Genomic resources of broomcorn millet: demonstration and application of a high-throughput BAC mapping pipeline.

BACKGROUND: With high-efficient water-use and drought tolerance, broomcorn millet has emerged as a candidate for food security. To promote its research process for molecular breeding and functional research, a comprehensive genome resource is of great importance. RESULTS: Herein, we constructed a BAC library for broomcorn millet, generated BAC end sequences based on the clone-array pooled shotgun sequencing strategy and Illumina sequencing technology, and integrated BAC clones into genome by a novel pipeline for BAC end profiling. The BAC library consisted of 76,023 clones with an average insert length of 123.48 Kb, covering about 9.9-fold of the 850 Mb genome. Of 9216 clones tested using our pipeline, 8262 clones were mapped on the broomcorn millet cultivar longmi4 genome. These mapped clones covered 308 of the 829 gaps left by the genome. To our knowledge, this is the only BAC resource for broomcorn millet. CONCLUSIONS: We constructed a high-quality BAC libraray for broomcorn millet and designed a novel pipeline for BAC end profiling. BAC clones can be browsed and obtained from our website ( http://eightstarsbio.com/gresource/JBrowse-1.16.5/index.html ). The high-quality BAC clones mapped on genome in this study will provide a powerful genomic resource for genome gap filling, complex segment sequencing, FISH, functional research and genetic engineering of broomcorn millet.

F-Box Gene D5RF Is Regulated by Agrobacterium Virulence Protein VirD5 and Essential for Agrobacterium-Mediated Plant Transformation.

We previously reported that the Agrobacterium virulence protein VirD5 possesses transcriptional activation activity, binds to a specific DNA element D5RE, and is required for Agrobacterium-mediated stable transformation, but not for transient transformation. However, direct evidence for a role of VirD5 in plant transcriptional regulation has been lacking. In this study, we found that the Arabidopsis gene D5RF (coding for VirD5 response F-box protein, At3G49480) is regulated by VirD5. D5RF has two alternative transcripts of 930 bp and 1594 bp that encode F-box proteins of 309 and 449 amino acids, designated as D5RF.1 and D5RF.2, respectively. D5RF.2 has a N-terminal extension of 140 amino acids compared to D5RF.1, and both of them are located in the plant cell nucleus. The promoter of the D5RF.1 contains two D5RE elements and can be activated by VirD5. The expression of D5RF is downregulated when the host plant is infected with virD5 deleted Agrobacterium. Similar to VirD5, D5RF also affects the stable but not transient transformation efficiency of Agrobacterium. Some pathogen-responsive genes are downregulated in the d5rf mutant. In conclusion, this study further confirmed Agrobacterium VirD5 as the plant transcription activator and identified Arabidopsis thalianaD5RF.1 as the first target gene of VirD5 in regulation.

NAD(P)H-dependent thioredoxin-disulfide reductase TrxR is essential for tellurite and selenite reduction and resistance in Bacillus sp. Y3.

Microbial reduction of selenite [Se(IV)] and tellurite [Te(IV)] to Se(0) and Te(0) can function as a detoxification mechanism and serve in energy conservation. In this study, Bacillus sp. Y3 was isolated and demonstrated to have an ability of simultaneous reduction of Se(IV) and Te(IV) during aerobic cultivation, with reduction efficiencies of 100% and 90%, respectively. Proteomics analysis revealed that the putative thioredoxin disulfide reductase (TrxR) and sulfate and energy metabolic pathway proteins were significantly upregulated after the addition of Se(IV) and Te(IV). qRT-PCR also showed an increased trxR transcription level in the presence of Se(IV) and Te(IV). Compared with a wild-type Escherichia coli strain, the TrxR-overexpressed E. coli strain showed higher Se(IV) and Te(IV) resistance levels and reduction efficiencies. Additionally, the TrxR showed in vitro Se(IV) and Te(IV) reduction activities when NADPH or NADH were present. When NADPH was used as the electron donor, the optimum conditions for enzyme activities were pH 8.0 and 37°C. The Km values of Te(IV) and Se(IV) were 16.31 and 2.91 mM, and the Vmax values of Te(IV) and Se(IV) were 12.23 and 11.20 µM min-1 mg-1, respectively. The discovery of the new reductive enzyme TrxR enriches the repertoire of the bacterial Se(IV) and Te(IV) resistance and reduction mechanisms. Bacillus sp. Y3 can efficiently reduce Se(IV) and Te(IV) simultaneously. Strain Y3 provides potential applications for selenite and tellurite bioremediation. The TrxR enzyme shows high catalytic activity for reducing Se(IV) and Te(IV). The discovery of TrxR enriches the bacterial Se(IV) and Te(IV) reduction mechanisms.

