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Deciphering the cell-type composition in the tumor immune microenvironment (TIME) can significantly increase the efficacy of cancer treatment and improve the prognosis of cancer. Such a task has benefited from microarrays and RNA sequencing technologies, which have been widely adopted in cancer studies, resulting in extensive expression profiles with clinical phenotypes across multiple cancers. Current state-of-the-art tools can infer cell-type composition from bulk expression profiles, providing the possibility of investigating the inter-heterogeneity and intra-heterogeneity of TIME across cancer types. Much can be gained from these tools in conjunction with a well-curated database of TIME cell-type composition data, accompanied by the corresponding clinical information. However, currently available databases fall short in data volume, multi-platform dataset integration, and tool integration. In this work, we introduce TIMEDB (https://timedb.deepomics.org), an online database for human tumor immune microenvironment cell-type composition estimated from bulk expression profiles. TIMEDB stores manually curated expression profiles, cell-type composition profiles, and the corresponding clinical information of a total of 39,706 samples from 546 datasets across 43 cancer types. TIMEDB comes readily equipped with online tools for automatic analysis and interactive visualization, and aims to serve the community as a convenient tool for investigating the human tumor microenvironment.

Penaeus japonicas is an important shrimp species, which is exposed to stressors including a variety of epidemic diseases. To date, little is known about the mechanisms involved in the response to white spot syndrome virus (WSSV) mediated by long non-coding RNAs (lncRNAs). A total of 6544 putative lncRNAs were identified in the hepatopancreas in P. japonicas, which provides a useful lncRNA reference resource for use in future studies. In addition, a total of 444 differentially expressed mRNAs and 457 differentially expressed lncRNAs were identified at 6, 12, and 24 h after WSSV infection in the hepatopancreas of P. japonicas. Functional enrichment analysis showed that the differentially expressed mRNAs were enriched in terms related to immune response and viral infectivity such as defense response, aminopeptidase activity, whereas the differentially expressed lncRNA partner genes were enriched in ubiquitin-dependent protein catabolic process, lipoprotein metabolic process, and antigen processing and presentation. Moreover, several lncRNAs were induced by WSSV infection, indicating these lncRNAs might participate in regulating many immune processes referring to their partner genes. Co-expression analysis of the lncRNAs and their partner genes identified some high lncRNA-mRNA correlations. These results suggest that WSSV stimulates the immune response in the hepatopancreas potentially through an important coding and non-coding gene network, thereby providing valuable information regarding non-coding responses to WSSV in Penaeus species.

Cadmium (Cd)-contaminated rice is a serious issue affecting food safety. Understanding the molecular regulatory mechanisms of Cd accumulation in rice grains is crucial to minimizing Cd concentrations in grains. We identified a member of the low-affinity cation transporter family, OsLCT2 in rice. It was a membrane protein. OsLCT2 was expressed in all tissues of the elongation and maturation zones in roots, with the strongest expression in pericycle and stele cells adjacent to the xylem. When grown in Cd-contaminated paddy soils, rice plants overexpressing OsLCT2 significantly reduced Cd concentrations in the straw and grains. Hydroponic experiment demonstrated its overexpression decreased the rate of Cd translocation from roots to shoots, and reduced Cd concentrations in xylem sap and in shoots of rice. Moreover, its overexpression increased Zn concentrations in roots by up-regulating the expression of OsZIP9, a gene responsible for Zn uptake. Overexpression of OsLCT2 reduces Cd accumulation in rice shoots and grains by limiting the amounts of Cd loaded into the xylem and restricting Cd translocation from roots to shoots of rice. Thus, OsLCT2 is a promising genetic resource to be engineered to reduce Cd accumulation in rice grains.

In nature, plants are colonized by various microbes that play essential roles in their growth and health. Heterosis is a natural genetic phenomenon whereby first-generation hybrids exhibit superior phenotypic performance relative to their parents. It remains unclear whether this concept can be extended to the 'hybridization' of microbiota from two parents in their descendants and what benefits the hybrid microbiota might convey. Here, we investigated the structure and function of the root microbiota from three hybrid rice varieties and their parents through amplicon sequencing analysis of bacterial 16S ribosomal DNA (rDNA) and fungal internal transcribed spacer (ITS) regions. We show that the bacterial and fungal root microbiota of the varieties are distinct from those of their parental lines and exhibit potential heterosis features in diversity and composition. Moreover, the root bacterial microbiota of hybrid variety LYP9 protects rice against soil-borne fungal pathogens. Systematic analysis of the protective capabilities of individual strains from a 30-member bacterial synthetic community derived from LYP9 roots indicated that community members have additive protective roles. Global transcription profiling analyses suggested that LYP9 root bacterial microbiota activate rice reactive oxygen species production and cell wall biogenesis, contributing to heterosis for protection. In addition, we demonstrate that the protection conferred by the LYP9 root microbiota is transferrable to neighboring plants, potentially explaining the observed hybrid-mediated superior effects of mixed planting. Our findings suggest that some hybrids exhibit heterosis in their microbiota composition that promotes plant health, highlighting the potential for microbiota heterosis in breeding hybrid crops. This article is protected by copyright. All rights reserved.

