Search

Key message: Cold-tolerant QTLqCSS12-regulated 14 hub genes are involved in the chloroplastic biological processes and in the protein synthesis and degradation processes injaponicarice. Low temperature is a main constraint factor for rice growth and production. To better understand the regulatory mechanisms underlying the cold tolerance phenotype in rice, here, we selected a cold-sensitive nearly isogenic line (NIL) NIL(qcss12) as materials to identify hub genes that are mediated by the cold-tolerant locus qCSS12 through weighted gene co-expression network analysis (WGCNA). Fourteen cold-responsive genes were identified, of which, 6 are involved in regulating biological processes in chloroplasts, including the reported EF-Tu, Prk, and ChlD, and 8 are involved in the protein synthesis and degradation processes. Differential expression of these genes between NIL(qcss12) and its controls under cold stress may be responsible for qCSS12-mediated cold tolerance in japonica rice. Moreover, natural variations in 12 of these hub genes are highly correlated with the cold tolerance divergence in two rice subspecies. The results provide deep insights into a better understanding of the molecular basis of cold adaptation in rice and provide a theoretical basis for molecular breeding. [ABSTRACT FROM AUTHOR]

Over half of the world's population relies on rice as their staple food. The brown planthopper (Nilaparvata lugens Stål, BPH) is a significant insect pest that leads to global reductions in rice yields. Breeding rice varieties that are resistant to BPH has been acknowledged as the most cost-effective and efficient strategy to mitigate BPH infestation. Consequently, the exploration of BPH-resistant genes in rice and the development of resistant rice varieties have become focal points of interest and research for breeders. In this review, we summarized the latest advancements in the localization, cloning, molecular mechanisms, and breeding of BPH-resistant rice. Currently, a total of 70 BPH-resistant gene loci have been identified in rice, 64 out of 70 genes/QTLs were mapped on chromosomes 1, 2, 3, 4, 6, 8, 10, 11, and 12, respectively, with 17 of them successfully cloned. These genes primarily encode five types of proteins: lectin receptor kinase (LecRK), coiled-coil-nucleotide-binding-leucine-rich repeat (CC-NB-LRR), B3-DNA binding domain, leucine-rich repeat domain (LRD), and short consensus repeat (SCR). Through mediating plant hormone signaling, calcium ion signaling, protein kinase cascade activation of cell proliferation, transcription factors, and miRNA signaling pathways, these genes induce the deposition of callose and cell wall thickening in rice tissues, ultimately leading to the inhibition of BPH feeding and the formation of resistance mechanisms against BPH damage. Furthermore, we discussed the applications of these resistance genes in the genetic improvement and breeding of rice. Functional studies of these insect-resistant genes and the elucidation of their network mechanisms establish a strong theoretical foundation for investigating the interaction between rice and BPH. Furthermore, they provide ample genetic resources and technical support for achieving sustainable BPH control and developing innovative insect resistance strategies. [ABSTRACT FROM AUTHOR]

Common wild rice (Oryza rufipogon Griff.) is invaluable genetic resource for rice resistance breeding. Whole-genome re-sequencing was conducted to systematically analyze the variations in two new inbred lines (Huaye 3 and Huaye 4) developed from a common wild rice. A total of 4,841,127 SNPs, 1,170,479 InDels, 24,080 structural variations (SVs), and 298 copy number variations (CNVs) were identified in three materials. Approximately 16.24 and 5.64% of the total SNPs and InDels of Huaye 3 and Huaye 4 were located in genic regions, respectively. Together, 12,486 and 15,925 large-effect SNPs, and 12,417 and 14,513 large-effect InDels, which affect the integrity of the encoded protein, were identified in Huaye 3 and Huaye 4, respectively. The distribution map of 194 and 245 NBS-LRR encoding homologs was constructed across 12 rice chromosomes. Further, GO enrichment analysis of the homologs with identical genotype variations in Huaye 3 and Huaye 4 revealed 67, 82, and 58 homologs involved in cell death, response to stress, and both terms, respectively. Comparative analysis displayed that 550 out of 652 SNPs and 129 out of 147 InDels were present in a widely used blast-susceptible rice variety (LTH). Protein-protein interaction analysis revealed a strong interaction between NBS-LRR candidates and several known R genes. One homolog of disease resistance protein (RPM1) was involved in the plant-pathogen interaction pathway. Artificial inoculation of disease/insect displayed resistance phenotypes against rice blast and brown planthopper in two lines. The results will provide allele-specific markers for rice molecular breeding. [ABSTRACT FROM AUTHOR]

Leaf rolling is a crucial agronomic trait to consider in rice (Oryza sativa L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The SEMI-ROLLED LEAF 1 (SRL1) gene plays a key role in regulating leaf rolling, as it encodes a glycosylphosphatidylinositol-anchored protein located on the plasma membrane. In this study, we used CRISPR/Cas9 to target the second and third exons of the SRL1 gene in the indica rice line GXU103, which resulted in the generation of 14 T0 transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T1 generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting SRL1 homozygous double-target knockout as srl1-103. This line exhibited defects in leaf development, leaf rolling in the mature upright leaves, and a compact nature of the fully grown plants. Compared with the wild type (WT), the T2 generation of srl1-103 varied in two key aspects: the width of flag leaf (12.6% reduction compared with WT) and the leaf rolling index (48.77% increase compared with WT). In order to gain a deeper understanding of the involvement of SRL1 in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T2 generation of srl1-103. A comparison of srl1-103 with WT revealed 459 differentially expressed genes (DEGs), including 388 upregulated genes and 71 downregulated genes. In terms of the function of the DEGs, there seemed to be a significant enrichment of genes associated with cell wall synthesis (LOC_Os08g01670, LOC_Os05g46510, LOC_Os04g51450, LOC_Os10g28080, LOC_Os04g39814, LOC_Os01g71474, LOC_Os01g71350, and LOC_Os11g47600) and vacuole-related genes (LOC_Os09g23300), which may partially explain the increased leaf rolling in srl1-103. Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene (LOC_Os08g44840) could be the main reason for the decreased leaf angle and the compact nature of the mutant plants. In summary, this study successfully elucidated the gene regulatory network in which SRL1 participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement. [ABSTRACT FROM AUTHOR]

