Cysteine protease RD21A regulated by E3 ligase SINAT4 is required for drought-induced resistance to Pseudomonas syringae in Arabidopsis.

Plants can be simultaneously exposed to multiple stresses. The interplay of abiotic and biotic stresses may result in synergistic or antagonistic effects on plant development and health. Temporary drought stress can stimulate plant immunity; however, the molecular mechanism of drought-induced immunity is largely unknown. In this study, we demonstrate that cysteine protease RD21A is required for drought-induced immunity. Temporarily drought-treated wild-type Arabidopsis plants became more sensitive to the bacterial pathogen-associated molecular pattern flg22, triggering stomatal closure, which resulted in increased resistance to Pseudomonas syringae pv. tomato DC3000 (Pst-DC3000). Knocking out rd21a inhibited flg22-triggered stomatal closure and compromised the drought-induced immunity. Ubiquitin E3 ligase SINAT4 interacted with RD21A and promoted its degradation in vivo. The overexpression of SINAT4 also consistently compromised the drought-induced immunity to Pst-DC3000. A bacterial type III effector, AvrRxo1, interacted with both SINAT4 and RD21A, enhancing SINAT4 activity and promoting the degradation of RD21A in vivo. Therefore, RD21A could be a positive regulator of drought-induced immunity, which could be targeted by pathogen virulence effectors during pathogenesis.

Identification and characterization of a novel Toll-like receptor 2 homologue in the large yellow croaker Larimichthys crocea.

Toll-like receptors (TLRs) are key components of innate immunity that play significant roles in immune defence against pathogen invasion. In the present study, we identified a novel TLR2 homologue (LycTLR2b) in large yellow croaker (Larimichthys crocea) that shared low sequence identity with the previously reported large yellow croaker TLR2 (tentatively named LycTLR2a). The full-length cDNA of LycTLR2b was 2926 nucleotides (nt) long and encoded a protein consisting of 797 amino acids (aa). The deduced LycTLR2b protein exhibited a typical TLR domain architecture including a signal peptide, seven leucine-rich repeats (LRRs) in the extracellular region, a transmembrane domain, and a Toll-Interleukin 1 receptor (TIR) domain in the cytoplasmic region. Phylogenetic analysis showed that both LycTLR2a and LycTLR2b fall into a major clade formed by all TLR2 sequences, and are divided into two distinct branches. Genomic organization revealed that the LycTLR2b gene lacks intron, which is similar to zebrafish and human TLR2 genes, whereas the LycTLR2a gene contains multiple introns, as found in damselfish TLR2a and Fugu TLR2 genes. Syntenic analysis suggested that the occurrence of LycTLR2a and LycTLR2b may result from a relatively recent genome duplication event. LycTLR2b mRNA was constitutively expressed in all tissues examined although at different levels. Following bacterial vaccine challenge, LycTLR2b expression levels were significantly up-regulated in both spleen and head kidney tissues. Taken together, these results indicated that two different TLR2 homologues, which may play roles in antibacterial immunity, exist in large yellow croaker.

Molecular characterization and biological effects of a CXCL8 homologue in large yellow croaker (Larimichthys crocea).

CXCL8 also called interleukin-8, is a CXC-type chemokine that plays a key role in promoting inflammation. Three subgroups of CXCL8 homologues have been reported in teleost fish, including CXCL8_L1, CXCL8_L2 and CXCL8_L3. In the present study, we identified a CXCL8 homologue belonging to CXCL8_L1 subgroup (LycCXCL8_L1) in large yellow croaker (Larimichthys crocea) that shares low identity to the previously reported large yellow croaker CXCL8 (LycCXCL8). The full-length cDNA of LycCXCL8_L1 is 716 nucleotides (nt) long and encodes a protein consisting of 99 amino acids (aa) with a putative molecular weight of 11.2 kDa. The deduced LycCXCL8_L1 protein contains a 22-aa signal peptide and a 77-aa mature polypeptide, which possesses an arrangement of four cysteines typical of other known CXC chemokines (C(34), C(36), C(60), and C(77)). Genomic analysis revealed that the LycCXCL8_L1 gene consisted of four exons and three introns and exhibited a similar exon-intron organization to LycCXCL8 and other species CXCL8 genes except for a different intron length. Phylogenetic analysis showed that both LycCXCL8_L1 and LycCXCL8 belong to CXCL8_L1 subgroup. LycCXCL8_L1 mRNA was constitutively expressed in all tissues examined although at different levels. Upon bacterial vaccine induction, LycCXCL8_L1 mRNA expression was rapidly increased in the spleen and head kidney tissues. Recombinant LycCXCL8_L1 and LycCXCL8 proteins produced in Escherichia coli both induced chemotaxis and superoxide production in peripheral blood leucocytes from large yellow croaker. These results indicate that two CXCL8_L1 molecules exist in large yellow croaker and play roles in inflammatory response.