High-throughput long paired-end sequencing of a Fosmid library by PacBio.

BACKGROUND: Large insert paired-end sequencing technologies are important tools for assembling genomes, delineating associated breakpoints and detecting structural rearrangements. To facilitate the comprehensive detection of inter- and intra-chromosomal structural rearrangements or variants (SVs) and complex genome assembly with long repeats and segmental duplications, we developed a new method based on single-molecule real-time synthesis sequencing technology for generating long paired-end sequences of large insert DNA libraries. RESULTS: A Fosmid vector, pHZAUFOS3, was developed with the following new features: (1) two 18-bp non-palindromic I-SceI sites flank the cloning site, and another two sites are present in the skeleton of the vector, allowing long DNA inserts (and the long paired-ends in this paper) to be recovered as single fragments and the vector (~ 8 kb) to be fragmented into 2-3 kb fragments by I-SceI digestion and therefore was effectively removed from the long paired-ends (5-10 kb); (2) the chloramphenicol (Cm) resistance gene and replicon (oriV), necessary for colony growth, are located near the two sides of the cloning site, helping to increase the proportion of the paired-end fragments to single-end fragments in the paired-end libraries. Paired-end libraries were constructed by ligating the size-selected, mechanically sheared pooled Fosmid DNA fragments to the Ampicillin (Amp) resistance gene fragment and screening the colonies with Cm and Amp. We tested this method on yeast and Setaria italica Yugu1. Fosmid-size paired-ends with an average length longer than 2 kb for each end were generated. The N50 scaffold lengths of the de novo assemblies of the yeast and S. italica Yugu1 genomes were significantly improved. Five large and five small structural rearrangements or assembly errors spanning tens of bp to tens of kb were identified in S. italica Yugu1 including deletions, inversions, duplications and translocations. CONCLUSIONS: We developed a new method for long paired-end sequencing of large insert libraries, which can efficiently improve the quality of de novo genome assembly and identify large and small structural rearrangements or assembly errors.

OsbZIP81, A Homologue of Arabidopsis VIP1, May Positively Regulate JA Levels by Directly Targetting the Genes in JA Signaling and Metabolism Pathway in Rice.

Rice (Oryza sativa L.) is one of the most important food crops in the world. In plants, jasmonic acid (JA) plays essential roles in response to biotic and abiotic stresses. As one of the largest transcription factors (TFs), basic region/leucine zipper motif (bZIP) TFs play pivotal roles through the whole life of plant growth. However, the relationship between JA and bZIP TFs were rarely reported, especially in rice. In this study, we found two rice homologues of Arabidopsis VIP1 (VirE2-interacting protein 1), OsbZIP81, and OsbZIP84. OsbZIP81 has at least two alternative transcripts, OsbZIP81.1 and OsbZIP81.2. OsbZIP81.1 and OsbZIP84 are typical bZIP TFs, while OsbZIP81.2 is not. OsbZIP81.1 can directly bind OsPIOX and activate its expression. In OsbZIP81.1 overexpression transgenic rice plant, JA (Jasmonic Acid) and SA (Salicylic acid) were up-regulated, while ABA (Abscisic acid) was down-regulated. Moreover, Agrobacterium, Methyl Jasmonic Acid (MeJA), and PEG6000 can largely induce OsbZIP81. Based on ChIP-Seq and Random DNA Binding Selection Assay (RDSA), we identified a novel cis-element OVRE (Oryza VIP1 response element). Combining ChIP-Seq and RNA-Seq, we obtained 1332 targeted genes that were categorized in biotic and abiotic responses, including α-linolenic acid metabolism and fatty acid degradation. Together, these results suggest that OsbZIP81 may positively regulate JA levels by directly targeting the genes in JA signaling and metabolism pathway in rice.