A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host uncharacterized endemic viruses with spillover potential to rice (Oryza sativa) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice (O. rufipogon) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector that is able to transmit RTIV and found that RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover. Author summary: Emerging and re-emerging dangerous viruses cause epidemics or pandemics to threaten human and economically important crops and animals. Some of the most dangerous human viruses have zoonotic origins from wild animals. Native plants may also host new endemic viruses that can spread to cause epidemics in cultivated rice, one of the most important staple plants in the world. In the research, we discovered Rice tiller inhibition virus (RTIV), a new insect-borne positive-strand RNA virus, from Asian wild rice plants through metagenomic sequencing, and demonstrated that RTIV could be transmitted by a wide-distributed specific species of aphids to cultivated rice and inhibited tillering, an important agronomic trait of rice plants. We further rescued RTIV by developing infectious clone and confirmed that it can be a dangerous aphid-transmitted viral pathogen of cultivated rice plants. Thus, not only a rare aphid-transmitted viral pathogen of rice plants was discovered in the research, our findings also indicate that novel pathogenic viruses could spread out from native plants to threaten important crop plants. [ABSTRACT FROM AUTHOR]

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important quarantine diseases in the world. Breeding disease-resistant varieties can solve the problem of prevention and treatment of BLS from the source. The discovery of the molecular mechanism of resistance is an important driving force for breeding resistant varieties. In this study, a BLS-resistant near isogenic line NIL-bls2 was used as the material. Guangxi Xoc strain gx01 (abbreviated as WT) and its mutant strain (abbreviated as MT) with a knockout type III effectors (T3Es) gene were used to infect rice material NIL-bls2. The molecular interaction mechanism of rice resist near isogenic lines in response to infection by different pathogenic strains was analyzed by transcriptome sequencing. The results showed that there were 415, 134 and 150 differentially expressed genes (DEGs) between the WT group and the MT group at 12, 24 and 48 h of post inoculation (hpi). Through GO and KEGG enrichment analysis, it was found that, compared with non-pathogenic strains, the T3Es secreted by pathogenic strains inhibited the signal transduction pathway mediated by ethylene (ET), jasmonic acid (JA) and salicylic acid (SA), and the MAPKK (MAPK kinase) and MAPKKK (MAPK kinase kinase) in the MAPK (mitogen-activated protein kinase) cascade reaction, which prevented plants from sensing extracellular stimuli in time and starting the intracellular immune defense mechanism; and inhibited the synthesis of lignin and diterpenoid phytochemicals to prevent plants from establishing their own physical barriers to resist the invasion of pathogenic bacteria. The inhibitory effect was the strongest at 12 h, and gradually weakened at 24 h and 48 h. To cope with the invasion of pathogenic bacteria, rice NIL-bls2 material can promote wound healing by promoting the synthesis of traumatic acid at 12 h; at 24 h, hydrogen peroxide was degraded by dioxygenase, which reduced and eliminated the attack of reactive oxygen species on plant membrane lipids; and at 48 h, rice NIL-bls2 material can resist the invasion of pathogenic bacteria by promoting the synthesis of lignin, disease-resistant proteins, monoterpene antibacterial substances, indole alkaloids and other substances. Through transcriptome sequencing analysis, the molecular interaction mechanism of rice resistance near isogenic lines in response to infection by different pathogenic strains was expounded, and 5 genes, Os01g0719300, Os02g0513100, Os03g0122300, Os04g0301500, and Os10g0575100 closely related to BLS, were screened. Our work provides new data resources and a theoretical basis for exploring the infection mechanism of Xoc strain gx01 and the resistance mechanism of resistance gene bls2. [ABSTRACT FROM AUTHOR]

Wild rice is a primary source of genes that can be utilized to generate rice cultivars with advantageous traits. Chromosome segment substitution lines (CSSLs) are consisting of a set of consecutive and overlapping donor chromosome segments in a recipient's genetic background. CSSLs are an ideal genetic population for mapping quantitative traits loci (QTLs). In this study, 59 CSSLs from the common wild rice (Oryza rufipogon Griff.) accession DP15 under the indica rice cultivar (O. sativa L. ssp. indica) variety 93-11 background were constructed through multiple backcrosses and marker-assisted selection (MAS). Through high-throughput whole genome re-sequencing (WGRS) of parental lines, 12,565 mapped InDels were identified and designed for polymorphic molecular markers. The 59 CSSLs library covered 91.72% of the genome of common wild rice accession DP15. The DP15-CSSLs displayed variation in six economic traits including grain length (GL), grain width (GW), thousand-grain weight (TGW), grain length-width ratio (GLWR), plant height (PH), and leaf margin color (LMC), which were finally attributed to 22 QTLs. A homozygous CSSL line and a purple leave margin CSSL line were selected to construct two secondary genetic populations for the QTLs mapping. Thus, the PH-controlling QTL qPH1.1 was mapped to a region of 4.31-Mb on chromosome 1, and the LMC-controlling QTL qLMC6.1 was mapped to a region of 370-kb on chromosome 6. Taken together, these identified novel QTLs/genes from common wild rice can potentially promote theoretical knowledge and genetic applications to rice breeders worldwide. [ABSTRACT FROM AUTHOR]

The type of soft rice with low amylose content (AC) is more and more favored by consumers for its better eating and cooking quality, as people's quality of life continuously improves in China. The Wx gene regulates the AC of rice grains, thus affecting the degree of softness of the rice. Mei Meng B (MMB), Tian Kang B (TKB), and DR462 are three indica rice maintained lines with good morphological characters, but also with undesirably high AC. Therefore, CRISPR/Cas9 technology was used to edit the Wx gene of these lines to create a batch of soft rice breeding materials. New gene-edited lines MMB-10-2, TKB-21-12, and DR462-9-9, derived from the above parental lines, respectively, were selected in the T2 generations, with an AC of 17.2%, 16.8%, and 17.8%, and gel consistency (GC) of 78.6 mm, 77.4 mm, and 79.6 mm, respectively. The rapid viscosity analysis (RVA) spectrum showed that the three edited lines had a better eating quality as compared to the corresponding wild type, and showing new characteristics, different from the high-quality soft rice popular in the market. There was no significant difference in the main agronomic traits in the three edited lines compared to the corresponding wild types. Moreover, the chalkiness of DR462-9-9 was reduced, resulting in an improved appearance of its polished rice. The present study created soft rice germplasms for breeding improved quality hybrid rice, without changing the excellent traits of their corresponding wild type varieties. [ABSTRACT FROM AUTHOR]