Two-line hybrid rice based on Photoperiod/thermo-sensitive genic male sterile (P/TGMS) lines has been developed and applied widely in agriculture due to the freedom in making hybrid combinations, less difficulty in breeding sterile lines, and simpler procedures for breeding and producing hybrid seed. However, there are certain risks associated with hybrid seed production; if the temperature during the P/TGMS fertility-sensitive period is lower than the critical temperature, seed production will fail due to self-pollination. In a previous study, we found that the issue of insufficient purity of two-line hybrid rice seed could be initially addressed by using the difference in tolerance to β-triketone herbicides (bTHs) between the female parent and the hybrid seeds. In this study, we further investigated the types of applicable herbicides, application methods, application time, and the effects on physiological and biochemical indexes and yield in rice. The results showed that this method could be used for hybrid purification by soaking seeds and spraying plants with the bTH benzobicylon (BBC) at safe concentrations in the range of 37.5-112.5 mg/L, and the seeds could be soaked in BBC at a treatment rate of 75.0 mg/L for 36–55 h without significant negative effects. The safe concentration for spraying in the field is 50.0-400.0 mg/L BBC at the three-leaf stage. The two methods of hybrid seed purification can reach 100% efficiency without compromising the nutritional growth and yield of hybrid rice. Unlike BBC, Mesotrione (MST) can only be sprayed to achieve hybrid purification at concentrations between 10.0 and 70.0 mg/L without affecting yield. bTH stress significantly increases the index of the reactive oxygen species (ROS) hydrogen peroxide (H2O2) in the hybrid LLY942 in the safe concentration range. The peroxidase (POD) enzyme activity showed a significant upward trend with increasing herbicide concentration, which kept the content of the lipid peroxide malondialdehyde (MDA) within a certain range that did not affect the normal growth of the rice hybrids. Our study establishes a foundation for the application of the bTH seed purification strategy and showed that it is the most economical, convenient, and effective way to improve the purity of two-line hybrid rice seeds.

Summary The development of embryo sacs is crucial for seed production in plants, but the genetic basis regulating the meiotic crossover formation in the macrospore and microspore mother cells remains largely unclear. Here, we report the characterization of a spontaneous rice female sterile variation 1 mutant (fsv1) that showed severe embryo sacs abortion with low seed‐setting rate. Through map‐based cloning and functional analyses, we isolated the causal gene of fsv1, OsMLH3 encoding a MutL‐homolog 3 protein, an ortholog of HvMLH3 in barley and AtMLH3 in Arabidopsis. OsMLH3 and OsMLH1 (MutL‐homolog 1) interact to form a heterodimer (MutLγ) to promote crossover formation in the macrospore and microspore mother cells and development of functional megaspore during meiosis, defective OsMLH3 or OsMLH1 in fsv1 and CRISPR/Cas9‐based knockout lines results in reduced type I crossover and bivalent frequency. The fsv1 and OsMLH3‐knockout lines are valuable germplasms for development of female sterile restorer lines for mechanized seed production of hybrid rice.

Obtaining genetic variation information from indica rice hybrid parents and identification of loci associated with heterosis are important for hybrid rice breeding. Here, we resequence 1,143 indica accessions mostly selected from the parents of superior hybrid rice cultivars of China, identify genetic variations, and perform kinship analysis. We find different hybrid rice crossing patterns between 3- and 2-line superior hybrid lines. By calculating frequencies of parental variation differences (FPVDs), a more direct approach for studying rice heterosis, we identify loci that are linked to heterosis, which include 98 in superior 3-line hybrids and 36 in superior 2-line hybrids. As a proof of concept, we find two accessions harboring a deletion in OsNramp5, a previously reported gene functioning in cadmium absorption, which can be used to mitigate rice grain cadmium levels through hybrid breeding. Resource of indica rice genetic variation reported in this study will be valuable to geneticists and breeders., Hybrid rice cultivars are widely planted around the world. Here, the authors resequence 1,143 indica accessions, focusing on the parents of superior hybrid rice lines in China, and reveal genetic loci that are associated with heterosis via measuring frequency of parental variation difference (FPVD).

Significance Rice breeding programs aim to develop cultivars with improved traits, including high grain yield and superior quality. In rice, OsPDCD5 encodes a programmed cell death 5 protein. Targeted mutagenesis of OsPDCD5 enhanced grain yield and plant architecture. Statistical analysis indicated that plot grain yield of OsPDCD5 knockout lines was enhanced by 6.25 to 20.13% in 11 popular or newly bred rice cultivars compared with the corresponding wild types. The OsPDCD5 knockout lines showed increases in milled rice percentage and gel consistency, and a decrease in amylose content. Our results provide insight into the molecular mechanism by which OsPDCD5 influences grain yield and plant architecture, and highlight a promising candidate gene for use in breeding programs designed to develop super rice cultivars.