A nucleotide-binding site-leucine-rich repeat receptor pair confers broad-spectrum disease resistance through physical association in rice.

Rice blast caused by Magnaporthe oryzae is the most destructive fungal disease in crops, greatly threatening rice production and food security worldwide. The identification and utilization of broad-spectrum resistance genes are considered to be the most economic and effective method to control the disease. In the past decade, many blast resistance ( R) genes have been identified, which mainly encode nucleotide-binding leucine-rich repeat (NLR) receptor family and confer limited race-specific resistance to the fungal pathogen. Resistance genes conferring broad-spectrum blast resistance are still largely lacking. In this study, we carried out a map-based cloning of the new blast R locus Pizh in variety ZH11. A bacterial artificial chromosome (BAC) clone of 165 kb spanning the Pizh locus was sequenced and identified 9 NLR genes, among which only Pizh-1 and Pizh-2 were expressed. Genetic complementation experiments indicated that Pizh-1 but not Pizh-2 alone could confer blast resistance. Intriguingly, both mutations on Pizh-1 and Pizh-2 by CRISPR-Cas9 abolished the Pizh-mediated resistance. We also observed that Pizh-1-mediated resistance was partially dependent on Pizh-2. Pizh-1 and Pizh-2 form a complex of NLRs through direct interaction. This suggests that Pizh-1 may function as the executor NLR and Pizh-2 as a 'helper' NLR that shares functional redundancy with other NLRs. Our current study provides not only a good tool for rice disease resistance breeding but also deep insight into NLR association and function in plant immunity. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

Construction of a Genomic Bacterial Artificial Chromosome (BAC) Library for the Prawn Macrobrachium rosenbergii and Initial Analysis of ZW Chromosome-Derived BAC Inserts.

Knowledge on sex determination has proven valuable for commercial production of the prawn Macrobrachium rosenbergii due to sex dimorphism of the male and female individuals. Previous studies indicated that prawn sex is determined by a ZW-ZZ chromosomal system, but no genomic information is available for the sex chromosome. Herein, we constructed a genomic bacterial artificial chromosome (BAC) library and identified the ZW-derived BAC clones for initial analysis of the sex chromosomal DNA sequence. The arrayed BAC library contains 200,448 clones with average insert size of 115.4 kb, corresponding to ∼ 4× coverage of the estimated 5.38 Gb genome. Based on a short female-specific marker, a Z- and a W-fragment were retrieved with the genomic walking method. Screening the BAC library using a ZW-specific marker as probe resulted in 12 positive clones. From these, a Z-derived (P331M17) and a W-derived (P122G2) BAC clones were randomly selected and sequenced by PacBio method. We report the construction of a large insert, deep-coverage, and high-quality BAC library for M. rosenbergii that provides a useful resource for positional cloning of target genes, genomic organization, and comparative genomics analysis. Our study not only confirmed the ZW/ZZ system but also discovered sex-linked genes on ZW chromosomes for the first time, contributing to a comprehensive understanding of the genomic structure of sex chromosomes in M. rosenbergii.

The rice "fruit-weight 2.2-like" gene family member OsFWL4 is involved in the translocation of cadmium from roots to shoots.

MAIN CONCLUSION: Heterogeneous expression of the rice genes "fruit-weight 2.2-like" (OsFWL) affects Cd resistance in yeast, and OsFWL4 mediates the translocation of Cd from roots to shoots. Cadmium (Cd) induces chronic and toxic effects in humans. In a previous study (Xu et al. in Planta 238:643-655, 2013), we cloned the rice genes, designated OsFWL1-8, homologous to the tomato fruit-weight 2.2. Here, we show that expression of genes OsFWL3-7 in yeast confers resistance to Cd. The Cd contents of OsFWL3-, -4-, -6- and -7-transformed Cd(II)-sensitive yeast mutant ycf1 cells were strongly decreased compared with those of empty vector, with the strongest resistance to Cd observed in cells expressing OsFWL4. Evaluation of truncated and site-directed mutation derivatives revealed that the CCXXG motifs near the second transmembrane region of OsFWL4 are involved in Cd resistance in yeast. Real-time PCR analysis showed that OsFWL4 expression was induced by CdCl2 stress in rice seedlings. Compared with WT plants, the Cd contents in the shoots of RNAi mediated OsFWL4 knockdown plants were significantly decreased, and Cd translocation from roots to shoots was reduced. According to bimolecular fluorescence complementation, yeast two-hybrid and Western-blotting assays, the OsFWL4 protein forms homo-oligomers. These results suggest that OsFWL4 might act directly as a transporter and is involved in the translocation of Cd from roots to shoots in rice.