The development of thermosensitive genic male sterile (TGMS) lines is the key to breeding two-line hybrid rice, which has been widely applied in China to increase grain yield. CRISPR/Cas9 has been widely used in genome editing to create novel mutants in rice. In the present study, a super grain quality line, GXU 47, was used to generate a new TGMS line with specific mutations in a major TGMS gene tms5 generated with CRISPR/Cas9-mediated genome editing in order to improve the rice quality of two-line hybrids. A mutagenesis efficiency level of 75% was achieved, and three homozygous T-DNA-free mutant lines were screened out. The mutants exhibited excellent thermosensitive male fertility transformation characteristics with complete male sterility at ≥24 °C and desirable male fertility at around 21 °C. Proteomic analysis based on isobaric tags for relative and absolute quantification (iTRAQ) was performed to unveil the subsequent proteomic changes. A total of 192 differentially expressed proteins (DEPs), including 35 upregulated and 157 downregulated, were found. Gene ontology (GO) analysis revealed that the DEPs were involved in a single-organism biosynthetic process, a single-organism metabolic process, oxidoreductase activity, and catalytic activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEPs were involved in ubiquinone and other terpenoid quinone biosynthesis, the biosynthesis of secondary metabolites, metabolic pathways, and phenylpropanoid biosynthesis. Our study shows that high mutation efficiency was achieved in both target sites, and T-DNA-free mutant lines were obtained in the T1 generation. The present study results prove that it is feasible and efficient to generate an excellent mutant line with CRISPR/Cas9, which provides a novel molecular mechanism of male sterility caused by the mutation of tms5. [ABSTRACT FROM AUTHOR]

Brown planthopper [BPH, Nilaparvata lugens (Stål)] is considered one of the most important pests of rice (Oryza sativa L.), which poses a serious threat to rice production. Identifying resistant Oryza germplasm can provide reliable accessions for breeding BPH resistant rice cultivars. In this study, the stem evaluation method (SEM) was first applied to identify the BPH resistance of 1,221 accessions of common wild rice (O. rufipogon Griff.) collected from three different regions of Guangxi Province, China. From this screening, 58 BPH resistant accessions were screened a second time, with 33 accessions ultimately identified as stable, highly resistant germplasm as confirmed by a third identification at the adult‐plant stage. The distribution of the 58 BPH‐resistant common wild rice accessions varies significantly from region to region. Genotypic analyses based on 42 simple sequence repeat (SSR) markers revealed that these 58 BPH‐resistant accessions were genetically diverse, reflecting the rich genetic diversity reported in Guangxi common wild rice. Furthermore, results verified that the SEM is efficient for rapid and accurate screening of BPH‐resistant germplasm, especially when a limited number of seeds are available or elite breeding lines need to be screened immediately. Also, SEM is the best method for evaluating BPH resistance at the adult stage because fewer insects are needed, and it is possible to repeat the evaluation in the same crop season. The 33 resistant rice accessions are a potential source of novel BPH resistance genes for developing cultivars with improved BPH resistance. Core Ideas: We used a new stem evaluation method (SEM) to evaluate rice accessions for resistance to BPH.The discovery of these resistant resources will provide more basic resources for further study.Genetic diversity of BPH‐resistant accessions in rice deserves further study.Discovery of resistant resources should focus on sampling from the lower reaches of Xijiang.Guangxi Oryza rufipogon was taken as experimental accessions, which shows superiority. [ABSTRACT FROM AUTHOR]

Background: Awn of rice is an important domestication trait closely associated with yield traits. Therefore, the identification of genes for awn development is of great significance for the elucidation of molecular mechanism of awn development and the genetic improvement of yield traits in rice. Results: In this study, using chromosome segment substitution lines (CSSLs) derived from a long-awned Guangxi common wild rice (GXCWR, Oryza rufipogon Griff.) and a short-awned indica cultivar 9311, we identified An-4, a potential quantitative trait locus (QTL) for awn development. Then, An-4 was fine mapped into a 56-kb region of chromosome 2, which contained four annotated genes. Among these four annotated genes, Os02g0594800 was concluded to be the potential candidate gene for An-4. An-4 exhibited pleiotropic effects on awn development and several yield traits. Scanning electron microscopy (SEM) analysis showed that An-4 significantly promoted awn development at Sp7 and Sp8 stage of spikelet development. Transcriptome analysis suggested that An-4 might influence the development of awn by regulating the expression of genes related to growth, developmental process, channel regulation and extracellular region. By contrast to those of 9311, the expression level of OsRR5 in CSSL128 was significantly down-regulated, whereas the expression levels of OsCKX2 and OsGA2ox5 in CSSL128 were significantly up-regulated. In addition, our study showed that An-4 had additive effects with other genes for awn development, such as An-1, An-2/LABA1 and An-3/GAD1/RAE2. Conclusions: The identification of An-4 lays a foundation for cloning of An-4 and further elucidation of the molecular mechanism of awn development. Moreover, the identification of favorable allelic variation of An-4 from 9311 will be useful to improve rice yield traits. [ABSTRACT FROM AUTHOR]

Little research on the effect of low nitrogen (N) and phosphorus (P) treatments, especially low P, on the resistance to brown planthopper (BPH, Nilaparvata lugens Stål) in Oryza sativa. Here, significantly fewer BPHs hatched on plants treated with low N or P. Furthermore, the treatments reduced BPH weight gain, survival rate, and honeydew excretion as well as the number of BPHs that settled on the plants. Similar to the observations in the control group, the average yield per plant under low N or P treatment decreased after infestation with BPH. Nevertheless, rice plants treated with low N or P showed obvious antibiotic and antixenotic effects on BPHs. Thus, these low-nutrient treatments could mitigate the reductions in rice yield after BPH infestation. This study clearly demonstrated that low N or P negatively affected BPH performance, indicating the potential application of these treatments as an alternative pest management strategy for BPHs. [ABSTRACT FROM AUTHOR]

