Chengcai An

SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem–loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples.

ABI4 Activates DGAT1 Expression in Arabidopsis Seedlings during Nitrogen Deficiency

Triacylglycerol (TAG) is the major seed storage lipid and is important for biofuel and other renewable chemical uses. Acyl-coenzyme A:diacylglycerol acyltransferase1 (DGAT1) is the rate-limiting enzyme in the TAG biosynthesis pathway, but the mechanism of its regulation is unknown. Here, we show that TAG accumulation in Arabidopsis (Arabidopsis thaliana) seedlings increased significantly during nitrogen deprivation (0.1 mm nitrogen) with concomitant induction of genes involved in TAG biosynthesis and accumulation, such as DGAT1 and OLEOSIN1. Nitrogen-deficient seedlings were used to determine the key factors contributing to ectopic TAG accumulation in vegetative tissues. Under low-nitrogen conditions, the phytohormone abscisic acid plays a crucial role in promoting TAG accumulation in Arabidopsis seedlings. Yeast one-hybrid and electrophoretic mobility shift assays demonstrated that ABSCISIC ACID INSENSITIVE4 (ABI4), an important transcriptional factor in the abscisic acid signaling pathway, bound directly to the CE1-like elements (CACCG) present in DGAT1 promoters. Genetic studies also revealed that TAG accumulation and DGAT1 expression were reduced in the abi4 mutant. Taken together, our results indicate that abscisic acid signaling is part of the regulatory machinery governing TAG ectopic accumulation and that ABI4 is essential for the activation of DGAT1 in Arabidopsis seedlings during nitrogen deficiency.

Overexpression of constitutively active OsCPK10 increases Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against Magnaporthe grisea.

Calcium-dependent protein kinases (CDPKs) are crucial calcium sensors involved in plant responses to pathogen infection. Here, we report isolation and functional characterization of the pathogen-responsive rice OsCPK10 gene. The expression of OsCPK10 was strongly induced following treatment with a Magnaporthe grisea elicitor. Kinase activity assay showed that the functional OsCPK10 protein not only autophosphorylated, but also phosphorylated Casein in a calcium-dependent manner. Overexpression of constitutively active OsCPK10 in Arabidopsis enhanced the resistance to infection with Pseudomonas syringae pv. tomato, associated with elevated expression of both SA- and JA-related defense genes. Similarly, transgenic rice plants containing constitutively active OsCPK10 exhibited enhanced resistance to blast fungus M. grisea. The enhanced resistance in the transgenic lines was associated with activated expression of SA- and JA-related defense genes. Collectively, our results indicate that rice OsCPK10 is a crucial regulator in plant immune responses, and that it may regulate disease resistance by activating both SA- and JA-dependent defense responses.

Soluble expression and one-step purification of a neurotoxin Huwentoxin-I in Escherichia coli

Huwentoxin-I (HWTX-I) is a small 33-amino acid neurotoxin from the venom of the Chinese bird spider Ornithoctonus huwena. HWTX-I selectively blocks N-type voltage-sensitive calcium channels (N-VSCCs) and has great potential for clinical application as a novel analgesic without inducing drug tolerance. However, there are still many unsolved issues for this peptide, such as its clinical efficacy in analgesia, anesthesia, and even its potential role in drug rehabilitation. Therefore, large amounts of active recombinant HWTX-I are urgently needed. In this report, we describe a novel and efficient way to produce large amounts of the valuable form in Escherichia coli. HWTX-I was expressed in soluble form as an N-terminal intein fusion product. After affinity purification, a pH shift-induced self-cleavage of the intein released HWTX-I, resulting in a single-column purification of the target protein. The whole-cell patch clamp assay showed that purified HWTX-I has activity similar to another commercialized N-VSCC blocker omega-conotoxin MVIIA. Production of HWTX-I by this method has the major advantages of high efficiency and low cost.

ABA-insensitive (ABI) 4 and ABI5 synergistically regulate DGAT1 expression in Arabidopsis seedlings under stress.