VirD5 is required for efficient Agrobacterium infection and interacts with Arabidopsis VIP2.

During Agrobacterium (Agrobacterium tumefaciens) infection, the translocated virulence proteins (VirD2, VirE2, VirE3, VirF and VirD5) play crucial roles. It is thought that, through protein-protein interactions, Agrobacterium uses and abuses host plant factors and systems to facilitate its infection. Although some molecular functions have been revealed, the roles of VirD5 still need to be further elucidated. Here, plant transformation and tumorigenesis mediated by genetically modified Agrobacterium strains were performed to examine VirD5 roles. In addition, protein-protein interaction-associated molecular and biochemistry technologies were used to reveal and elucidate VirD5 interaction with Arabidopsis VirE2 interacting protein 2 (VIP2). Our results showed that deleting virD5 from Agrobacterium reduced its tumor formation ability and stable transformation efficiency but did not affect the transient transformation efficiency. We also found that VirD5 can interact with Arabidopsis VIP2. Further experiments demonstrated that VirD5 can affect VIP2 binding to cap-binding proteins (CBP20 and CBP80). The tumorigenesis efficiency for cbp80 mutant was not significantly changed, but that for cbp20, cbp20cbp80 mutants were significantly increased. This work demonstrates experimentally that VirD5 is required for efficient Agrobacterium infection and may promote this process by competitive interaction with Arabidopsis VIP2. CBP20 is involved in the Agrobacterium infection process and its effect can be synergistically enhanced by CBP80.

The asparagus genome sheds light on the origin and evolution of a young Y chromosome.

Sex chromosomes evolved from autosomes many times across the eukaryote phylogeny. Several models have been proposed to explain this transition, some involving male and female sterility mutations linked in a region of suppressed recombination between X and Y (or Z/W, U/V) chromosomes. Comparative and experimental analysis of a reference genome assembly for a double haploid YY male garden asparagus (Asparagus officinalis L.) individual implicates separate but linked genes as responsible for sex determination. Dioecy has evolved recently within Asparagus and sex chromosomes are cytogenetically identical with the Y, harboring a megabase segment that is missing from the X. We show that deletion of this entire region results in a male-to-female conversion, whereas loss of a single suppressor of female development drives male-to-hermaphrodite conversion. A single copy anther-specific gene with a male sterile Arabidopsis knockout phenotype is also in the Y-specific region, supporting a two-gene model for sex chromosome evolution.

Heterologous expression of Avermectins biosynthetic gene cluster by construction of a Bacterial Artificial Chromosome library of the producers.

Avermectins, a group of polyketide natural products, are widely used as anthelmintics in agriculture. Metabolic engineering and combinatorial biosynthesis were extensively employed to improve Avermectins production and create novel Avermectin derivatives, including Ivermectin and Doramectin. It is labor intensive and time cost to genetically manipulate Avermectins producer Streptomyces avermitilis in vivo. Cloning and heterologous expression of Avermectins biosynthetic gene cluster will make it possible to tailor the cluster in vitro. We constructed a Bacterial Artificial Chromosome (BAC) library of S. avermitilis ATCC 31267 with inserted DNA fragments ranged from 100 to 130 Kb. Five recombinant BAC clones which carried the Avermectins biosynthetic gene cluster ave (81 Kb in size) were screened out from the library. Then, ave was hetero-expressed in S. lividans. Three Avermectin components, A2a, B1a and A1a were detected from the cell extracts of recombinant strains. It will facilitate the development of Avermectin derivatives by polyketide synthase domain swapping and provide functional element for Avermectins synthetic biology study.