Chloroplasts are the organelles responsible for photosynthesis and regulate normal plant growth. Although translation elongation factors play important roles in chloroplast development, functional studies of chloroplast translation elongation factors in higher plants remain very sparse. Here, we obtained a rice mutant exhibiting seedling-lethal albino phenotype and named it albino and lethal seedling 1 (als1). Consistently, low content of photosynthetic pigments, malformed chloroplasts and defective photosynthesis were observed in als1 mutant leaves. Map-based cloning experiment showed that als1 mutant had a T base insertion in Os02g0595700 , causing a frame shift and premature stop codon. ALS1 encoded a GTP-binding protein EF-Tu, which acts as a translation elongation factor in chloroplast protein translation. ALS1 was found to be expressed throughout plant with highest expression level in young leaves. Moreover, ALS1 was located in chloroplast, whereas the truncated als1 could not normally be located in chloroplast. Additionally, the ALS1 mutation significantly influenced the expression of downstream genes, such as genes relevant to chlorophyll biosynthesis, photosynthesis as well as chloroplast development. These results show that ALS1 acts as a key regulator of chloroplast development and plant growth. • We identified and characterized a novel rice mutant with seedling-lethal albino phenotype and named the mutant als1. • ALS1 plays a crucial role in chlorophyll biosynthesis, photosynthesis and chloroplast development. • als1 protein cannot be localized in chloroplasts, which may lead to seedling lethal albino phenotype in als1 mutants. [ABSTRACT FROM AUTHOR]

Background: Drought stress is an adverse factor with deleterious effects on several aspects of rice growth. However, the mechanism underlying drought resistance in rice remains unclear. To understand the molecular mechanism of the drought response in rice, drought-sensitive CSSL (Chromosome Single-substitution Segment Line) PY6 was used to map QTLs of sensitive phenotypes and to reveal the impact of the QTLs on transcriptional profiling. Results: The QTL dss-1 was mapped onto the short arm of chromosome 1 of rice. According to transcriptomic analysis, the identified differentially expressed genes (DEGs) exhibited a downregulated pattern and were mainly enriched in photosynthesis-related GO terms, indicating that photosynthesis was greatly inhibited under drought. Further, according to weighted gene coexpression network analysis (WGCNA), specific gene modules (designating a group of genes with a similar expression pattern) were strongly correlated with H2O2 (4 modules) and MDA (3 modules), respectively. Likewise, GO analysis revealed that the photosynthesis-related GO terms were consistently overrepresented in H2O2-correlated modules. Functional annotation of the differentially expressed hub genes (DEHGs) in the H2O2 and MDA-correlated modules revealed cross-talk between abiotic and biotic stress responses for these genes, which were annotated as encoding WRKYs and PR family proteins, were notably differentially expressed between PY6 and PR403. Conclusions: We speculated that drought-induced photosynthetic inhibition leads to H2O2 and MDA accumulation, which can then trigger the reprogramming of the rice transcriptome, including the hub genes involved in ROS scavenging, to prevent oxidative stress damage. Our results shed light on and provide deep insight into the drought resistance mechanism in rice. [ABSTRACT FROM AUTHOR]

As organelles for photosynthesis in green plants, chloroplasts play a vital role in solar energy capture and carbon fixation. The maintenance of normal chloroplast physiological functions is essential for plant growth and development. Low temperature is an adverse environmental stress that affects crop productivity. Low temperature severely affects the growth and development of plants, especially photosynthesis. To date, many studies have reported that chloroplasts are not only just organelles of photosynthesis. Chloroplasts can also perceive chilling stress signals via membranes and photoreceptors, and they maintain their homeostasis and promote photosynthesis by regulating the state of lipid membranes, the abundance of photosynthesis-related proteins, the activity of enzymes, the redox state, and the balance of hormones and by releasing retrograde signals, thus improving plant resistance to low temperatures. This review focused on the potential functions of chloroplasts in fine tuning photosynthesis processes under low-temperature stress by perceiving stress signals, modulating the expression of photosynthesis-related genes, and scavenging excess reactive oxygen species (ROS) in chloroplasts to survive the adverse environment. [ABSTRACT FROM AUTHOR]

The brown planthopper (Nilaparvata lugens Stål; BPH) is the most destructive rice pests worldwide. Identification and application of major resistance genes and cultivation of resistant varieties are the most efficient methods to control this insect. In this study, the rice variety, ARC5984, was detected to be highly resistant to BPH due to the presence of a major resistance gene, which has primarily been mapped in a 6-cM region on the long arm of chromosome 4. Fine mapping of the resistance gene was performed using the recombinants derived from BC1F2 individuals; the target gene was described to be a 46-kb region in the Nipponbare genome. Based on the information on the genomic sequences of the varieties Nipponbare, ZS97 and MH63, six, seven and 11 candidate genes were identified in the finally located region, respectively. The relative expression of the candidate genes Os04g35190 and Os04g35210 was significantly different between the resistant and susceptible plants after BPH infestation. The sequencing results indicated the coding domain sequence (CDS) of Os04g35210 from ARC5984 to be identical to that of Bph6. But the CDS of Os04g35190 was different between in ARC5984 and 9311. Moreover, the preliminary-near-isogenic lines (pre-NILs; the lines carrying resistance genes) with genetic background of the 9311 showed significant antixenosis to BPH, and thus, restricted its growth and development when compared with that in the susceptible line of 9311. Finally, a marker-assisted selection (MAS) technique was developed for the markers, RM5635 and BF9; its efficiency has been confirmed by applying it in screening of backcrossed individuals. The development in the fine mapping of the major gene will facilitate efforts of cloning this important resistance gene and to apply it in breeding of BPH-resistance rice varieties. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is a highly toxic heavy metal in nature, which causes severe damage to plant growth. The molecular mechanisms for Cd detoxification are poorly understood. Here, we report that a G-type ATP-binding cassette transporter, OsABCG36, is involved in Cd tolerance in rice. OsABCG36 was expressed in both roots and shoots at a low level, but expression in the roots rather than the shoots was greatly up-regulated by a short exposure to Cd. A spatial expression analysis showed that Cd-induced expression of OsABCG36 was found in both the root tip and the mature root region. Transient expression of OsABCG36 in rice protoplast cells showed that it was localized to the plasma membrane. Immunostaining showed that OsABCG36 was localized in all root cells except the epidermal cells. Knockout of OsABCG36 resulted in increased Cd accumulation in root cell sap and enhanced Cd sensitivity, but did not affect tolerance to other metals including Al, Zn, Cu, and Pb. The concentration of Cd in the shoots was similar between the knockout lines and wild-type rice. Heterologous expression of OsABCG36 in yeast showed an efflux activity for Cd, but not for Zn. Taken together, our results indicate that OsABCG36 is not involved in Cd accumulation in the shoots, but is required for Cd tolerance by exporting Cd or Cd conjugates from the root cells in rice. [ABSTRACT FROM AUTHOR]