Spaceflight environment-induced variation in root yield and active constituents of Salvia miltiorrhiza.

Salvia miltiorrhiza is a significant source of bioactive compounds providing human health effects. Here, we surveyed root yield and the active constituents' divergences of second generation S. miltiorrhiza (SP2) responding to a spaceflight environment. High-performance liquid chromatography was conducted for the comprehensive constituents' characterizations of 28 SP2 lines (224 individuals) and the ground control (eight individuals). The results showed that the mean fresh and dry weight of roots ranged from 116 to 172 g and 25 to 119 g, respectively, in SP2 lines. In addition, the mean contents of four tanshinone compounds (tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I) of 28 SP2 lines varied from 0.32 to 1.04 mg ·â€Šg(-1), 0.47 to 2.39 mg ·â€Šg(-1), 0.25 to 1.60 mg ·â€Šg(-1), and 0.53 to 1.67 mg ·â€Šg(-1), respectively. Except for salvianolic acid B, which varied drastically from 72 % to 201 % of the ground control treatment, the other six phenolic acid contents of the 28 SP2 lines all increased after spaceflight. Principal component analysis was performed to obtain an overview of the distribution of all samples, and score plots clearly separated the SP2 accessions from ground controls. Moreover, a positive relationship was observed between tanshinone I and tanshinone IIA (r = 0.790, p < 0.01), and rosmarinic acid was positively correlated with salvianolic acid B (r = 0.728, p < 0.01). In conclusion, this study demonstrated that a spaceflight environment induced SP2 accessions remarkably in the variation of root yield and active constituent content.

Molecular characterization and expression analysis of TLR 7 and TLR 8 homologs in large yellow croaker (Pseudosciaena crocea).

The two toll-like receptor (TLR) genes, LycTLR7 and LycTLR8, were cloned from large yellow croaker (Pseudosciaena crocea), an economically important marine fish in China. The full-length cDNAs of LycTLR7 and LycTLR8 are 3544 and 3593 bp, with an open reading frame (ORF) of 3165 and 3093 bp, encoding 1053 and 1030 amino acids, respectively. The TLR family motifs, such as leucine rich repeat (LRR) and Toll/interleukin (IL)-1 receptor (TIR) domain, are conserved in the LycTLR7 and LycTLR8, with 17 and 14 LRRs, and with a TIR domain, respectively. It is also noted that an LRR N-terminal domain (LRR-NT, residues 24-60) is present in the LycTLR7 but not in the LycTLR8. Both LycTLR7 and LycTLR8 contain a conserved extracellular CxRCxxxxxPCxxC motif, which was found in TLR7/TLR8 of other species and required for stimulus-induced signal transduction. Homology comparison shows that LycTLR7 has 79%, 71.9%, 65.9% and 65.8% identity to fugu, rainbow trout, carp and catfish TLR7, while LycTLR8 has 67.1%, 60.7%, 60.6%, 52.4%, and 51.5% identity to fugu TLR8, rainbow trout TLR8a1, rainbow trout TLR8a2, catfish TLR8-2, and catfish TLR8-1, respectively. Subcellular localization analysis revealed that both LycTLR7 and LycTLR8 are located in the endoplasmic reticulum of epithelioma papulosum cyprini (EPC) cells, which is similar to TLR7/TLR8 in mammals. The two TLRs were constitutively expressed in all tissues tested, especially in immune-related tissues such as spleen, head kidney and gills. An increased expression of LycTLR7 and LycTLR8 was observed in head kidney and spleen of large yellow croakers stimulated by poly (I:C), a viral mimic. In head kidney, their mRNA expression was up-regulated more than 10 times compared to the controls at 12 h after poly (I:C) stimulation. These results suggested that LycTLR7 and LycTLR8 may play a role in the defense against viral infection like their mammalian homologs.

Molecular characterization and expression analysis of toll-like receptor 1 from large yellow croaker (Pseudosciaena crocea).

