzyme activities and bacterial community structure is crucial. We investigated two planting methods (direct seeding and transplanting) of paddy rice (Oryza sativa) and organic amendments with rice straw and biochar on crop yield and soil biological and physicochemical properties. Rhizosphere bacterial communities at the maturity stage of rice growth were characterized through high-throughput 16S rRNA sequencing. Soil biochemical properties and enzyme activity levels were analyzed. Grain yield of paddy rice with transplanting increased 10.6% more than that with direct seeding. The application of rice straw increased grain yield by 7.1 and 8.2%, more than with biochar and the control, respectively. Compared to biochar and the control, the application of rice straw significantly increased sucrase, cellulase, protease, organic carbon, available phosphorus, nitrate, and ammonium. The application of biochar increased microbial biomass nitrogen and carbon, urease, pH, available nitrogen, and available potassium compared to the application of rice straw and the control. Principal coordinate analysis and dissimilarity distances confirmed significant differences among the microbial communities associated with planting methods and organic amendments. Bacteroidetes, Nitrospirae, Firmicutes, and Gemmatimonadetes abundance increased with rice straw relative to biochar and the control. The biochar addition was associated with significant increases in Chloroflexi, Patescibacteria, Proteobacteria, and Actinobacteria abundance. Pearson's correlation analyzes showed that Chloroflexi, Bacteroidetes and Nitrospirae abundance was positively correlated with grain yield. The relative abundance of these bacteria in soil may be beneficial for improving grain yield. These results suggest that planting methods and organic amendments impact soil biochemical characteristics, enzyme activity levels, and microbial community composition.

QTL Detection for Rice Grain Length and Fine Mapping of a Novel Locus qGL6.1.

BACKGROUND: Grain length (GL) that is directly associated with appearance quality is a key target of selection in rice breeding. Although abundant quantitative trait locus (QTL) associated with GL have been identified, it was still relatively weak to identify QTL for GL from japonica genetic background, as the shortage of japonica germplasms with long grains. We performed QTLs analysis for GL using a recombinant inbred lines (RILs) population derived from the cross between japonica variety GY8 (short grains) and LX1 (long grains) in four environments. RESULTS: A total of 197 RILs were genotyped with 285 polymorphic SNP markers. Three QTLs qGL5.3, qGL6.1 and qGL11 were detected to control GL by individual environmental analyses and multi-environment joint analysis. Of these, a major-effect and stable QTL qGL6.1 was identified to be a novel QTL, and its LX1 allele had a positive effect on GL. For fine-mapping qGL6.1, a BC1F2 population consisting of 2,487 individuals was developed from a backcross between GY8 and R176, one line with long grain. Eight key informative recombinants were identified by nine kompetitive allele specific PCR (KASP) markers. By analyzing key recombinants, the qGL6.1 locus was narrowed down to a 40.41 kb genomic interval on chromosome 6. One candidate gene LOC_Os06g43304.1 encoding cytochrome P450 (CYP71D55) was finally selected based on the difference in the transcriptional expression and variations in its upstream and downstream region. CONCLUSIONS: Three QTLs qGL5.3, qGL6.1 and qGL11 were identified to control grain length in rice. One novel QTL qGL6.1 was fine mapped within 40.41 kb region, and LOC_Os06g43304.1 encoding cytochrome P450 (CYP71D55) may be its candidate gene. We propose that the further cloning of the qGL6.1 will facilitate improving appearance quality in japonica varieties.

Soil bacterial community as impacted by addition of rice straw and biochar.

The application of straw and biochar can effectively improve soil quality, but whether such application impacts paddy soil bacterial community development remains to be clarified. Herein, the impacts of three different field amendment strategies were assessed including control (CK) treatment, rice straw (RS) application (9000 kg ha-1), and biochar (BC) application (3150 kg ha-1). Soil samples were collected at five different stages of rice growth, and the bacterial communities therein were characterized via high-throughput 16S rDNA sequencing. The results of these analyses revealed that soil bacterial communities were dominated by three microbial groups (Chloroflexi, Proteobacteria and Acidobacteria). Compared with the CK samples, Chloroflexi, Actinobacteria, Nitrospirae and Gemmatimonadetes levels were dominated phyla in the RS treatment, and Acidobacteria, Actinobacteria, Nitrospirae and Patescibacteria were dominated phyla in the BC treatment. Compared with the RS samples, Chloroflexi, Acidobacteria, Actinobacteria, and Verrucomicrobia levels were increased, however, Proteobacteria, Gemmatimonadetes, Nitrospirae, and Firmicute levels were decreased in the BC samples. Rhizosphere soil bacterial diversity rose significantly following RS and BC amendment, and principal component analyses confirmed that there were significant differences in soil bacterial community composition among treatment groups when comparing all stages of rice growth other than the ripening stage. Relative to the CK treatment, Gemmatimonadaceae, Sphingomonadaceae, Thiovulaceae, Burkholderiaceae, and Clostridiaceae-1 families were dominant following the RS application, while Thiovulaceae and uncultured-bacterium-o-C0119 were dominant following the BC application. These findings suggest that RS and BC application can improve microbial diversity and richness in paddy rice soil in Northeast China.