1-N-histidine phosphorylation of ChlD by the AAA+ ChlI2 stimulates magnesium chelatase activity in chlorophyll synthesis.

Magnesium chelatase (Mg-chelatase) inserts magnesium into protoporphyrin during the biosynthesis of chlorophyll and bacteriochlorophyll. Enzyme activity is reconstituted by forming two separate preactivated complexes consisting of a GUN4/ChlH/protoporphyrin IX substrate complex and a ChlI/ChlD enzyme 'motor' complex. Formation of the ChlI/ChlD complex in both Chlamydomonas reinhardtii and Oryza sativa is accompanied by phosphorylation of ChlD by ChlI, but the orthologous protein complex from Rhodobacter capsulatus, BchI/BchD, gives no detectable phosphorylation of BchD. Phosphorylation produces a 1-N-phospho-histidine within ChlD. Proteomic analysis indicates that phosphorylation occurs at a conserved His residue in the C-terminal integrin I domain of ChlD. Comparative analysis of the ChlD phosphorylation with enzyme activities of various ChlI/ChlD complexes correlates the phosphorylation by ChlI2 with stimulation of Mg-chelatase activity. Mutation of the H641 of CrChlD to E641 prevents both phosphorylation and stimulation of Mg-chelatase activity, confirming that phosphorylation at H641 stimulates Mg-chelatase. The properties of ChlI2 compared with ChlI1 of Chlamydomonas and with ChlI of Oryza, shows that ChlI2 has a regulatory role in Chlamydomonas.

An integrated analysis of QTL mapping and RNA sequencing provides further insights and promising candidates for pod number variation in rapeseed (Brassica napus L.).

BACKGROUND: As the most important yield component in rapeseed (Brassica napus L.), pod number is determined by a series of successive growth and development processes. Pod number shows extensive variation in rapeseed natural germplasm, which is valuable for genetic improvement. However, the genetic and especially the molecular mechanism for this kind of variation are poorly understood. In this study, we conducted QTL mapping and RNA sequencing, respectively, using the BnaZNRIL population and its two parental cultivars Zhongshuang11 and No.73290 which showed significant difference in pod number, primarily due to the difference in floral organ number. RESULT: A total of eight QTLs for pod number were identified using BnaZNRIL population with a high-density SNP linkage map, each was distributed on seven linkage groups and explained 5.8-11.9% of phenotypic variance. Then, they were integrated with those previously detected in BnaZNF2 population (deriving from same parents) and resulted in 15 consensus-QTLs. Of which, seven QTLs were identical to other studies, whereas the other eight should be novel. RNA sequencing of the shoot apical meristem (SAM) at the formation stage of floral bud primordia identified 9135 genes that were differentially expressed between the two parents. Gene ontology (GO) analysis showed that the top two enriched groups were S-assimilation, providing an essential nutrient for the synthesis of diverse metabolites, and polyamine metabolism, serving as second messengers that play an essential role in flowering genes initiation. KEGG analysis showed that the top three overrepresented pathways were carbohydrate (707 genes), amino acid (390 genes) and lipid metabolisms (322 genes). In silico mapping showed that 647 DEGs were located within the confidence intervals of 15 consensus QTLs. Based on annotations of Arabidopsis homologs corresponding to DEGs, nine genes related to meristem growth and development were considered as promising candidates for six QTLs. CONCLUSION: In this study, we discovered the first repeatable major QTL for pod number in rapeseed. In addition, RNA sequencing was performed for SAM in rapeseed, which provides new insights into the determination of floral organ number. Furthermore, the integration of DEGs and QTLs identified promising candidates for further gene cloning and mechanism study.

pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping.