OsNRAMP5 is a transporter responsible for cadmium (Cd) and manganese (Mn) uptake and root-to-shoot translocation of Mn in rice plants. Knockout of OsNRAMP5 is regarded as an effective approach to minimize Cd uptake and accumulation in rice. It is vital to evaluate the effects of knocking out OsNRAMP5 on Cd and Mn accumulation, as well as Cd tolerance of rice plants in response to varying environmental Cd concentrations, and to uncover the underlying mechanism, which until now, has remained largely unexplored. This study showed that knockout of OsNRAMP5 decreased Cd uptake, but simultaneously facilitated Cd translocation from roots to shoots. The effect of OsNRAMP5 knockout on reducing root Cd uptake weakened, however its effect on improving root-to-shoot Cd translocation was constant with increasing environmental Cd concentrations. As a result, its mutation dramatically reduced Cd accumulation in shoots under low and moderate Cd stress, but inversely increased that under high Cd conditions. Interestingly, Cd tolerance of its knockout mutants was persistently enhanced, irrespective of lower or higher Cd concentrations in shoots, compared with that of wild-type plants. Knockout of OsNRAMP5 mitigated Cd toxicity by dramatically diminishing Cd uptake at low or moderate external Cd concentrations. Remarkably, its knockout effectively complemented deficient mineral nutrients in shoots, thereby indirectly enhancing rice tolerance to severe Cd stress. Additionally, its mutation conferred preferential delivery of Mn to young leaves and grains. These results have important implications for the application of the OsNRAMP5 mutation in mitigating Cd toxicity and lowering the risk of excessive Cd accumulation in rice grains.

Additional file 3: Fig. S1. PCR-based genotyping for the 28-bp deletion in indica accessions (partially). M: DL2000 DNA marker; lane 1–24: IR64, Nipponbare, Bing 1A, Digu, Yuetai A, 9311, Zhenxian 97A, Fengyuan A, Mianhui 725, II-32A, Tianfeng A, Fenghuazan, Yuanhui 2 hao, Yuetai B, IR36, Gui 99, Fengyuan A, Shuhui 498, Miyang 46, IR64, D62A, G46A, CDR22, Wufeng A. Fig. S2. Pfam motifs of HIS1 and amino acid variation of haplotypes. The shaded parts are predicted amino acids due to the 28-bp deletion in H5. Fig. S3. Protein sequence alignment of 2OGDs. (A) Protein sequence alignment and conserved site analysis of 91 experimentally characterized 2OGDs in plants. (B) Protein sequence alignment and conserved site analysis of 114 putative 2OGDs in rice. Fig. S4. RNA expression of HIS1 in some accessions harboring H1, H2, H3, and H5. The qRT-PCR results were normalized with the actin reference gene. Error bars represent the standard error of the mean (SEM) of three replicates. Fig. S5. The T1510G mutation is completely co-segregated with the susceptibility to BBC (partially). Rice accessions Nongxiang42 and Xianhui207 carry HIS1 haplotypes H2 and H3, respectively. The upper electrophoretic band represents the PCR products of HIS1, and the lower band shows the PCR products treated with the restriction enzyme Dde1. Fig. S6. BBC is effective when the seeds are mixed. (A). BBC can effectively kill hybrid sterile lines (MingS) without harming hybrids (Mingliangyou143). The red arrow indicates the sterile line that was killed. (B). Random selection of 10 normal and severely affected seedlings. Through the detection of 28-bp deletion primers, it was found that the normal materials were all in a heterozygous state, and the sterile line materials lacking 28-bp were killed.

Background Benzobicyclon (BBC) is a β-triketone herbicide (bTH) used in rice paddy fields. It has the advantages of high efficiency, low toxicity, high crop safety, and good environmental compatibility, and shows efficacy against paddy weeds resistant to other types of herbicides. However, as some important indica rice varieties are susceptible to BBC, BBC is currently only registered and applied in japonica rice cultivation areas. Results By analyzing haplotypes of the bTHs broad-spectrum resistance gene HIS1 and phenotypes for BBC in 493 major indica rice accessions in China, we identified a novel non-functional allelic variant of HIS1 in addition to the previously reported 28-bp deletion. Through detection with markers specific to the two non-functional mutations, it was clear that 25.4% of indica conventional varieties, 59.9% of fertility restorers, and 15.9% of sterile lines were susceptible to BBC. In addition, due to natural allelic variations of the HIS1 gene in the sterile and restorer lines, some two-line hybrid sterile lines were sensitive to bTHs, and the corresponding restorers were resistant. We showed the potential effectiveness of using bTHs to address the issue of two-line hybrid rice seed purity stemming from the self-crossing of sterile lines during hybrid rice seed production. Finally, allelic variations of the HIS1 gene may also play an important role in the mechanized seed production of hybrid rice. Conclusions Our findings offer guidance for the application of BBC in indica rice areas and provide a non-transgenic approach to address the seed purity issue of two-line hybrid rice.

