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Six circular mitochondrial genomes of multi-, bi-, and uninucleate Rhizoctonia isolates were assembled and found that all the genomes contain 14 conserved protein-coding genes, one ribosomal protein (rps3), and 23 tRNA in the same order. The mitogenome sizes of uninucleate isolates were relatively smaller than binucleate and multinucleate stains. The size variations between uninucleate and multinucleate isolates were from both intergenic and intronic regions, whereas the differences between uninucleate and binucleate isolates were predominantly from intergenic regions. The phylogenetic analysis revealed that Rhizoctonia strains of the same nucleate types had a closer relationship.

The WRKY transcription factor (TF) gene family expanded greatly in the evolutionary process from green algae to flowering plants through whole genome, segmental, and tandem duplications. Genomic sequences from diverse plant species provide valuable information about the origin and evolution of WRKY domain-containing proteins. Accumulating data indicate that WRKY TFs bind W-box and/or other cis-elements, revealing the specificity of cis-element recognition based on the structure of the WRKY protein as well as the nucleotides adjacent to the core sequences. Physical interactions among WRKY proteins or between WRKY proteins and other regulatory factors afford important insights into the regulation of this TF family. WRKY proteins are essential players in the kinase signaling network. The interaction of plant resistance (R) proteins with WRKY TFs and the existence of unusual chimeric R-WRKY proteins suggest diversity in signaling pathways for rapid immune responses. In this review, we focus mainly on progress in understanding the function of WRKY TFs in response to biotic stresses and focus on their multiple roles in Arabidopsis and rice plants.

The basidiomycetous fungal genus, Rhizoctonia, can cause severe damage to many plants and is composed of multinucleate, binucleate, and uninucleate species differing in pathogenicity. Here we generated chromosome-scale genome assemblies of the three nuclear types of Rhizoctonia isolates. The genomic comparisons revealed that the uninucleate JN strain likely arose by somatic hybridization of two binucleate isolates, and maintained a diploid nucleus. Homeolog gene pairs in the JN genome have experienced both decelerated or accelerated evolution. Homeolog expression dominance occurred between JN subgenomes, in which differentially expressed genes show potentially less evolutionary constraint than the genes without. Analysis of mating-type genes suggested that Rhizoctonia maintains the ancestral tetrapolarity of the Basidiomycota. Long terminal repeat-retrotransposons displayed a reciprocal correlation with the chromosomal GC content in the three chromosome-scale genomes. The more aggressive multinucleate XN strain had more genes encoding enzymes for host cell wall decomposition. These findings demonstrate some evolutionary changes of a recently derived hybrid and in multiple nuclear types of Rhizoctonia., Li et al. generate chromosome-scale genome assemblies of the three nuclear types of the basidiomycetous fungi Rhizoctonia isolates. Genomic comparisons implicate that uninucleate genomes were hybrids derived potentially from binucleate ancestors, suggesting evolutionary changes of a recently derived hybrid and in multiple nuclear types of Rhizoctonia.

Selenium is an essential trace element and micronutrient for human health. Application of organic selenium in plants and microorganisms as trace element supplement is attracting more and more attention. In this study, Bacillus cereus, an important probiotic, was used for selenium enrichment with sodium selenite as selenium source. The growth curve of B. cereus was investigated, and 150 μg/ml was selected as the concentration of selenium for B. cereus fermentation. With application of response surface methodology, the optimal fermentation conditions were obtained as follows: inoculation quantity of 7%, culture temperature of 33°C, and shaking speed of 170 rpm, leading to the maximal selenium conversion ratio of 94.3 ± 0.2%. Field emission scanning electron microscope and energy dispersive spectrometry evidenced that inorganic selenium had been successfully transformed. This study may contribute to get a strain with high Se conversion ratio, so as to extract organic selenium in the form of selenoprotein to be used for further application.

Background Ketamine has been reported to cause neonatal neurotoxicity in a variety of developing animal models. Various studies have been conducted to study the mechanism of neurotoxicity for general anesthetic use during the neonatal period. Previous experiments have suggested that developmentally generated granule neurons in the hippocampus dentate gyrus (DG) supported hippocampus-dependent memory. Therefore, this study aimed to investigate whether ketamine affects the functional integration of developmentally generated granule neurons in the DG. For this purpose,the postnatal day 7 (PND-7) Sprague-Dawley (SD) rats were divided into the control group and the ketamine group (rats who received 4 injections of 40 mg/kg ketamine at 1 h intervals). To label dividing cells, BrdU was administered for three consecutive days after the ketamine exposure; NeuN+/BrdU+cells were observed by using immunofluorescence. To evaluate the developmentally generated granule neurons that support hippocampus-dependent memory, spatial reference memory was tested by using Morris Water Maze at 3 months old, after which the immunofluorescence was used to detect c-Fos expression in the NeuN+/BrdU+ cells. The expression of caspase-3 was measured by western blot to detect the apoptosis in the hippocampal DG. Results The present results showed that the neonatal ketamine exposure did not influence the survival rate of developmentally generated granule neurons at 2 and 3 months old, but ketamine interfered with the integration of these neurons into the hippocampal DG neural circuits and caused a deficit in hippocampal-dependent spatial reference memory tasks. Conclusions In summary, these findings may promote more studies to investigate the neurotoxicity of ketamine in the developing brain.