Triacylglycerol (TAG) accumulation is essential for seed maturation in plants. Diacylglycerol acyltransferase 1 (DGAT1) is the rate‐limiting enzyme in TAG biosynthesis. In this study, we show that TAG accumulation in Arabidopsis seedlings is correlated with environmental stress, and both ABI4 and ABI5 play important roles in regulating DGAT1 expression. Tobacco transient assays revealed the synergistic effect of ABI4 with ABI5 in regulating DGAT1 expression. Taken together, our findings indicate ABI5 is an important accessory factor with ABI4 in the activation of DGAT1 in Arabidopsis seedlings under stress.

Identification of a bHLH-type G-box binding factor and its regulation activity with G-box and Box I elements of the PsCHS1 promoter.

With the use of in vivo recombination theory, the screening time of yeast one-hybrid system was decreased in the present study. A basic helix-loop-helix (bHLH) protein PsGBF was successfully obtained from a glutathione (GSH)-induced pea cDNA library using the G-box cis-element of the PsCHS1 promoter as a bait. Electrophoretic mobility shift assay (EMSA) and β-galactosidase assay results suggested that PsGBF possesses both G-box-specific binding and transcription-activating activities. The specific interaction of PsGBF with G-box was further confirmed by in vivo transient expression assays in tobacco. The current study examined the combination effect of G-box with Box I elements in the interaction with PsGBF or OsMYC. The results indicated that PsGBF bound with the G-box, but not the Box I element. Moreover, this combination effect of G-box and Box I only associated with PsGBF but not with other bHLH-type proteins such as OsMYC.

Ubiquitin Ligases RGLG1 and RGLG5 Regulate Abscisic Acid Signaling by Controlling the Turnover of Phosphatase PP2CA

Two RING E3 ligases mediate the degradation of the phosphatase PP2CA, a repressor of abscisic acid signaling, thereby modulating the activation of the ABA pathway in Arabidopsis. Abscisic acid (ABA) is an essential hormone for plant development and stress responses. ABA signaling is suppressed by clade A PP2C phosphatases, which function as key repressors of this pathway through inhibiting ABA-activated SnRK2s (SNF1-related protein kinases). Upon ABA perception, the PYR/PYL/RCAR ABA receptors bind to PP2Cs with high affinity and biochemically inhibit their activity. While this mechanism has been extensively studied, how PP2Cs are regulated at the protein level is only starting to be explored. Arabidopsis thaliana RING DOMAIN LIGASE5 (RGLG5) belongs to a five-member E3 ubiquitin ligase family whose target proteins remain unknown. We report that RGLG5, together with RGLG1, releases the PP2C blockade of ABA signaling by mediating PP2CA protein degradation. ABA promotes the interaction of PP2CA with both E3 ligases, which mediate ubiquitination of PP2CA and are required for ABA-dependent PP2CA turnover. Downregulation of RGLG1 and RGLG5 stabilizes endogenous PP2CA and diminishes ABA-mediated responses. Moreover, the reduced response to ABA in germination assays is suppressed in the rglg1 amiR (artificial microRNA)-rglg5 pp2ca-1 triple mutant, supporting a functional link among these loci. Overall, our data indicate that RGLG1 and RGLG5 are important modulators of ABA signaling, and they unveil a mechanism for activation of the ABA pathway by controlling PP2C half-life.

The LSD1-Interacting Protein GILP Is a LITAF Domain Protein That Negatively Regulates Hypersensitive Cell Death in Arabidopsis