To make better use of global germplasm resources for improving the eating quality of hybrid rice, using the resequencing data from the 3,000 rice genomes project (3K RGP), the allelic variations of the rice Wx locus were analysed. With the exception of five rare alleles discovered for the first time in our study, most of these alleles were known alleles of Wx. Furthermore, a set of Kompetitive allele-specific PCR (KASP) markers based on these Wx alleles have been developed, and thirty-six main parents of hybrid rice from 1976 to 2018 were selected for Wx genotyping. The results showed that only three Wx alleles existed in the main parents of hybrids, and the allelic combination of the hybrids changed from Wxa/Wxb and Wxlv/Wxb to Wxb/Wxb with the development of hybrid rice. Wxb is widely used in the male parents of hybrid rice. Wxa and Wxlv were used in the female parents of early hybrid rice, and they were gradually replaced by Wxb. In the future, more favourable Wx alleles from cultivated rice should be identified, introduced, and effectively used to improve hybrid rice quality.

In this study, we compared the physicochemical properties and starch structures of hybrid rice varieties with similar apparent amylose content but different taste values. In addition to the apparent amylose content, gel permeation chromatography analysis showed that the higher proportions of amylopectin short chains and relatively lower proportions of amylopectin long chains, which could lead to higher peak viscosity and breakdown value, as well as a softer and stickier texture of cooked rice, were the key factors in determining the eating quality of hybrid rice. High-performance anion-exchange chromatography analyses showed that the proportion of amylopectin short chains (degree of polymerization 6-10) and intermediate chains (degree of polymerization 13-24), which might affect the gelatinisation enthalpy and crystallinity, also contributed greatly to the eating quality of hybrid rice. Moreover, this study indicated that a greater diversity of forms and sizes of starch granules might influence the eating quality of hybrid rice.

Rice grain with excessive cadmium (Cd) is a major source of dietary Cd intake and a serious threat to health for people who consume rice as a staple food. The development of elite rice cultivars with consistently low Cd content is challenging for conventional breeding approaches, and new strategies urgently need to be developed. Here, we report the development of new indica rice lines with low Cd accumulation and no transgenes by knocking out the metal transporter gene OsNramp5 using CRISPR/Cas9 system. Hydroponic culture showed that Cd concentrations in shoots and roots of osnramp5 mutants were dramatically decreased, resulting in rescue of impaired growth in high Cd condition. Cd-contaminated paddy field trials demonstrated that Cd concentration in osnramp5 grains was consistently less than 0.05 mg/kg, in contrast to high Cd concentrations from 0.33 mg/kg to 2.90 mg/kg in grains of Huazhan (the wild-type indica rice). In particular, the plant yield was not significantly affected in osnramp5 mutants. Furthermore, we developed promising hybrid rice lines with extremely low Cd content in grains. Our work supplies a practical approach to developing Cd pollution-safe indica rice cultivars that minimizes Cd contamination risk in grains.

The female gametophyte is an important participant in the sexual reproduction of plants. The molecular mechanism of its development has received much attention in recent years. As important regulators of gene expression, miRNAs have been certified to play a significant role in many biological processes of plants, including sexual reproduction. In this study, to investigate the potential regulatory effects of miRNAs on rice female gametophyte abortion, we used the high-throughput sequencing method to compare the miRNA transcriptome in ovules of a high frequency female-sterile line (fsv1) and a rice wild-type line (Gui 99) during ovule development. As a result, 522 known miRNAs and 295 novel miRNAs were expressed in the developing ovule of rice, while 100 known miRNAs were significantly differentially expressed between these two rice lines during ovule development. Combining with gene expression information, a total of 627 coherent target genes of these differential expressed known miRNAs between fsv1 and Gui 99 were identified. The functional analyses of these coherent target genes revealed that the coherent target genes of differential expressed known miRNAs between the two rice lines are involved in many biological pathways, such as protein degradation, auxin signal transduction, and transcription factor regulation. These results provide us with important clues to investigate the regulatory roles of miRNAs in rice female gametophyte abortion.

Background As important female reproductive tissues, the rice (Oryza sativa L.) ovule and female gametophyte is significant in terms of their fertility. Long noncoding RNAs (lncRNAs) play important and wide-ranging roles in the growth and development of plants and have become a major research focus in recent years. Therefore, we explored the characterization and expression change of lncRNAs during ovule development and female gametophytic abortion. Results In our study, whole-transcriptome strand-specific RNA sequencing (ssRNA-seq) was performed in the ovules of a high-frequency female-sterile rice line (fsv1) and a wild-type rice line (Gui99) at the megaspore mother cell meiosis stage (stage 1), functional megaspore mitosis stage (stage 2) and female gametophyte mature stage (stage 3). By comparing two rice lines, we identified 152, 233, and 197 differentially expressed lncRNAs at the three ovule developmental stages. Functional analysis of the coherent target genes of these differentially expressed lncRNAs indicated that many lncRNAs participate in multiple pathways such as hormone and cellular metabolism and signal transduction. Moreover, there were many differentially expressed lncRNAs acting as the precursors of some miRNAs that are involved in the development of ovules and female gametophytes. In addition, we have found that lncRNAs can act as decoys, competing with mRNAs for binding to miRNAs to maintain the normal expression of genes related to ovule and female gametophyte development. Conclusion These results provide important clues for elucidating the female gametophyte abortion mechanism in rice. This study also expands our understanding about the biological functions of lncRNAs and the annotation of the rice genome. Electronic supplementary material The online version of this article (10.1186/s12864-019-5442-6) contains supplementary material, which is available to authorized users.