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns associated with microbial pathogens (PAMP) and induce antimicrobial immune responses. Here we report the molecular cloning and characterization of a TLR1 homologue from the large yellow croaker (LycTLR1). The complete cDNA of LycTLR1 is 3487 nucleotides long, encoding a protein of 802 amino acids. The deduced LycTLR1 has a typical TLR domain architecture including 4 leucine-rich repeats (LRRs) (residues 42-491), one C-terminal LRR domain (residues 527-583) at the extracellular region and a TIR domain (residues 646-791) in the cytoplasmic region. Homology comparison shows that LycTLR1 has 76.8%-47.6% amino acid identity to known fish TLR1. Genomic analysis revealed that LycTLR1 consisted of only one exon in the coding region, which is conserved among other TLR1 from different mammalian species and fish analyzed to date, except the zebrafish. The mRNA of LycTLR1 was constitutively expressed in spleen, head kidney, blood, liver, heart, gills, intestine, brains and muscle, with the highest levels in spleen and blood. Upon stimulation with LPS, the LycTLR1 expression obviously increased in the anterior kidney cells of large yellow croaker, suggesting a role for LycTLR1 in the immune response to LPS.

Using innovative tools for in vitro absorption, distribution, metabolism and excretion studies to efficiently and effectively support drug development.

Drug development is a complicated and lengthy process requiring a significant amount of intellectual and capital input, as well as extensive collaborations among various organizations and institutions. Contract research organizations play important roles at some or even all stages of the drug development process. To provide better service in in vitro drug absorption, disposition, metabolism and excretion studies, maintain data accuracy and promote work efficiency, we developed an integrated information system termed the 'Drug Metabolism Information System', and it is being used routinely by our drug metabolism department. The Drug Metabolism Information System assists scientists with assay design, data analysis and report drafting and thus reduces human error.

Evaluate the guide RNA effectiveness via Agrobacterium-mediated transient assays in Nicotiana benthamiana.

CRISPR/Cas9-based genome editing system is a powerful tool for plant genetic improvement. However, the variable efficiency of guide RNA(s) (gRNA) represents a key limiting factor that hampers the broad application of the CRISPR/Cas9 system in crop improvement. Here, we employed the Agrobacterium-mediated transient assays to evaluate the effectiveness of gRNAs for editing genes in Nicotiana benthamiana and soybean. We designed a facile screening system based on indels that can be introduced by CRISPR/Cas9-mediated gene editing. A gRNA binding sequence (23 nucleotides) was inserted into the open reading frame of yellow fluorescent protein (YFP) gene (gRNA-YFP), which disrupted the YFP reading frame and results in no fluorescent signal when it was expressed in plant cells. Transiently co-expression of Cas9 and a gRNA targeting the gRNA-YFP gene in plant cells could restore the YFP reading frame and recover the YFP signals. We evaluated five gRNAs targeting Nicotiana benthamiana and soybean genes and confirmed the reliability of the gRNA screening system. The effective gRNAs targeting NbEDS1, NbWRKY70, GmKTI1, and GmKTI3 had been used to generate transgenic plants and resulted in expected mutations on each gene. While a gRNA targeting NbNDR1 was confirmed to be ineffective in transient assays. This gRNA indeed failed to trigger target gene mutations in stable transgenic plants. Thus, this new transient assay system can be used to validate the effectiveness of gRNAs before generating stable transgenic plants.

Characterization of large yellow croaker (Pseudosciaena crocea) ß-actin promoter supports ß-actin gene as an internal control for gene expression modulation and its potential application in transgenic studies in fish.

As a housekeeping gene, ß-actin is one of the most commonly used reference gene and its promoter is widely used in transgenic studies in mammals and fish. In this study, we used genomic walker technology to clone the ß-actin gene (Lycß-actin) promoter sequence from large yellow croaker, an economically important marine fish in China. The Lycß-actin promoter region spans 3350 nucleotides (nt) and contains several transcription factor binding sites and a conserved enhancer motif (ATGGTAATAA) in the first intron. A promoter activity assay showed that this promoter region can drive enhanced green fluorescent protein (EGFP) gene expression in the fish cell line, EPC. Luciferase activity analysis demonstrated that the activity of the Lycß-actin promoter is not affected by poly(I:C) or lipopolysaccharide (LPS) stimulation. Absolute real-time PCR analysis of various tissues revealed that Lycß-actin expression levels are not significantly altered by poly(I:C) or inactivated trivalent bacterial vaccine (P > 0.05). These results suggest that ß-actin can be used as a suitable internal control for gene expression modulation in response to immune stimulations in large yellow croaker. In vivo transgenic experiments showed that the Lycß-actin promoter region can drive efficient EGFP expression in large yellow croaker fries or fertilized zebrafish eggs, supporting its potential application in transgenic studies in fish.