Research and Development Strategies for Hybrid japonica Rice.

The utilization of heterosis has resulted in significant breakthroughs in rice breeding. However, the development of hybrid japonica has been slow in comparison with that of hybrid indica. The present review explores the history and current status of hybrid japonica breeding. With the creation of japonica cytoplasmic male sterility and photo-thermo-sensitive genic male sterile lines, both three-line and two-line systems of hybrid rice have been created, and a series of hybrid japonica rice varieties have been developed and cultivated widely. At the same time, some progress has been made in genetic research of molecular mechanism for heterosis and QTL mapping for traits such as fertility, stigma exposure and flower time. In addition, genomics and transcriptome have been widely used in the research of hybrid rice, which provides a strong support for its development. Although the research on hybrid japonica has made many advances, there are still some restrictive problems. Based on the research and production of hybrid japonica rice, the prospect and development strategies of hybrid japonica rice are analyzed.

iTRAQ proteomics reveals the regulatory response to Magnaporthe oryzae in durable resistant vs. susceptible rice genotypes.

Rice blast disease caused by Magnaporthe oryzae (M. oryzae) is one of the most serious diseases. Although previous research using two-dimensional gel-based proteomics to assess the proteins related to the rice blast resistance had been done, few proteins were identified. Here, we used the iTRAQ method to detect the differentially expressed proteins (DEPs) in the durable resistant rice variety Gangyuan8 (GY8) and the susceptible rice variety Lijiangxintuanheigu (LTH) in response to M. oryzae invasion, and then transcriptome sequencing was used to assist analysis A total of 193 and 672 DEPs were specifically identified in GY8 and LTH, respectively, with only 46 similarly expressed DEPs being shared by GY8 and LTH.39 DEPs involved in plant-pathogen interaction, plant hormone signal transduction, fatty acid metabolism and peroxisome biosynthesis were significantly different between compatible interaction (LTH) and incompatible interaction (GY8). Some proteins participated in peroxide signal transduction and biosynthesis was down-regulated in GY8 but up-regulated in LTH. A lot of genes encoding pathogenesis-related gene (PR), such as chitinase and glucanase, were significantly up-regulated at both the transcriptome and proteome levels at 24 hours post-inoculation in GY8, but up-regulated at the transcriptome level and down-regulated at the proteome level in LTH. Our study reveals that the pathogen-associated molecular pattern (PAMP)-triggered immunity defense system may be activated at the transcriptome level but was inhibited at the protein level in susceptible rice varieties after inoculation. The results may facilitate future studies of the molecular mechanisms of rice blast resistance.

DEP1 is involved in regulating the carbon-nitrogen metabolic balance to affect grain yield and quality in rice (Oriza sativa L.).

The DEP1 (dense and erect panicle 1) gene, which corresponds to the erect panicle architecture, shows a pleiotropic effect in increasing grain yield and nitrogen use efficiency (NUE) in rice. Nevertheless, it remains unclear whether the carbon-nitrogen metabolic balance changes as the dep1 allele enhances nitrogen uptake and assimilation. In this study, we generated transgenic Akitakomati plants by overexpressing dep1 and analyzed the carbon-nitrogen metabolic status, gene expression profiles, and grain yield and quality. Under either low or high nitrogen growth conditions, the carbon-nitrogen metabolic balance of dep1-overexpressed lines was broken in stem sheaths and leaves but not in grains; the dep1-overexpressed plants showed higher expressions of glutamine synthetase (GS) and glutamate synthase (GOGAT) genes than the wildtype, along with increased total nitrogen and soluble protein content in the straw at maturity. However, the ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and phosphoenolpyruvate carboxylase (PEPC) genes were downregulated in dep1-overexpressed plants, leading to a decreased carbohydrate content and carbon/nitrogen ratio. Although the unbalanced carbon-nitrogen metabolism decreased the grain-filling rate, grain setting percentage, 1000 grain weight, and grain quality in dep1-overexpressed lines, it led to increased grain numbers per panicle and consequently increased grain yield. Our results suggest that an unbalanced carbon-nitrogen metabolic status is a major limiting factor for further improving grain yield and quality in erect panicle varieties.