Citric acid (CA) can regulate the balance of anions and cations in plants, and improve their resistance to heavy metals. It is not clear if foliar application with CA has any effect on migration of Cd and Mn in rice plant. In this work, a low-Cd-accumulating indica rice line (P7) and a high-Cd-accumulating line (HZ) were used to investigate the influence of CA on the transport of Cd and Mn as well as amino acid metabolism in grains. Content of Cd in grains and other organs increased with the increase of Cd content (0.1–2.4 mg kg−1) in soil, while decreased with the foliar application with CA. With the increase of Cd content in rice grains, the content of most amino acids in HZ, P7, HZ + CA and P7 + CA showed an obvious decline trend. Foliar application with CA efficiently raised the Mn:Cd ratio in grains and nodes of both HZ and P7. Meanwhile, the expression levels of OsNramp2, 3 and 5 in panicles were efficiently enhanced by CA application when plants grew in soil with Cd content of 0.6–2.4 mg kg−1. The increasing effect of CA on the content of 4 amino acids (i.e., Glu, Phe, Thr and Ala) in grains was related to varieties and Cd pollution. These results indicate that foliar application with CA can regulate the transport of Cd and Mn in the opposite directions in tissues and inhibit Cd accumulation in grains by enhancing expression of OsNRAMP 2, 3 or 5 and triggering the defense response of some amino acids in Cd-contaminated environment.

The comprehensive transcriptome analysis of rice female-sterile line and wild-type line ovule provides an important clue for exploring the regulatory network of the formation of rice fertile female gametophyte. Ovules are the female reproductive tissues of rice (Oryza sativa L.) and play a major role in sexual reproduction. To investigate the potential mechanism of rice female gametophyte fertility, we used RNA sequencing, combined with genetic subtraction, to compare the transcriptome of the ovules of a high-frequency female-sterile line (fsv1) and a rice wild-type line (Gui 99) during ovule development. Ovules were harvested at three developmental stages: ovule containing megaspore mother cell in meiosis process (stage 1), ovule containing functional megaspore in mitosis process (stage 2), and ovule containing mature female gametophyte (stage 3). Six cDNA libraries generated a total of 42.2 million high-quality clean reads that aligned with 30,204 genes. The comparison between the fsv1 and Gui 99 ovules identified a large number of differentially expressed genes (DEGs), i.e., 45, 495, and 932 DEGs at the three ovule developmental stages, respectively. From the comparison of the two rice lines, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and MapMan analyses indicated that a large number of DEGs associated with starch and sucrose metabolism, plant hormone signal transduction, protein modification and degradation, oxidative phosphorylation, and receptor kinase. These DEGs might play roles in ovule development and fertile female gametophyte formation. Many transcription factor genes and epigenetic-related genes also exhibit different expression patterns and significantly different expression levels in two rice lines during ovule development, which might provide important information regarding the abortive mechanism of the female gametophyte in rice.

Biofilms cause persistent bacterial infections and are extremely recalcitrant to antimicrobials, due in part to reduced penetration of antimicrobials into biofilms that allows bacteria residing in the depth of a biofilm to survive antimicrobial treatment. Here, we describe the preparation of surface-adaptive, Triclosan-loaded micellar nanocarriers showing (1) enhanced biofilm penetration and accumulation, (2) electrostatic targeting at acidic pH toward negatively charged bacterial cell surfaces in a biofilm, and (3) antimicrobial release due to degradation of the micelle core by bacterial lipases. First, it was established that mixed-shell-polymeric-micelles (MSPM) consisting of a hydrophilic poly(ethylene glycol) (PEG)-shell and pH-responsive poly(beta-amino ester) become positively charged at pH 5.0, while being negatively charged at physiological pH. This is opposite to single-shell-polymeric-micelles (SSPM) possessing only a PEG-shell and remaining negatively charged at pH 5.0. The stealth properties of the PEG-shell combined with its surface adaptive charge allow MSPMs to penetrate and accumulate in staphylococcal biofilms, as demonstrated for fluorescent Nile red loaded micelles using confocal-laser-scanning-microscopy. SSPMs, not adapting a positive charge at pH 5.0, could not be demonstrated to penetrate and accumulate in a biofilm. Once micellar nanocarriers are bound to a staphylococcal cell surface, bacterial enzymes degrade the MSPM core to release its antimicrobial content and kill bacteria over the depth of a biofilm. This constitutes a highly effective pathway to control blood-accessible staphylococcal biofilms using antimicrobials, bypassing biofilm recalcitrance to antimicrobial penetration.