Applications that use Bacterial Artificial Chromosome (BAC) libraries often require paired-end sequences and knowledge of the physical location of each clone in plates. To facilitate obtaining this information in high-throughput, we generated pBACode vectors: a pool of BAC cloning vectors, each with a pair of random barcodes flanking its cloning site. In a pBACode BAC library, the BAC ends and their linked barcodes can be sequenced in bulk. Barcode pairs are determined by sequencing the empty pBACode vectors, which allows BAC ends to be paired according to their barcodes. For physical clone mapping, the barcodes are used as unique markers for their linked genomic sequence. After multi-dimensional pooling of BAC clones, the barcodes are sequenced and deconvoluted to locate each clone. We generated a pBACode library of 94,464 clones for the flounder Paralichthys olivaceus and obtained paired-end sequence from 95.4% of the clones. Incorporating BAC paired-ends into the genome preassembly improved its continuity by over 10-fold. Furthermore, we were able to use the barcodes to map the physical locations of each clone in just 50 pools, with up to 11 808 clones per pool. Our physical clone mapping located 90.2% of BAC clones, enabling targeted characterization of chromosomal rearrangements.

Whole Genome Mapping with Feature Sets from High-Throughput Sequencing Data.

A good physical map is essential to guide sequence assembly in de novo whole genome sequencing, especially when sequences are produced by high-throughput sequencing such as next-generation-sequencing (NGS) technology. We here present a novel method, Feature sets-based Genome Mapping (FGM). With FGM, physical map and draft whole genome sequences can be generated, anchored and integrated using the same data set of NGS sequences, independent of restriction digestion. Method model was created and parameters were inspected by simulations using the Arabidopsis genome sequence. In the simulations, when ~4.8X genome BAC library including 4,096 clones was used to sequence the whole genome, ~90% of clones were successfully connected to physical contigs, and 91.58% of genome sequences were mapped and connected to chromosomes. This method was experimentally verified using the existing physical map and genome sequence of rice. Of 4,064 clones covering 115 Mb sequence selected from ~3 tiles of 3 chromosomes of a rice draft physical map, 3,364 clones were reconstructed into physical contigs and 98 Mb sequences were integrated into the 3 chromosomes. The physical map-integrated draft genome sequences can provide permanent frameworks for eventually obtaining high-quality reference sequences by targeted sequencing, gap filling and combining other sequences.

Building two indica rice reference genomes with PacBio long-read and Illumina paired-end sequencing data.

Over the past 30 years, we have performed many fundamental studies on two Oryza sativa subsp. indica varieties, Zhenshan 97 (ZS97) and Minghui 63 (MH63). To improve the resolution of many of these investigations, we generated two reference-quality reference genome assemblies using the most advanced sequencing technologies. Using PacBio SMRT technology, we produced over 108 (ZS97) and 174 (MH63) Gb of raw sequence data from 166 (ZS97) and 209 (MH63) pools of BAC clones, and generated ~97 (ZS97) and ~74 (MH63) Gb of paired-end whole-genome shotgun (WGS) sequence data with Illumina sequencing technology. With these data, we successfully assembled two platinum standard reference genomes that have been publicly released. Here we provide the full sets of raw data used to generate these two reference genome assemblies. These data sets can be used to test new programs for better genome assembly and annotation, aid in the discovery of new insights into genome structure, function, and evolution, and help to provide essential support to biological research in general.

Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63.

Asian cultivated rice consists of two subspecies: Oryza sativa subsp. indica and O. sativa subsp. japonica Despite the fact that indica rice accounts for over 70% of total rice production worldwide and is genetically much more diverse, a high-quality reference genome for indica rice has yet to be published. We conducted map-based sequencing of two indica rice lines, Zhenshan 97 (ZS97) and Minghui 63 (MH63), which represent the two major varietal groups of the indica subspecies and are the parents of an elite Chinese hybrid. The genome sequences were assembled into 237 (ZS97) and 181 (MH63) contigs, with an accuracy >99.99%, and covered 90.6% and 93.2% of their estimated genome sizes. Comparative analyses of these two indica genomes uncovered surprising structural differences, especially with respect to inversions, translocations, presence/absence variations, and segmental duplications. Approximately 42% of nontransposable element related genes were identical between the two genomes. Transcriptome analysis of three tissues showed that 1,059-2,217 more genes were expressed in the hybrid than in the parents and that the expressed genes in the hybrid were much more diverse due to their divergence between the parental genomes. The public availability of two high-quality reference genomes for the indica subspecies of rice will have large-ranging implications for plant biology and crop genetic improvement.

GUN4-Protoporphyrin IX Is a Singlet Oxygen Generator with Consequences for Plastid Retrograde Signaling.