The original version of this article unfortunately contained mistake in Fig. 3 caption. The authors changed the figure color to improve quality during the publication process but missed to change the the legend at Figure caption. [ABSTRACT FROM AUTHOR]

Low temperature at the seedling stage is a major damaging factor for rice production in southern China. To better understand the cold response of cultivated and wild rice, cold-sensitive cultivar 93–11 (Oryza sativa L. ssp. Indica) and cold-resistant hybrid wild rice DC907 with a 93–11 genetic background were used for a quantitative proteomic analysis with tandem mass tags (TMT) in parallel. Rice seedlings grown for four weeks at a normal temperature (25°C) were treated at 8–10°C for 24, 72 and 120 h. The number of differentially expressed proteins increased gradually over time in the cold-exposed rice in comparison with the untreated rice. A total of 366 unique proteins involved in ATP synthesis, photosystem, reactive oxygen species, stress response, cell growth and integrity were identified as responding to cold stress in DC907. While both DC907 and 93–11 underwent similar alterations in proteomic profiles in response to cold stress, DC907 responded in a prompter manner in terms of expressing cold-responding proteins, maintained a higher level of photosynthesis to power the cells, and possessed a stable and higher level of DIR proteins to prevent the plant from obtaining irreversible cell structure damage. The observations made in this study may lay a new foundation for further investigation of cold sensitivity or tolerance mechanisms in rice. [ABSTRACT FROM AUTHOR]

Low temperature can greatly restrict the growth and development of rice. The rice seedlings show growth retardation, lamina wrap, and part of blade even died under the condition of low temperature. In order to get more information about cold stress responses in rice, two dimensional electrophoresis and bioinformatics analysis of mass spectrometry were used to preliminary survey the cold tolerance of cold sensitive line 9311 and cold resistance variety Fujisaka 5 under cold stress. Two dimensional electrophoresis maps of 9311 and Fujisaka 5 were established under cold treatment. With analysis of bioinformation, the proteins were found involve in many aspects of rice development. The largest category of proteins is functioning on metabolism. By comparing the proteins from the two varieties, it can be found that most proteins from 9311 were down-regulated and were up-regulated in Fujisaka 5. The results showed that the membrane composition and structure were damaged, metabolism changed dramatically and rice defense system was activated under the cold stimulation. Fifty-nine proteins related to the resistance of cold stress were identified in our study, and we have investigated and classified all of their biological functions. The importance of our study are providing some conduct for the research of rice resistant to cold stress, supporting auxiliary technique for rice varieties and widening the search field of cold tolerance in plants. [ABSTRACT FROM AUTHOR]

[Objectives] Rice materials with broad-spectrum resistance against several pathogenic strains of Xaruhomans oryzae pv. Oryzicola (Xooc) at multiple growth stages were screened, in order to provide reliable resistance sources for variety breeding against rice bacterial leaf streak. [Methods] A total of 1100 rice lines with rich genetic background were offered as selective objects, and highly susceptible cultivar Jingang 30 was used as the control. Inoculation was carried out with five strong pathogenic strains of Xooc through acupuncture method at various growth stages for resistance identification. [Results] Fourteen disease-resistant materials were obtained through preliminary screening, accounting for 1.27% of the total materials. Nine materials moderately resistant to bacterial leaf streak were obtained via secondary screening, accounting for 0.82% of the total materials. Besides, three of them (RL6, RL9 and RL14) were resistant to a number of pathogenic strains of Xooc at three growth stages. In particular, RL16 presented broad-spectrum resistance to multi-strains with high resistance levels. Six of them (RL2, RL4, RL5, RL8, RL11 and RL12) were resistant to single strain at single growth stage. [Conclusions] The obtained three materials have broad-spectrum resistance to bacterial leaf streak at multiple growth stages, which can be served as an important source to cultivate disease-resistant rice varieties. RL6 maintains high broad-spectrum resistance at all growth stages, which can be used as a prior resistance source for rice variety breeding. Six materials obtained with resistance against bacterial leaf streak at particular growth stages can be used as candidate resistance sources. [ABSTRACT FROM AUTHOR]

Brown planthopper, blast, and bacterial blight are the main biotic stressors of rice and can cause a massive loss in rice production. Aroma is an important character of rice quality. It is of far-reaching significance to breed resistant and high-quality varieties using germplasms with objective genes. In this study, the introgression and pyramiding of brown planthopper (BPH), blast, and bacterial leaf blight (BLB) resistance genes and aroma genes into elite rice maintainers and restorers were conducted through conventional cross-breeding coupled with the marker-assisted selection (MAS) breeding method. Single-plant selection was performed from F2 onwards to select desirable recombinants possessing alleles of interest with suitable phenotypes. Respective linked markers were used in each generation from intercrossing to the F7 generation for tracking the presence of targeted genes. A total of 74 improved lines (ILs) have been developed which possess a combination of 1 to 4 genes for BPH, blast, and BLB resistance and aroma. These ILs showed moderate to high resistance to multiple biotic stresses (BPH, blast and BLB) or aromatic fragrance without obvious negative effects on agronomic traits. As multiple resistance and aromatic traits have become significant objectives in rice breeding, these resistance and/or aroma gene introgressed or pyramided lines have important application prospects. Core ideas: (1) marker-assisted breeding was used to pyramid multiple genes for an elite breeding line; (2) improved lines with the introgression of 1–4 genes were developed to achieve high resistance against various biotic stresses and aroma; (3) new lines were used as donor parents to introgress multiple genes in other genetic backgrounds. [ABSTRACT FROM AUTHOR]