Bacterial infections are mostly due to bacteria in their biofilm mode-of-growth, making them recalcitrant to antibiotic penetration. In addition, the number of bacterial strains intrinsically resistant to available antibiotics is alarmingly growing. This study reports that micellar nanocarriers with a poly(ethylene glycol) shell fully penetrate staphylococcal biofilms due to their biological invisibility. However, when the shell is complemented with poly(beta-amino ester), these mixed-shell micelles become positively charged in the low pH environment of a biofilm, allowing not only their penetration but also their accumulation in biofilms without being washed out, as do single-shell micelles lacking the pH-adaptive feature. Accordingly, bacterial killing of multidrug resistant staphylococcal biofilms exposed to proto-porphyrin IX-loaded mixed-shell micelles and after light-activation is superior compared with single-shell micelles. Subcutaneous infections in mice, induced with vancomycin-resistant, bioluminescent staphylococci can be eradicated by daily injection of photoactivatable protoporphyrin IX-loaded, mixed-shell micelles in the bloodstream and light-activation at the infected site. Micelles, which are not degraded by bacterial enzymes in the biofilm, are degraded in the liver and spleen and cleared from the body through the kidneys. Thus, adaptive micellar nanocarriers loaded with light-activatable antimicrobials constitute a much-needed alternative to current antibiotic therapies.

Genetic diversity within parental lines of hybrid rice is the foundation of heterosis utilization and yield improvement. Previous studies have suggested that genetic diversity was narrow in cytoplasmic male sterile (CMS/A line) and restorer lines (R line) for Three-line hybrid rice. However, the genetic diversity within maintainer lines (B line), especially at a genome-wide scale, remains largely unknown. In the present study, we performed deep re-sequencing of the elite maintainer line V20B (Oryza sativa L. ssp. indica). We then compared the V20B sequence with the 93-11 (Oryza sativa L. ssp. indica) genome sequence. 112.1 × 106 paired-end reads (PE reads) were generated with approximately 30-fold sequencing depth. The V20B PE reads uniquely covered 87.6 % of the 93-11 genome sequence. Overall, a total of 660,778 single-nucleotide polymorphism (SNPs) and 266,301 insertions and deletions (InDels) were identified, yielding an average of 2.1 SNPs/kb and 0.8 InDels/kb. Genome-wide distribution of the SNPs and InDels was non-random, and variation-rich and variation-poor regions were identified in all chromosomes. A total of 20,562 non-synonymous SNPs spanning 8,854 genes were annotated. Our results identified DNA polymorphisms at the genome-wide scale and uncovered the high level of genetic diversity between V20B and 93-11. Our results proved that next-generation sequencing technologies can be powerful tools to study genome-wide DNA polymorphisms, to query genetic diversity, and to enable molecular improvement efforts with Three-line hybrid rice. Further, our results also indicated that 93-11 could be used as core germplasm for the improvement of wild-abortive CMS lines and the maintainer lines.

Hybrid rice is the dominant form of rice planted in China, and its use has extended worldwide since the 1970s. It offers great yield advantages and has contributed greatly to the world’s food security. However, the molecular mechanisms underlying heterosis have remained a mystery. In this study we integrated genetics and omics analyses to determine the candidate genes for yield heterosis in a model two-line rice hybrid system, Liang-you-pei 9 (LYP9) and its parents. Phenomics study revealed that the better parent heterosis (BPH) of yield in hybrid is not ascribed to BPH of all the yield components but is specific to the BPH of spikelet number per panicle (SPP) and paternal parent heterosis (PPH) of effective panicle number (EPN). Genetic analyses then identified multiple quantitative trait loci (QTLs) for these two components. Moreover, a number of differentially expressed genes and alleles in the hybrid were mapped by transcriptome profiling to the QTL regions as possible candidate genes. In parallel, a major QTL for yield heterosis, rice heterosis 8 (RH8), was found to be the DTH8/Ghd8/LHD1 gene. Based on the shared allelic heterozygosity of RH8 in many hybrid rice cultivars, a common mechanism for yield heterosis in the present commercial hybrid rice is proposed.

Hybrid rice breeding offers an important strategy to improve rice production, in which the cultivation of a male sterile line is the key to the success of cross-breeding. CRISPR/Cas9 systems have been widely used in target-site genome editing, whereas their application for crop genetic improvement has been rarely reported. Here, using the CRISPR/Cas9 system, we induced specific mutations in TMS5, which is the most widely applied thermo-sensitive genic male sterility (TGMS) gene in China, and developed new “transgene clean” TGMS lines. We designed 10 target sites in the coding region of TMS5 for targeted mutagenesis using the CRISPR/Cas9 system and assessed the potential rates of on- and off-target effects. Finally, we established the most efficient construct, the TMS5ab construct, for breeding potentially applicable “transgene clean” TGMS lines. We also discussed factors that affect the editing efficiency according to the characteristics of different target sequences. Notably, using the TMS5ab construct, we developed 11 new “transgene clean” TGMS lines with potential applications in hybrid breeding within only one year in both rice subspecies. The application of our system not only significantly accelerates the breeding of sterile lines but also facilitates the exploitation of heterosis.