Background Hypersensitive cell death, a form of avirulent pathogen-induced programmed cell death (PCD), is one of the most efficient plant innate immunity. However, its regulatory mechanism is poorly understood. AtLSD1 is an important negative regulator of PCD and only two proteins, AtbZIP10 and AtMC1, have been reported to interact with AtLSD1. Methodology/Principal Findings To identify a novel regulator of hypersensitive cell death, we investigate the possible role of plant LITAF domain protein GILP in hypersensitive cell death. Subcellular localization analysis showed that AtGILP is localized in the plasma membrane and its plasma membrane localization is dependent on its LITAF domain. Yeast two-hybrid and pull-down assays demonstrated that AtGILP interacts with AtLSD1. Pull-down assays showed that both the N-terminal and the C-terminal domains of AtGILP are sufficient for interactions with AtLSD1 and that the N-terminal domain of AtLSD1 is involved in the interaction with AtGILP. Real-time PCR analysis showed that AtGILP expression is up-regulated by the avirulent pathogen Pseudomonas syringae pv. tomato DC3000 avrRpt2 (Pst avrRpt2) and fumonisin B1 (FB1) that trigger PCD. Compared with wild-type plants, transgenic plants overexpressing AtGILP exhibited significantly less cell death when inoculated with Pst avrRpt2, indicating that AtGILP negatively regulates hypersensitive cell death. Conclusions/Significance These results suggest that the LITAF domain protein AtGILP localizes in the plasma membrane, interacts with AtLSD1, and is involved in negatively regulating PCD. We propose that AtGILP functions as a membrane anchor, bringing other regulators of PCD, such as AtLSD1, to the plasma membrane. Human LITAF domain protein may be involved in the regulation of PCD, suggesting the evolutionarily conserved function of LITAF domain proteins in the regulation of PCD.

Efficient LEC2 activation of OLEOSIN expression requires two neighboring RY elements on its promoter

As the main structural protein of oil body, OLEOSIN is highly expressed only during seed development. OLEOSIN promoter is a very useful tool for seed-specific gene engineering and seed bioreactor designing. The B3 domain transcription factor leafy cotyledon2 (LEC2) plays an important role in regulating seed development and seed-specific gene expression. Here, we first report how seed-specific B3 domain transcription factor leafy cotyledon2 (LEC2) efficiently activates OLEOSIN expression. The central promoter region of OLEOSIN, responsible for seed specificity and LEC2 activation, was determined by 5′-deletion analysis. Binding experiments in yeast cells and electrophoretic mobility shift assays showed that LEC2 specifically bound to two conserved RY elements in this region. In transient expression assays, mutation in either RY element dramatically reduced LEC2 activation of OLEOSIN promoter activity, while double mutation abolished it. Analysis of the distribution of RY elements in seed-specific genes activated by LEC2 also supported the idea that genes containing neighboring RY elements responded strongly to LEC2 activation. Therefore, we conclude that two neighboring RY elements are essential for efficient LEC2 activation of OLEOSIN expression. These findings will help us better utilize seed-specific promoter activity.

Two Novel RING-Type Ubiquitin Ligases, RGLG3 and RGLG4, Are Essential for Jasmonate-Mediated Responses in Arabidopsis

Jasmonates (JAs) regulate various stress responses and development processes in plants, and the JA pathway is tightly controlled. In this study, we report the functional characterization of two novel RING-type ubiquitin ligases, RING DOMAIN LIGASE3 (RGLG3) and RGLG4, in modulating JA signaling. Both RGLG3 and RGLG4 possessed ubiquitin ligase activities and were widely distributed in Arabidopsis (Arabidopsis thaliana) tissues. Altered expression of RGLG3 and RGLG4 affected methyl JA-inhibited root growth and JA-inductive gene expression, which could be suppressed by the coronatine insensitive1 (coi1) mutant. rglg3 rglg4 also attenuated the inhibitory effect of JA-isoleucine-mimicking coronatine on root elongation, and consistently, rglg3 rglg4 was resistant to the coronatine-secreting pathogen Pseudomonas syringae pv tomato DC3000, suggesting that RGLG3 and RGLG4 acted in response to the coronatine and promoted JA-mediated pathogen susceptibility. In addition, rglg3 rglg4 repressed wound-stunted plant growth, wound-stimulated expression of JA-responsive genes, and wound-induced JA biosynthesis, indicating their roles in JA-dependent wound response. Furthermore, both RGLG3 and RGLG4 responded to methyl JA, P. syringae pv tomato DC3000, and wounding in a COI1-dependent manner. Taken together, these results indicate that the ubiquitin ligases RGLG3 and RGLG4 are essential upstream modulators of JA signaling in response to various stimuli.