The genomes uncoupled 4 (GUN4) protein is a nuclear-encoded, chloroplast-localized, porphyrin-binding protein implicated in retrograde signaling between the chloroplast and nucleus, although its exact role in this process is still unclear. Functionally, it enhances Mg-chelatase activity in the chlorophyll biosynthesis pathway. Because GUN4 is present only in organisms that carry out oxygenic photosynthesis and because it binds protoporphyrin IX (PPIX) and Mg-PPIX, it has been suggested that it prevents production of light- and PPIX- or Mg-PPIX-dependent reactive oxygen species. A chld-1/GUN4 mutant with elevated PPIX has a light-dependent up-regulation of GUN4, implicating this protein in light-dependent sensing of PPIX, with the suggestion that GUN4 reduces PPIX-generated singlet oxygen, O2(a(1)Δg), and subsequent oxidative damage (Brzezowski, P., Schlicke, H., Richter, A., Dent, R. M., Niyogi, K. K., and Grimm, B. (2014) Plant J. 79, 285-298). In direct contrast, our results show that purified GUN4 and oxidatively damaged ChlH increase the rate of PPIX-generated singlet oxygen production in the light, by a factor of 5 and 10, respectively, when compared with PPIX alone. Additionally, the functional GUN4-PPIX-ChlH complex and ChlH-PPIX complexes generate O2(a(1)Δg) at a reduced rate when compared with GUN4-PPIX. As O2(a(1)Δg) is a potential plastid-to-nucleus signal, possibly through second messengers, light-dependent O2(a(1)Δg) generation by GUN4-PPIX is proposed to be part of a signal transduction pathway from the chloroplast to the nucleus. GUN4 thus senses the availability and flux of PPIX through the chlorophyll biosynthetic pathway and also modulates Mg-chelatase activity. The light-dependent O2(a(1)Δg) generation from GUN4-PPIX is thus proposed as the first step in retrograde signaling from the chloroplast to the nucleus.

Genomic information of the arsenic-resistant bacterium Lysobacter arseniciresistens type strain ZS79(T) and comparison of Lysobacter draft genomes.

Lysobacter arseniciresistens ZS79(T) is a highly arsenic-resistant,rod-shaped, motile, non-spore-forming, aerobic, Gram-negative bacterium. In this study, four Lysobacter type strains were sequenced and the genomic information of L. arseniciresistens ZS79(T) and the comparative genomics results of the Lysobacter strains were described. The draft genome sequence of the strain ZS79(T) consists of 3,086,721 bp and is distributed in 109 contigs. It has a G+C content of 69.5 % and contains 2,363 protein-coding genes including eight arsenic resistant genes.

High correlation between genotypes and phenotypes of environmental bacteria Comamonas testosteroni strains.

BACKGROUND: Members of Comamonas testosteroni are environmental microorganisms that are usually found in polluted environment samples. They utilize steroids and aromatic compounds but rarely sugars, and show resistance to multiple heavy metals and multiple drugs. However, comprehensive genomic analysis among the C. testosteroni strains is lacked. RESULTS: To understand the genome bases of the features of C. testosteroni, we sequenced 10 strains of this species and analyzed them together with other related published genome sequences. The results revealed that: 1) the strains of C. testosteroni have genome sizes ranging from 5.1 to 6.0 Mb and G + C contents ranging from 61.1% to 61.8%. The pan-genome contained 10,165 gene families and the core genome contained 3,599 gene families. Heap's law analysis indicated that the pan-genome of C. testosteroni may be open (α = 0.639); 2) by analyzing 31 phenotypes of 11 available C. testosteroni strains, 99.4% of the genotypes (putative genes) were found to be correlated to the phenotypes, indicating a high correlation between phenotypes and genotypes; 3) gene clusters for nitrate reduction, steroids degradation and metal and multi-drug resistance were found and were highly conserved among all the genomes of this species; 4) the genome similarity of C. testosteroni may be related to the geographical distances. CONCLUSIONS: This work provided an overview on the genomes of C. testosteroni and new genome resources that would accelerate the further investigations of this species. Importantly, this work focused on the analysis of potential genetic determinants for the typical characters and found high correlation between the phenotypes and their corresponding genotypes.