Rice (Oryza sativa L.) is one of the major crops in the world and significant increase in grain yield is constant demand for breeders to meet the needs of a rapidly growing population. The size of grains is one of major components determining rice yield and a vital trait for domestication and breeding. To increase the grain size in rice, OsSPL16/qGW8 was mutagenized through CRISPR/Cas9, and proteomic analysis was performed to reveal variations triggered by mutations. More specifically, mutants were generated with two separate guide RNAs targeting recognition sites on opposite strands and genomic insertions and deletions were characterized. Mutations followed Mendelian inheritance and homozygous and heterozygous mutants lacking any T-DNA and off-target effects were screened. The mutant lines showed a significant increase in grain yield without any change in other agronomic traits in T0, T1, and T2 generations. Proteomic screening found a total of 44 differentially expressed proteins (DEPs), out of which 33 and 11 were up and downregulated, respectively. Most of the DEPs related to pyruvate kinase, pyruvate dehydrogenase, and cell division and proliferation were upregulated in the mutant plants. Pathway analysis revealed that DEPs were enriched in the biosynthesis of secondary metabolites, pyruvate metabolism, glycolysis/gluconeogenesis, carbon metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and citrate cycle. Gene Ontology (GO) analysis presented that most of the DEPs were involved in the pyruvate metabolic process and pyruvate dehydrogenase complex. Proteins related to pyruvate dehydrogenase E1 component subunit alpha-1 displayed higher interaction in the protein-protein interaction (PPI) network. Thus, the overall results revealed that CRISPR/Cas9-guided OsSPL16 mutations have the potential to boost the grain yield of rice. Additionally, global proteome analysis has broad applications for discovering molecular components and dynamic regulation underlying the targeted gene mutations. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via the original article. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) is involved in regulating drought tolerance, and pyrabactin resistance-like (PYL) proteins are known as ABA receptors. To elucidate the role of one of the ABA receptors in rice, OsPYL9 was mutagenized through CRISPR/Cas9 in rice. Homozygous and heterozygous mutant plants lacking any off-targets and T-DNA were screened based on site-specific sequencing and used for morpho-physiological, molecular, and proteomic analysis. Mutant lines appear to accumulate higher ABA, antioxidant activities, chlorophyll content, leaf cuticular wax, and survival rate, whereas a lower malondialdehyde level, stomatal conductance, transpiration rate, and vascular bundles occur under stress conditions. Proteomic analysis found a total of 324 differentially expressed proteins (DEPs), out of which 184 and 140 were up and downregulated, respectively. The OsPYL9 mutants showed an increase in grain yield under both drought and well watered field conditions. Most of the DEPs related to circadian clock rhythm, drought response, and reactive oxygen species were upregulated in the mutant plants. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEPs were only involved in circadian rhythm and Gene Ontology (GO) analysis showed that most of the DEPs were involved in response to abiotic stimulus, and abscisic acid-activated signaling pathways. Protein GIGANTEA, Adagio-like, and Pseudo-response regulator proteins showed higher interaction in protein–protein interaction (PPI) network. Thus, the overall results showed that CRISPR/Cas9-generated OsPYL9 mutants have potential to improve both drought tolerance and the yield of rice. Furthermore, global proteome analysis provides new potential biomarkers and understandings of the molecular mechanism of rice drought tolerance. [ABSTRACT FROM AUTHOR]

In rice, semi-dwarfism is among the most required characteristics, as it facilitates better yields and offers lodging resistance. Here, semi-dwarf rice lines lacking any residual transgene-DNA and off-target effects were generated through CRISPR/Cas9-guided mutagenesis of the OsGA20ox2 gene in a high yielding Basmati rice line, and the isobaric tags for relative and absolute quantification (iTRAQ) strategy was utilized to elucidate the proteomic changes in mutants. The results indicated the reduced gibberellins (GA1 and GA4) levels, plant height (28.72%), and flag leaf length, while all the other traits remained unchanged. The OsGA20ox2 expression was highly suppressed, and the mutants exhibited decreased cell length, width, and restored their plant height by exogenous GA3 treatment. Comparative proteomics of the wild-type and homozygous mutant line (GXU43_9) showed an altered level of 588 proteins, 273 upregulated and 315 downregulated, respectively. The identified differentially expressed proteins (DEPs) were mainly enriched in the carbon metabolism and fixation, glycolysis/gluconeogenesis, photosynthesis, and oxidative phosphorylation pathways. The proteins (Q6AWY7, Q6AWY2, Q9FRG8, Q6EPP9, Q6AWX8) associated with growth-regulating factors (GRF2, GRF7, GRF9, GRF10, and GRF11) and GA (Q8RZ73, Q9AS97, Q69VG1, Q8LNJ6, Q0JH50, and Q5MQ85) were downregulated, while the abscisic stress-ripening protein 5 (ASR5) and abscisic acid receptor (PYL5) were upregulated in mutant lines. We integrated CRISPR/Cas9 with proteomic screening as the most reliable strategy for rapid assessment of the CRISPR experiments outcomes. [ABSTRACT FROM AUTHOR]