Sub-gingival anaerobic pathogens can colonize an implant surface to compromise osseointegration of dental implants once the soft tissue seal around the neck of an implant is broken. In vitro evaluations of implant materials are usually done in monoculture studies involving either tissue integration or bacterial colonization. Co-culture models, in which tissue cells and bacteria battle simultaneously for estate on an implant surface, have been demonstrated to provide a better in vitro mimic of the clinical situation. Here we aim to compare the surface coverage by U2OS osteoblasts cells prior to and after challenge by two anaerobic sub-gingival pathogens in a co-culture model on differently modified titanium (Ti), titanium-zirconium (TiZr) alloys and zirconia surfaces. Monoculture studies with either U2OS osteoblasts or bacteria were also carried out and indicated significant differences in biofilm formation between the implant materials, but interactions with U2OS osteoblasts were favourable on all materials. Adhering U2OS osteoblasts cells, however, were significantly more displaced from differently modified Ti surfaces by challenging sub-gingival pathogens than from TiZr alloys and zirconia variants. Combined with previous work employing a co-culture model consisting of human gingival fibroblasts and supra-gingival oral bacteria, results point to a different material selection to stimulate the formation of a soft tissue seal as compared to preservation of osseointegration under the unsterile conditions of the oral cavity.

The size and shape of the plant leaf is an important agronomic trait. To understand the molecular mechanism governing plant leaf shape, we characterized a classic rice (Oryza sativa) dwarf mutant named narrow leaf1 (nal1), which exhibits a characteristic phenotype of narrow leaves. In accordance with reduced leaf blade width, leaves of nal1 contain a decreased number of longitudinal veins. Anatomical investigations revealed that the culms of nal1 also show a defective vascular system, in which the number and distribution pattern of vascular bundles are altered. Map-based cloning and genetic complementation analyses demonstrated that Nal1 encodes a plant-specific protein with unknown biochemical function. We provide evidence showing that Nal1 is richly expressed in vascular tissues and that mutation of this gene leads to significantly reduced polar auxin transport capacity. These results indicate that Nal1 affects polar auxin transport as well as the vascular patterns of rice plants and plays an important role in the control of lateral leaf growth.

The future of rice breeding will likely be built on the basis of the further utilization of heterosis between elite cultivars and genetic resources from distant subspecies of rice. Previous studies have proved that exogenous genomic DNA transformation methods can be used to transfer genetic information from distant relatives (donor) into cultivated rice (recipient). However, the mechanism underlying this form of genetic transfer is poorly characterized, and the genes that cause the phenotypic changes in these variants are typically difficult to identify. This study examined YVB, a stable variant line with greatly improved grain quality traits that was derived from an indica variety (V20B) by transferring genomic DNA of O.minuta through the "spike-stalk injection method (SIM)". We used restriction-site associated DNA sequencing technology (RAD-seq) to evaluate a population of BC1F5 backcross lines (YVB × V20B); the RAD-seq data were used to construct a genetic linkage map with high-density SNPs for use in association analysis exploring genotype-phenotype relationships at the whole-genome level. A total of 17 quantitative trait loci (QTLs) for rice quality traits were mapped to chromosomes 3, 5, 6, 8, and 9. 8 major QTLs controlling different phenotypic variations were mapped to the same region of chromosome 5. This region contained the GS5 gene for grain weight and the qSW5/GW5 gene for grain width. This study provides new resources and insights into the molecular mechanisms of grain trait phenotypic variation and the transmission of genetic information via the introduction of genomic DNA to a distantly related crop relative species.

Biomaterial-implants are frequently used to restore function and form of human anatomy. However, the presence of implanted biomaterials dramatically elevates infection risk. Paradoxically, dental-implants placed in a bacteria-laden milieu experience moderate failure-rates, due to infection (0.0-1.1 %), similar to the ones of joint-arthroplasties placed in a near-sterile environment (0.1-1.3 %). Transcutaneous bone-fixation pins breach the immune-barrier of the epidermis, exposing underlying sterile-tissue to an unsterile external environment. In contrast to dental-implants, also placed in a highly unsterile environment, these pins give rise to relatively high infection-associated failure-rates of up to 23.0 %. Herein, we attempt to identify causes as to why dental-implants so often succeed, where others fail. The major part of all implants considered are metal-made, with similar surface-finishes. Material choice was therefore discarded as underlying the paradox. Antimicrobial activity of saliva has also been suggested as a cause for the success of dental-implants, but was discarded because saliva is the implant-site-fluid from which viable bacteria adhere. Crevicular fluid was discarded as it is largely analogous to serum. Instead, we attribute the relative success of dental-implants to (1) ability of oral tissues to heal rapidly in the continuous presence of commensal bacteria and opportunistic pathogens, and (2) tolerance of the oral immune-system. Inability of local tissue to adhere, spread and grow in presence of bacteria and an intolerant immune-system are identified as the likely main causes explaining the susceptibility of other implants to infection-associated failure. In conclusion, it is the authors' belief that new anti-infection strategies for a wide range of biomaterial-implants may be derived from the relative success of dental-implants.

