Deletion of a target gene in Indica rice via CRISPR/Cas9.

KEY MESSAGE: Using CRISPR/Cas9, we successfully deleted large fragments of the yield-related gene DENSE AND ERECT PANICLE1 in Indica rice at relatively high frequency and generated gain-of-function dep1 mutants. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a rapidly developing technology used to produce gene-specific modifications in both mammalian and plant systems. Most CRISPR-induced modifications in plants reported to date have been small insertions or deletions. Few large target gene deletions have thus far been reported, especially for Indica rice. In this study, we designed multiple CRISPR sgRNAs and successfully deleted DNA fragments in the gene DENSE AND ERECT PANICLE1 (DEP1) in the elite Indica rice line IR58025B. We achieved deletion frequencies of up to 21% for a 430 bp target and 9% for a 10 kb target among T0 events. Constructs with four sgRNAs did not generate higher full-length deletion frequencies than constructs with two sgRNAs. The multiple mutagenesis frequency reached 93% for four targets, and the homozygous mutation frequency reached 21% at the T0 stage. Important yield-related trait characteristics, such as dense and erect panicles and reduced plant height, were observed in dep1 homozygous T0 mutant plants produced by CRISPR/Cas9. Therefore, we successfully obtained deletions in DEP1 in the Indica background using the CRISPR/Cas9 editing tool at relatively high frequency.

Genome sequence and transcriptome analysis of the radioresistant bacterium Deinococcus gobiensis: insights into the extreme environmental adaptations.

The desert is an excellent model for studying evolution under extreme environments. We present here the complete genome and ultraviolet (UV) radiation-induced transcriptome of Deinococcus gobiensis I-0, which was isolated from the cold Gobi desert and shows higher tolerance to gamma radiation and UV light than all other known microorganisms. Nearly half of the genes in the genome encode proteins of unknown function, suggesting that the extreme resistance phenotype may be attributed to unknown genes and pathways. D. gobiensis also contains a surprisingly large number of horizontally acquired genes and predicted mobile elements of different classes, which is indicative of adaptation to extreme environments through genomic plasticity. High-resolution RNA-Seq transcriptome analyses indicated that 30 regulatory proteins, including several well-known regulators and uncharacterized protein kinases, and 13 noncoding RNAs were induced immediately after UV irradiation. Particularly interesting is the UV irradiation induction of the phrB and recB genes involved in photoreactivation and recombinational repair, respectively. These proteins likely include key players in the immediate global transcriptional response to UV irradiation. Our results help to explain the exceptional ability of D. gobiensis to withstand environmental extremes of the Gobi desert, and highlight the metabolic features of this organism that have biotechnological potential.

Corynebacterium deserti sp. nov., isolated from desert sand.

A novel coryneform bacterium, designated strain GIMN1.010T, was isolated from a sand sample collected in the desert in the west of China. Cells were Gram-stain-positive, non-spore-forming, catalase-positive, irregular rods. Comparative 16S rRNA gene sequence analysis demonstrated that strain GIMN1.010T belonged to the genus Corynebacterium and was related closely to Corynebacterium glutamicum ATCC 13032T (98.4 % similarity). However, the level of DNA-DNA relatedness between strain GIMN1.010T and C. glutamicum ATCC 13032T was only 22.4±1.72 %, showing that strain GIMN1.010T represented a genomic species distinct from C. glutamicum. On the basis of phenotypic and phylogenetic data, strain GIMN1.010T is considered to represent a novel species of the genus Corynebacterium, for which the name Corynebacterium deserti sp. nov. is proposed. The type strain is GIMN1.010T (=CCTCC AB 2010341T=NRRL B-59552).

Complete genome sequence of the nitrogen-fixing and rhizosphere-associated bacterium Pseudomonas stutzeri strain DSM4166.

We present here the analysis of the whole-genome sequence of Pseudomonas stutzeri strain DSM4166, a diazotrophic isolate from the rhizosphere of a Sorghum nutans cultivar. To our knowledge, this is the second genome to be sequenced for P. stutzeri. The availability and analysis of the genome provide insight into the evolution of the nitrogen fixation property and identification of rhizosphere competence traits required in interactions with host plants.

Genome-wide transcriptome and proteome analysis of Escherichia coli expressing IrrE, a global regulator of Deinococcus radiodurans.

Gram-negative bacterium Escherichia coli and the Gram-positive Deinococcus radiodurans fundamentally differ in their cell structures and gene regulations. We have previously reported that IrrE, a Deinococcus genus-specific global regulator, confers significantly enhanced tolerance to various abiotic stresses. To better understand the global effects of IrrE on the regulatory networks, we carried out combined transcriptome and proteome analysis of E. coli expressing the IrrE protein. Our analysis showed that 216 (4.8%) of all E. coli genes were induced and 149 (3.3%) genes were repressed, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon source utilization, amino acid utilization, acid resistance, a hydrogenase and an oxidase. Also regulated were the EvgSA two-component system, the GadE, GadX and PurR master regulators, and 10 transcription factors (AppY, GadW, YhiF, AsnC, BetI, CynR, MhpR, PrpR, TdcA and KdgR). These results demonstrated that IrrE acts as global regulator and consequently improves abiotic stress tolerances in the heterologous host E. coli. The implication of our findings is discussed in relation to the evolutionary role of horizontal gene transfer in bacterial regulatory networks and environmental adaptation.