Summary Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up‐regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)‐limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)‐limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)‐limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination.

The skeletal system is a dynamic organ that continuously undergoes coupled trabeculae and blood vessels remodeling, indicating the possible existence of molecular crosstalk between endothelial and osteoblastic cells. Since the cross-talk between bone-forming osteoblasts (OBs) and vessel-forming endothelial cells (ECs) have progressively gained investigators' attention, few studies focused on the regulatory function of extracellular vesicles derived from OBs on ECs. In this study, the effect of the exosomes derived from mature osteoblasts (MOBs) on the ECs was investigated. Firstly, exosomes derived from mature osteoblasts (MOB-Exos) were isolated and identified by NanoSight light scatter technology, electron microscopy and Western bolting. Fluorescent labeling of MOB-Exos revealed its internalization by ECs. RNA interference technique was used to knock down matrix metalloproteinase-2 (MMP2) in MOB-Exos. Then ECs were co-cultured with MOB-Exos and MMP2 knockdown MOB-Exos. Wound healing migration assay, transwell migration assay, CCK-8 assay and tube formation assay of ECs were conducted to determine the angiogenic capability of ECs. Then the VEGF/Erk1/2 pathway markers were detected by Western blot. Our results showed that MOB-Exos could promote the proliferation, migration and tube formation of ECs. Meanwhile, the promoted angiogenetic capacities of ECs were impaired when MMP2 in MOB-Exos was knocked down. In addition, immunoblotting indicated that MOB-Exos could promote the activation of the VEGF/Erk1/2 pathway of ECs; whereas the activation of the VEGF/Erk1/2 pathway was attenuated when the ECs were co-cultured with the MMP2 knockdown MOB-Exos. In conclusion, the MMP-2 existing in exosomes derived from MOBs could promote the angiogenesis of ECs in vitro, which might be realized through VEGF/Erk1/2 signaling pathway.

Maize sheath blight is a destructive disease of maize world-wide. The causal pathogens of the disease include multinucleate, binucleate and uninucleate Rhizoctonia isolates according to the latest reports. In this study, the virulence and virulence factors of one uninucleate, two binucleate and nine multinucleate Rhizoctonia isolates were compared. The results showed that there was significant difference in the virulence of the tested Rhizoctonia isolates on maize, with multinucleate Rhizoctonia isolates the most virulent, binucleate Rhizoctonia isolates moderate and uninucleate Rhizoctonia isolate the lowest. The cell wall-degrading enzymes (CWDEs) and phytotoxins of the tested isolates were extracted and their enzymatic or biological activities were detected. The correlation coefficients between the enzymatic activity of polymethylgalacturonase (PMG), polygalacturonase (PG) and endo-β-1,4-glucanase (Cx) and pathogenicity of the isolates were 0.8210, 0.7314 and 0.8497, respectively. The correlation coefficients between the inhibition rate of corn seed germination and root growth caused by the phytotoxins and pathogenicity of the isolates were 0.9257 and 0.9690, respectively. These results indicated that CWDEs and phytotoxins likely act as virulence factors during infection. In conclusion, the lowest virulence of the uninucleate Rhizoctonia isolate JN on maize is possibly due to the low activities of CADEs and phytotoxins.

Plant-pathogenic Rhizoctonia spp. are soil-borne fungi with world-wide distribution. Most Rhizoctonia spp. are multinuclear and heterokaryotic fungi, which makes it difficult to build a stable transformation system. In this study, one uninucleate Rhizoctonia isolate, JN, was recovered from a field maize plant. Single nuclear status was observed under fluorescent microscopy after the hyphae were stained with Hoechst33258 solution. The optimal growth conditions of JN mycelia were 28 °C at pH7.0 on potato dextrose agar (PDA) media. The mycelial color of JN cultured on PDA turned from white to light brown with aging. Analysis of the internal transcribed spacer (ITS) sequence of the ribosomal DNA (rDNA) showed that there were two types of ITS sequences within the single isolate and the nucleotide identity of the two sequences is only 95 %. JN is more sensitive to SDS, PEG4000, Sorbitol, hydrogen peroxide (H2O2) and sodium chloride (NaCl) than a multinucleate Rhizoctonia solani, XN5, cultured on PDA. Given the single nucleus, JN has great potential to be used in fungal transformation.