To study the assembly of phycocyanin β subunit, the gene cpcT was first cloned from Arthrospira platensis FACHB314. To explore the function of cpcT, the DNA of phycocyanin β subunit and cpcT were transformed into Escherichia coli BL21 with the plasmid pET-hox1-pcyA, which contained the genes hemeoxygenase 1 (Hox1) and ferredoxin oxidoreductase (PcyA) needed to produce phycocyanobilin. The transformed strains showed specific phycocyanin fluorescence, and the fluorescence intensity was stronger than the strains with only phycocyanin β subunit, indicating that CpcT can promote the assembly of phycocyanin to generate fluorescence. To study the possible binding sites of apo-phycocyanin and phycocyanobilin, the Cys-82 and Cys-153 of the β subunit were individually mutated, giving two kinds of mutants. The results show that Cys-153 maybe the active site for β subunit binding to phycocyanobilins, which is catalyzed by CpcT in A. platensis FACHB314.

OBJECTIVE: Dental implants anchor in bone through a tight fit and osseo-integratable properties of the implant surfaces, while a protective soft tissue seal around the implants neck is needed to prevent bacterial destruction of the bone-implant interface. This tissue seal needs to form in the unsterile, oral environment. We aim to identify surface properties of dental implant materials (titanium, titanium-zirconium alloy and zirconium-oxides) that determine the outcome of this "race-for-the-surface" between human-gingival-fibroblasts and different supra-gingival bacterial strains.METHODS: Biofilms of three streptococcal species or a Staphylococcus aureus strain were grown in mono-cultures on the different implant materials in a parallel-plate-flow-chamber and their biovolume evaluated using confocal-scanning-laser-microscopy. Similarly, adhesion, spreading and growth of human-gingival-fibroblasts were evaluated. Co-culture experiments with bacteria and human-gingival-fibroblasts were carried out to evaluate tissue interaction with bacterially contaminated implant surfaces. Implant surfaces were characterized by their hydrophobicity, roughness and elemental composition.RESULTS: Biofilm formation occurred on all implant materials, and neither roughness nor hydrophobicity had a decisive influence on biofilm formation. Zirconium-oxide attracted most biofilm. All implant materials were covered by human-gingival-fibroblasts for 80-90% of their surface areas. Human-gingival-fibroblasts lost the race-for-the-surface against all bacterial strains on nearly all implant materials, except on the smoothest titanium variants.SIGNIFICANCE: Smooth titanium implant surfaces provide the best opportunities for a soft tissue seal to form on bacterially contaminated implant surfaces. This conclusion could only be reached in co-culture studies and coincides with the results from the few clinical studies carried out to this end.

The phytohormone cytokinin (CK) positively regulates the activity and function of the shoot apical meristem (SAM), which is a major parameter determining seed production. The rice ( Oryza sativa L.) Gn1a / OsCKX2 ( Grain number 1a / Cytokinin oxidase 2 ) gene, which encodes a cytokinin oxidase, has been identified as a major quantitative trait locus contributing to grain number improvement in rice breeding practice. However, the molecular mechanism of how the expression of OsCKX2 is regulated in planta remains elusive. Here, we report that the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST) directly regulates OsCKX2 expression in the reproductive meristem. DST-directed expression of OsCKX2 regulates CK accumulation in the SAM and, therefore, controls the number of the reproductive organs. We identify that DST reg1 , a semidominant allele of the DST gene, perturbs DST-directed regulation of OsCKX2 expression and elevates CK levels in the reproductive SAM, leading to increased meristem activity, enhanced panicle branching, and a consequent increase of grain number. Importantly, the DST reg1 allele provides an approach to pyramid the Gn1a -dependent and Gn1a -independent effects on grain production. Our study reveals that, as a unique regulator of reproductive meristem activity, DST may be explored to facilitate the genetic enhancement of grain production in rice and other small grain cereals.

The transformation of distally related genomic DNAs into plant was proposed as a novel technique to breed new cultivars. For example, a restorer rice line, RB207, was successfully developed and stabilized through the transformation of genomic DNAs of Echinochloa crusgalli (E. crusgalli) into a rice line, R207. Although the phenotypes of this variant line are apparently different from its receptor, the molecular bases are not elucidated yet. Herein, we have systematically studied the differential proteomes from the tissues of E. crusgalli, R207, and RB207 in an attempt to find an explanation regarding the phenotypic changes of RB207. The 2-DE method was employed to separate the leaf and embryo proteins of these plants followed by protein identification with mass spectrometry. In the leaf, 953 +/- 15, 1084 +/- 11, and 1091 +/- 11 silver-stained spots were detected, whereas in the embryo, 986 +/- 3, 884 +/- 10, and 892 +/- 14 spots were found from E. crusgalli, R207, and RB207, respectively. In comparison to the 2-DE images of the two rice lines, which showed many similarities, the ones of the E. crusgalli and rice were found to be so different that they were incomparable. There were some differentially expressed 2-DE spots between the two rice cultivars, 72 in leaf and 53 in embryo, respectively. The results of protein identification suggested that, regardless of leaves or embryos, none of the E. crusgalli genes were encoded in the new rice cultivar, RB207. The fact that 60% of the differentially expressed spots between R207 and RB207, however, were verified as the proteins involved in metabolism and photosynthesis makes a rather convincing argument that the DNA fragments transferred from E. crusgalli to rice are responsible for exerting the unknown influence to the expression of rice genes.