Genome sequence of Acinetobacter calcoaceticus PHEA-2, isolated from industry wastewater.

Genome analysis of Acinetobacter calcoaceticus PHEA-2 was undertaken because of the importance of this bacterium for bioremediation of phenol-polluted water and because of the close phylogenetic relationship of this species with the human pathogen Acinetobacter baumannii. To our knowledge, this is the first strain of A. calcoaceticus whose genome has been sequenced.

Microbacterium radiodurans sp. nov., a UV radiation-resistant bacterium isolated from soil.

Strain GIMN 1.002(T), a UV radiation-tolerant bacterium, was isolated from the upper sand layers of the Gobi desert, Xinjiang, China and characterized in order to determine its taxonomic position. Cells were Gram-reaction-positive, heterotrophic, strictly aerobic, short rods. 16S rRNA gene sequence analysis revealed that strain GIMN 1.002(T) belonged to the genus Microbacterium and was closely related to Microbacterium arborescens DSM 20754(T) (98.8 % 16S rRNA gene sequence similarity) and Microbacterium imperiale DSM 20530(T) (98.7 %). However, strain GIMN 1.002(T) had low DNA-DNA relatedness with M. arborescens DSM 20754(T) (17.1 %) and M. imperiale DSM 20530(T) (12.89 %). Strain GIMN 1.002(T) possessed chemotaxonomic markers that were consistent with its classification in the genus Microbacterium, i.e. MK-11, MK-12 and MK-10 as major menaquinones and anteiso-C(15 : 0) (38.67 %), iso-C(16 : 0) (18.16 %) and iso-C(15 : 0) (17.46 %) as predominant cellular fatty acids. The DNA G+C content was 67.74 mol%. The cell-wall sugar was rhamnose. On the basis of the data from this study, strain GIMN 1.002(T) represents a novel species of the genus Microbacterium, for which the name Microbacterium radiodurans sp. nov. is proposed. The type strain is GIMN 1.002(T) (=CCTCC M208212(T) =NRRL B-24799(T)).

Deinococcus gobiensis sp. nov., an extremely radiation-resistant bacterium.

A Gram-positive, non-motile, spherical, red-pigmented and facultatively anaerobic bacterium, designated strain I-0(T), was isolated from a sand sample of the Gobi desert in Xinjiang Autonomous Region, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this isolate represents a novel member of the genus Deinococcus, with low sequence similarities (<94 %) to recognized Deinococcus species. The major cellular fatty acids were C(16 : 1)omega7c and C(16 : 0). Its polar lipid profile contained several unidentified glycolipids, phosphoglycolipids, phospholipids, pigments and an aminophospholipid. The peptidoglycan type was Orn-Gly(2) (A3beta) and the predominant respiratory quinone was MK-8. The DNA G+C content was 65.4 mol%. DNA-DNA relatedness between strain I-0(T) and Deinococcus radiodurans ACCC 10492(T) was 37 %. The strain was shown to be extremely resistant to gamma radiation (>15 kGy) and UV light (>600 J m(-2)). On the basis of the phylogenetic, chemotaxonomic and phenotypic data presented, strain I-0(T) represents a novel species of the genus Deinococcus, for which the name Deinococcus gobiensis sp. nov. is proposed. The type strain is I-0(T) (=DSM 21396(T) =CGMCC 1.7299(T)).

IrrE, a global regulator of extreme radiation resistance in Deinococcus radiodurans, enhances salt tolerance in Escherichia coli and Brassica napus.

BACKGROUND: Globally, about 20% of cultivated land is now affected by salinity. Salt tolerance is a trait of importance to all crops in saline soils. Previous efforts to improve salt tolerance in crop plants have met with only limited success. Bacteria of the genus Deinococcus are known for their ability to survive highly stressful conditions, and therefore possess a unique pool of genes conferring extreme resistance. In Deinococcus radiodurans, the irrE gene encodes a global regulator responsible for extreme radioresistance. METHODOLOGY/PRINCIPAL FINDINGS: Using plate assays, we showed that IrrE protected E. coli cells against salt shock and other abiotic stresses such as oxidative, osmotic and thermal shocks. Comparative proteomic analysis revealed that IrrE functions as a switch to regulate different sets of proteins such as stress responsive proteins, protein kinases, glycerol-degrading enzymes, detoxification proteins, and growth-related proteins in E. coli. We also used quantitative RT-PCR to investigate expression of nine selected stress-responsive genes in transgenic and wild-type Brassica napus plants. Transgenic B. napus plants expressing the IrrE protein can tolerate 350 mM NaCl, a concentration that inhibits the growth of almost all crop plants. CONCLUSIONS: Expression of IrrE, a global regulator for extreme radiation resistance in D. radiodurans, confers significantly enhanced salt tolerance in both E. coli and B. napus. We thus propose that the irrE gene might be used as a potentially promising transgene to improve abiotic stress tolerances in crop plants.