Heading date is one of the most important agronomic traits in rice. Here, we analyzed an early flowering mutant ds9 under long day (LD) conditions and found that the early flowering phenotype was controlled by a single dominant gene. Mapping the Ds9 revealed that it was located near SSR marker RM5432 on chromosome 8, close to the known heading date-related DTH8/Ghd8 locus. Sequence analysis indicated that Ds9 was allelic to Ghd8, encoding the OsHAP3H subunit of CCAAT-box binding proteins (HAPs), and transformation of ds9 mutant with the OsHAP3H gene from wild type XS11 plants significantly increased the heading date under LD conditions. Yeast two hybrid and three hybrid assays revealed that the C-terminus of OsHAP3H is required for its interactions with OsHAP2 and OsHAP5. The data demonstrated that OsHAP3H plays a critical role as a repressor of photoperiodic flowering in rice.

Metabolomic and transcriptomic approaches were used to dissect the enhanced disease resistance in the plants harbouring a RNA interfering construct of OsWRKY62 and OsWRKY76 (dsOW62/76) genes. The primary metabolic pathways were activated in dsOW62/76 compared with wild-type (ZH17) plants, revealed by increased accumulation of amino acids and constituents of citric acid cycle etc. Contents of phenolic acids derived from phenylpropanoid pathway were elevated in dsOW62/76 plants. Importantly, phenolamides, conjugates of the phenolic acids with amines, were detected in large number and mostly at higher levels in dsOW62/76 compared with ZH17 plants; however, the free pools of flavonoids were mostly decreased in dsOW62/76. Salicylic acid (SA) and jasmonic acid (JA)/JA-Ile contents were increased in dsOW62/76 and knockout lines of individual OsWRKY62 and OsWRKY76 genes. Transcription of isochorismate synthase (OsICS1) gene was suppressed in dsOW62/76 and in MeJA-treated rice plants, whereas the transcription level of cinnamoyl-CoA hydratase-dehydrogenase (OsCHD) gene for β-oxidation in peroxisome was increased. The calli with OsCHD mutation showed markedly decreased SA accumulation. These results indicate that OsWRKY62 and OsWRKY76 function as negative regulators of biosynthetic defense-related metabolites and provide evidence for an important role of phenylpropanoid pathway in SA production in rice.

Rice OsERF922, encoding an APETELA2/ethylene response factor (AP2/ERF) type transcription factor, is rapidly and strongly induced by abscisic acid (ABA) and salt treatments, as well as by both virulent and avirulent pathovars of Magnaporthe oryzae, the causal agent of rice blast disease. OsERF922 is localized to the nucleus, binds specifically to the GCC box sequence, and acts as a transcriptional activator in plant cells. Knockdown of OsERF922 by means of RNAi enhanced resistance against M. oryzae. The elevated disease resistance of the RNAi plants was associated with increased expression of PR, PAL, and the other genes encoding phytoalexin biosynthetic enzymes and without M. oryzae infection. In contrast, OsERF922-overexpressing plants showed reduced expression of these defence-related genes and enhanced susceptibility to M. oryzae. In addition, the OsERF922-overexpressing lines exhibited decreased tolerance to salt stress with an increased Na(+)/K(+) ratio in the shoots. The ABA levels were found increased in the overexpressing lines and decreased in the RNAi plants. Expression of the ABA biosynthesis-related genes, 9-cis-epoxycarotenoid dioxygenase (NCED) 3 and 4, was upregulated in the OsERF922-overexpressing plants, and NCED4 was downregulated in the RNAi lines. These results suggest that OsERF922 is integrated into the cross-talk between biotic and abiotic stress-signalling networks perhaps through modulation of the ABA levels.

Osmotin promoter binding protein 1 (OPBP1), an AP2/ERF transcription factor of tobacco, has been demonstrated to function in disease resistance and salt tolerance in tobacco. To increase stress tolerant capability of rice, we generated rice plants with an OPBP1 overexpressing construct. Salinity shock treatment with 250 mM NaCl indicated that most of the OPBP1 transgenic plants can survive, whereas the control seedlings cannot. Similar recovery was found by using the seedlings grown in 200 mM NaCl for two weeks. The OPBP1 transgenic and control plants were also studied for oxidative stress tolerance by treatment with paraquat, showing the transgenic lines were damaged less in comparison with the control plants. Further, the OPBP1 overexpression lines exhibited enhanced resistance to infections of Magnaporthe oryzae and Rhizoctonia solani pathogens. Gene expressing analysis showed increase in mRNA accumulation of several stress related genes. These results suggest that expression of OPBP1 gene increase the detoxification capability of rice.