Common wild rice contains valuable resources of novel alleles for rice improvement. It is well known that genetic populations provide the basis for a wide range of genetic and genomic studies. In particular, chromosome segment substitution lines (CSSLs) ais a powerful tool for fine mapping of quantitative traits, new gene discovery and marker-assisted breeding. In this study, 132 CSSLs were developed from a cultivated rice (Oryza sativa) cultivar (93-11) and common wild rice (Oryza rufipogon Griff. DP30) by selfing-crossing, backcrossing and marker-assisted selection (MAS). Based on the high-throughput sequencing of the 93-11 and DP30, 285 pairs of Insertion-deletions (InDel) markers were selected with an average distance of 1.23 Mb. The length of this DP30-CSSLs library was 536.4 cM. The coverage rate of substitution lines cumulatively overlapping the whole genome of DP30 was about 91.55%. DP30-CSSLs were used to analyze the variation for 17 traits leading to the detection of 36 quantitative trait loci (QTLs) with significant phenotypic effects. A cold-tolerant line (RZ) was selected to construct a secondary mapping F2 population, which revealed that qCT2.1 is in the 1.7 Mb region of chromosome 2. These CSSLs may, therefore, provide powerful tools for genome wide large-scale gene discovery in wild rice. This research will also facilitate fine mapping and cloning of QTLs and genome-wide study of wild rice. Moreover, these CSSLs will provide a foundation for rice variety improvement. [ABSTRACT FROM AUTHOR]

Rice blast (Magnaporthe oryzae) is a devastating disease affecting rice production globally. The development of cultivars with host resistance has been proved to be the best strategy for disease management. Several rice-resistance genes (R) have been recognized which induce resistance to blast in rice but R gene-mediated mechanisms resulting in defense response still need to be elucidated. Here, mutant lines generated through CRISPR/Cas9 based targeted mutagenesis to investigate the role of Pi21 against blast resistance and 17 mutant plants were obtained in T0 generation with the mutation rate of 66% including 26% bi-allelic, 22% homozygous, 12% heterozygous, and 3% chimeric and 17 T-DNA-free lines in T1 generation. The homozygous mutant lines revealed enhanced resistance to blast without affecting the major agronomic traits. Furthermore, comparative proteome profiling was adopted to study the succeeding proteomic regulations, using iTRAQ-based proteomic analysis. We identified 372 DEPs, among them 149 up and 223 were down-regulated, respectively. GO analysis revealed that the proteins related to response to stimulus, photosynthesis, carbohydrate metabolic process, and small molecule metabolic process were up-regulated. The most of DEPs were involved in metabolic, ribosomal, secondary metabolites biosynthesis, and carbon metabolism pathways. 40S ribosomal protein S15 (P31674), 50S ribosomal protein L4, L5, L6 (Q10NM5, Q9ZST0, Q10L93), 30S ribosomal protein S5, S9 (Q6YU81, Q850W6, Q9XJ28), and succinate dehydrogenase (Q9S827) were hub-proteins. The expression level of genes related to defense mechanism, involved in signaling pathways of jasmonic acid (JA), salicylic acid (SA), and ethylene metabolisms were up-regulated in mutant line after the inoculation of the physiological races of M. oryzae as compared to WT. Our results revealed the fundamental value of genome editing and expand knowledge about fungal infection avoidance in rice. [ABSTRACT FROM AUTHOR]

Rice leaf morphology is an essential agronomic trait to develop drought-tolerant genotypes for adequate and stable crop production in drought-prone areas. Here, rolled leaf mutant plants were acquired by CRISPR/Cas9-based mutagenesis of Semi-rolled leaf1,2 (SRL1 and SRL2) genes, and isobaric tags for relative and absolute quantification (iTRAQ) based proteomic analysis was performed to analyze the subsequent proteomic regulation events. Homozygous mutants exhibit decreased chlorophyll content, transpiration rate, stomatal conductance, vascular bundles (VB), stomatal number, and agronomic traits with increased panicle number and bulliform cells (BCs). Under drought stress, mutant plants displayed lower malondialdehyde (MDA) content while higher survival rate, abscisic acid (ABA) content, superoxide dismutase (SOD), catalase (CAT) activities, and grain filling percentage compare with their wild type (WT). Proteomic results revealed that 270 proteins were significantly downregulated, and 107 proteins were upregulated in the mutant line compared with WT. Proteins related to lateral organ boundaries' (LOB) domain (LBD) were downregulated, whereas abiotic stress-responsive proteins were upregulated in the CRISPR mutant. LBD proteins (Q5KQR7, Q6K713, Q7XGL4, Q8LQH4), probable indole-3-acetic acid-amido synthetase (Q60EJ6), putative auxin transporter-like protein 4 (Q53JG7), Monoculm 1 (Q84MM9) and AP2 (Apetala2) domain-containing protein (Q10A97) were found to be hub-proteins. The hybrids developed from mutant restorers showed a semi-rolled leaf phenotype with increased panicle number, grain number per panicle, and yield per plant. Our findings reveal the intrinsic value of genome editing and expand the knowledge about the network of proteins for leaf rolling and drought avoidance in rice. [ABSTRACT FROM AUTHOR]

Plant height (PH) is one of the most important agronomic traits of rice, as it directly affects the yield potential and lodging resistance. Here, semi-dwarf mutant lines were developed through CRISPR/Cas9-based editing of OsGA20ox2 in an indica rice cultivar. Total 24 independent lines were obtained in T0 generation with the mean mutation rate of 73.5% including biallelic (29.16%), homozygous (47.91%) and heterozygous (16.66%) mutations, and 16 T-DNA-free lines (50%) were obtained in T1 generation without off-target effect in four most likely sites. Mutations resulted in a changed amino acid sequence of mutant plants and reduced gibberellins (GA) level and PH (22.2%), flag leaf length (FLL) and increased yield per plant (YPP) (6.0%), while there was no effect on other agronomic traits. Mutants restored their PH to normal by exogenous GA3 treatment. The expression of the OsGA20ox2 gene was significantly suppressed in mutant plants, while the expression level was not affected for other GA biosynthesis (OsGA2ox3 and OsGA3ox2) and signaling (D1, GIDI and SLR1) genes. The mutant lines showed decreased cell length and width, abnormal cell elongation, while increased cell numbers in the second internode sections at mature stage. Total 30 protein spots were exercised, and 24 proteins were identified, and results showed that OsGA20ox2 editing altered protein expression. Five proteins including, glyceraldehyde-3-phosphate dehydrogenase, putative ATP synthase, fructose-bisphosphate aldolase 1, S-adenosyl methionine synthetase 1 and gibberellin 20 oxidase 2, were downregulated in dwarf mutant lines which may affect the plant growth. Collectively, our results provide the insights into the role of OsGA20ox2 in PH and confirmed that CRISPR–Cas9 is a powerful tool to understand the gene functions. [ABSTRACT FROM AUTHOR]