Nicotiana species as surrogate host for studying the pathogenicity of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is one of the most important bacterial diseases of cucurbits worldwide. However, the mechanisms associated with A. citrulli pathogenicity and genetics of host resistance have not been extensively investigated. We idenitfied Nicotiana benthamiana and Nicotiana tabacum as surrogate hosts for studying A. citrulli pathogenicity and non-host resistance triggered by type III secreted (T3S) effectors. Two A. citrulli strains, M6 and AAC00-1, that represent the two major groups amongst A. citrulli populations, induced disease symptoms on N. benthamiana, but triggered a hypersensitive response (HR) on N. tabacum plants. Transient expression of 19 T3S effectors from A. citrulli in N. benthamiana leaves revealed that three effectors, Aave_1548, Aave_2708, and Aave_2166, trigger water-soaking-like cell death in N. benthamiana. Aave_1548 knockout mutants of M6 and AAC00-1 displayed reduced virulence on N. benthamiana and melon (Cucumis melo L.). Transient expression of Aave_1548 and Aave_2166 effectors triggered a non-host HR in N. tabacum, which was dependent on the functionality of the immune signalling component, NtSGT1. Hence, employing Nicotiana species as surrogate hosts for studying A. citrulli pathogenicity may help characterize the function of A. citrulli T3S effectors and facilitate the development of new strategies for BFB management.

A tyrosine aminotransferase involved in rosmarinic acid biosynthesis in Prunella vulgaris L.

Rosmarinic acid (RA) and its derivants are medicinal compounds that comprise the active components of several therapeutics. We isolated and characterised a tyrosine aminotransferase of Prunella vulgaris (PvTAT). Deduced PvTAT was markedly homologous to other known/putative plant TATs. Cytoplasmic localisation of PvTAT was observed in tobacco protoplasts. Recombinantly expressed and purified PvTAT had substrates preference for L-tyrosine and phenylpyruvate, with apparent K m of 0.40 and 0.48 mM, and favoured the conversion of tyrosine to 4-hydroxyphenylpyruvate. In vivo activity was confirmed by functional restoration of the Escherichia coli tyrosine auxotrophic mutant DL39. Agrobacterium rhizogenes-mediated antisense/sense expression of PvTAT in hairy roots was used to evaluate the contribution of PvTAT to RA synthesis. PvTAT were reduced by 46-95% and RA were decreased by 36-91% with low catalytic activity in antisense transgenic hairy root lines; furthermore, PvTAT were increased 0.77-2.6-fold with increased 1.3-1.8-fold RA and strong catalytic activity in sense transgenic hairy root lines compared with wild-type counterparts. The comprehensive physiological and catalytic evidence fills in the gap in RA-producing plants which didn't provide evidence for TAT expression and catalytic activities in vitro and in vivo. That also highlights RA biosynthesis pathway in P. vulgaris and provides useful information to engineer natural products.

Genomic features of bacterial adaptation to plants.

Plants intimately associate with diverse bacteria. Plant-associated bacteria have ostensibly evolved genes that enable them to adapt to plant environments. However, the identities of such genes are mostly unknown, and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterial genomes to identify thousands of plant-associated gene clusters. Genomes of plant-associated bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant-associated genomes do. We experimentally validated candidates from two sets of plant-associated genes: one involved in plant colonization, and the other serving in microbe-microbe competition between plant-associated bacteria. We also identified 64 plant-associated protein domains that potentially mimic plant domains; some are shared with plant-associated fungi and oomycetes. This work expands the genome-based understanding of plant-microbe interactions and provides potential leads for efficient and sustainable agriculture through microbiome engineering.

Isolation and functional analysis of the promoter of the amphioxus Hsp70a gene.

Amphioxus is a promising laboratorial model animal for studying the evolutionary and developmental mechanisms that appeared during the invertebrate-chordate to vertebrate transition. However, the main drawback for the use of amphioxus as a model organism is the lack of well-developed technical approaches. Conditional gene expression, as performed with thermal control, is a very useful strategy in gene function studies. To make this method possible in amphioxus studies, here we report the isolation and characterization of an amphioxus Hsp70 gene (Hsp70a) and its promoter in Chinese amphioxus (Branchiostoma belcheri). Hsp70a showed very low expression at normal temperatures but was robustly induced in animals upon heat shock. The basal cis-acting elements (CAAT and TATA), as well as four heat shock elements (HSEs), were found within the regulatory region (-1031 to -11 upstream from the start codon), but surprisingly most of the elements were located in the 5'UTR region (-252 to -10). Reporter constructs, including sequences from both the transcription start site (TSS) and ATG were tested for transient expression in EPC cells and microinjected zebrafish embryos. Results suggested that the 5'UTR region, which includes a TATA box at -92bp, a CAAT box at -152bp, and three HSE elements (-212 to -106), represents the core hsp promoter sequence of the B. belcheri Hsp70a gene. Therefore in this study we identified an effectively thermo-inducible promoter in amphioxus that could be used for the establishment of a conditional gene expression system in which the target gene can be regulated in a temporal- or tissue-specific way in amphioxus.