Many bacterial pathogens of plants and animals deliver effector proteins into host cells to promote infection. Elucidation of how pathogen effector proteins function not only is critical for understanding bacterial pathogenesis but also provides a useful tool in discovering the functions of host genes. In this study, we characterized the Pseudomonas syringae pv tomato DC3000 effector protein Avirulence Protein E (AvrE), the founding member of a widely distributed, yet functionally enigmatic, bacterial effector family. We show that AvrE is localized in the plasma membrane (PM) and PM-associated vesicle-like structures in the plant cell. AvrE contains two physically interacting domains, and the amino-terminal portion contains a PM-localization signal. Genome-wide microarray analysis indicates that AvrE, as well as the functionally redundant effector Hypersensitive response and pathogenicity-dependent Outer Protein M1, down-regulates the expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13) gene in Arabidopsis (Arabidopsis thaliana). Mutational analysis shows that NHL13 is required for plant immunity, as the nhl13 mutant plant displayed enhanced disease susceptibility. Our results defined the action site of one of the most important bacterial virulence proteins in plants and the antibacterial immunity function of the NHL13 gene.

Cryptogein (Crypt), an elicitin secreted fromPhytophthora cryptogea, was used for genetic engineering of biotic and abiotic resistance plants. We generated transgenic tobacco plants harboring a rice phenylalanine ammonia-lyase (PAL) promoter andCrypt fusion gene (PAL::Crypt) or the mutatedCrypt (mutation of the lysine at the position 13 to valine) under the controlCaMV35S promoter (CaMV35S::CryK13V). T2 progeny of the transgenic plants showed significantly enhanced disease resistance to pathogens of fungalPhytophthora parasitica varnicotiana (Ppn) andAlternaria alternata, and bacterialPseudomonas syringae pvtabaci. The amount of mRNA accumulation ofCrypt andCryK13V was quite low in the transgenic lines analyzed by Northern blot, and was detected by a reverse transcription PCR method. Plants harboringPAL::Crypt construct showed faster and stronger induction ofPR1a gene afterPpn inoculation than that in the wild-type plants. The results suggested that the induciblePAL promoter could rapidly respond to pathogen attack and efficiently suppress the pathogen infection. Furthermore, the enhanced tolerance to salt stress in both of theCrypt andCryK13V expressing tobacco plants was also observed compared with that in the control plants. The constitutive expression ofPR and transcription factor genes in the transformants was probably associated with the salt tolerance. The above observations suggested that a cross-talk between biotic and abiotic stresses existed in tobacco plants.

The WRKY family of transcription factors (TFs) functions as transcriptional activators or repressors in various signaling pathways. In this study, we discovered that OsWRKY62 and OsWRKY76, two genes of the WRKY IIa subfamily, undergo constitutive and inducible alternative splicing. The full-length OsWRKY62.1 and OsWRKY76.1 proteins formed homocomplexes and heterocomplexes, and the heterocomplex dominates in the nuclei when analyzed in Nicotiana benthamiana leaves. Transgenic overexpression of OsWRKY62.1 and OsWRKY76.1 in rice (Oryza sativa) enhanced plant susceptibility to the blast fungus Magnaporthe oryzae and the leaf blight bacterium Xanthomonas oryzae pv oryzae, whereas RNA interference and loss-of-function knockout plants exhibited elevated resistance. The dsOW62/76 and knockout lines of OsWRKY62 and OsWRKY76 also showed greatly increased expression of defense-related genes and the accumulation of phytoalexins. The ratio of full-length versus truncated transcripts changed in dsOW62/76 plants as well as in response to pathogen infection. The short alternative OsWRKY62.2 and OsWRKY76.2 isoforms could interact with each other and with full-length proteins. OsWRKY62.2 showed a reduced repressor activity in planta, and two sequence determinants required for the repressor activity were identified in the amino terminus of OsWRKY62.1. The amino termini of OsWRKY62 and OsWRKY76 splice variants also showed reduced binding to the canonical W box motif. These results not only enhance our understanding of the DNA-binding property, the repressor sequence motifs, and the negative feedback regulation of the IIa subfamily of WRKYs but also provide evidence for alternative splicing of WRKY TFs during the plant defense response.

In order to identify natural products for plant disease control, the essential oil of star anise (Illicium verum Hook. f.) fruit was investigated for its antifungal activity on plant pathogenic fungi. The fruit essential oil obtained by hydro-distillation was analyzed for its chemical composition by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). trans-Anethole (89.5%), 2-(1-cyclopentenyl)-furan (0.9%) and cis-anethole (0.7%) were found to be the main components among 22 identified compounds, which accounted for 94.6% of the total oil. The antifungal activity of the oil and its main component trans-anethole against plant pathogenic fungi were determined. Both the essential oil and trans-anethole exhibited strong inhibitory effect against all test fungi indicating that most of the observed antifungal properties was due to the presence of trans-anethole in the oil, which could be developed as natural fungicides for plant disease control in fruit and vegetable preservation.