Proline-rich proteins (PRPs) play multiple physiological and biochemical roles in plant growth and stress response. In this study, we reported that the knockout of OsPRP1 induced cold sensitivity in rice. Mutant plants were generated by CRISPR/Cas9 technology to investigate the role of OsPRP1 in cold stress and 26 mutant plants were obtained in T0 generation with the mutation rate of 85% including 15% bi-allelic, 53.3% homozygous, and 16.7% heterozygous and 16 T-DNA-free lines in T1 generation. The conserved amino acid sequence was changed and the expression level of OsPRP1 was reduced in mutant plants. The OsPRP1 mutant plants displayed more sensitivity to cold stress and showed low survival rate with decreased root biomass than wild-type (WT) and homozygous mutant line with large fragment deletion was more sensitive to low temperature. Mutant lines accumulated less antioxidant enzyme activity and lower levels of proline, chlorophyll, abscisic acid (ABA), and ascorbic acid (AsA) content relative to WT under low-temperature stress. The changes of antioxidant enzymes were examined in the leaves and roots with exogenous salicylic acid (SA) treatment which resulted in increased activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress, while enzyme antioxidant activity was lower in untreated seedlings which showed that exogenous SA pretreatment could alleviate the low-temperature stress in rice. Furthermore, the expression of three genes encoding antioxidant enzyme activities (SOD4, POX1, and OsCAT3) was significantly down-regulated in the mutant lines as compared to WT. These results suggested that OsPRP1 enhances cold tolerance by modulating antioxidants and maintaining cross talk through signaling pathways. Therefore, OsPRP1 gene could be exploited for improving cold tolerance in rice and CRISPR/Cas9 technology is helpful to study the function of a gene by analyzing the phenotypes of knockout mutants generated. [ABSTRACT FROM AUTHOR]

Background: The brown planthopper (Nilaparvata lugens Stål; BPH), one of the most destructive pests of rice, has proven to be a substantial threat, conferring enormous production losses in Asia and becoming a difficult challenge to manipulate and control under field conditions. The continuous use of insecticides promotes the resurgence of BPH, which results in resistant varieties adapting through the upgrading of new BPH biotypes. To overcome resistance acquired by BPH against resistance varieties, different forms of novel resistant gene fusions act as functional domains for breeding to enhance insect resistance. Results: The current study reports on the novel BPH resistance gene Bph36 derived from two introgression lines (RBPH16 and RBPH17) developed from wild rice GX2183 which was previously reported to be resistant to BPH. Using two F2 crossing populations (Kangwenqizhan × RBPH16 and Huanghuazhan × RBPH17) in a bulked segregant analysis (BSA) for identification of resistant genes and QTL analysis, two QTLs for BPH resistance were generated on the long and short arms of chromosome 4, which was further confirmed by developing BC1F2:3 populations by backcrossing via marker assisted selection (MAS) approach. One BPH resistance locus on the short arm of chromosome 4 was mapped to a 38-kb interval flanked by InDel markers S13 and X48, and then was named Bph36, whereas another locus on the long arm of chromosome 4 was also detected in an interval flanked by RM16766 and RM17033, which was the same as that of Bph27. An evaluation analysis based on four parameters (BPH host selection, honeydew weight, BPH survival rate and BPH population growth rate) shows that Bph36 conferred high levels antibiosis and antixenosis to BPH. Moreover, Bph36 pyramided with Bph3, Bph27, and Bph29 through MAS into elite cultivars 9311 and MH511 (harbored Xa23), creating different background breeding lines that also exhibited strong resistance to BPH in the seedling or tillering stage. Conclusion: Bph36 can be utilized in BPH resistance breeding programs to develop high resistant rice lines and the high-resolution fine mapping will facilitate further map-based cloning and marker-assisted gene pyramiding of resistant gene. MAS exploited to pyramid with Bph3, Bph27, Bph29, and Xa23 was confirmed the effectiveness for BPH resistance breeding in rice and provided insights into the molecular mechanism of defense to control this devastating insect. [ABSTRACT FROM AUTHOR]

Background: Rice (Oryza sativa L.) is a thermophilic crop vulnerable to chilling stress. However, common wild rice (Oryza rufipogon Griff.) in Guangxi (China) has the ability to tolerate chilling stress. To better understand the molecular mechanisms underlying chilling tolerance in wild rice, iTRAQ-based proteomic analysis was performed to examine CTS-12, a major chilling tolerance QTL derived from common wild rice, mediated chilling and recovery-induced differentially expressed proteins (DEPs) between the chilling-tolerant rice line DC90 and the chilling-sensitive 9311. Results: Comparative analysis identified 206 and 155 DEPs in 9311 and DC90, respectively, in response to the whole period of chilling and recovery. These DEPs were clustered into 6 functional groups in 9311 and 4 in DC90. The majority were enriched in the ‘structural constituent of ribosome’, ‘protein-chromophore linkage’, and ‘photosynthesis and light harvesting’ categories. Short Time-series Expression Miner (STEM) analysis revealed distinct dynamic responses of both chloroplast photosynthetic and ribosomal proteins between 9311 and DC90. Conclusion: CTS-12 might mediate the dynamic response of chloroplast photosynthetic and ribosomal proteins in DC90 under chilling (cold acclimation) and recovery (de-acclimation) and thereby enhancing the chilling stress tolerance of this rice line. The identified DEPs and the involvement of CTS-12 in mediating the dynamic response of DC90 at the proteomic level illuminate and deepen the understanding of the mechanisms that underlie chilling stress tolerance in wild rice. [ABSTRACT FROM AUTHOR]