PlaD: A Transcriptomics Database for Plant Defense Responses to Pathogens, Providing New Insights into Plant Immune System / 基因组蛋白质组与生物信息学报·英文版

High-throughput transcriptomics technologies have been widely used to study plant transcriptional reprogramming during the process of plant defense responses, and a large quantity of gene expression data have been accumulated in public repositories. However, utilization of these data is often hampered by the lack of standard metadata annotation. In this study, we curated 2444 public pathogenesis-related gene expression samples from the model plant Arabidopsis and three major crops (maize, rice, and wheat). We organized the data into a user-friendly database termed as PlaD. Currently, PlaD contains three key features. First, it provides large-scale curated data related to plant defense responses, including gene expression and gene functional annotation data. Second, it provides the visualization of condition-specific expression profiles. Third, it allows users to search co-regulated genes under the infections of various pathogens. Using PlaD, we conducted a large-scale transcriptome analysis to explore the global landscape of gene expression in the curated data. We found that only a small fraction of genes were differentially expressed under multiple conditions, which might be explained by their tendency of having more network connections and shorter network distances in gene networks. Collectively, we hope that PlaD can serve as an important and comprehensive knowledgebase to the community of plant sciences, providing insightful clues to better understand the molecular mechanisms underlying plant immune responses. PlaD is freely available at http://systbio.cau.edu.cn/plad/index.php or http://zzdlab.com/plad/index.php.

Cloning and expression analysis of polygalacturonase-inhibiting protein gene from Panax notoginseng / 中草药

Objective: To clone the full-length cDNA sequence of PnPGIP gene encoding polygalacturonase-inhibiting protein (PGIP) from Panax notoginseng and analyze the expression levels of PnPGIP. Methods: Based on P. notoginseng expressed sequence tag (EST) encoding PGIP, specific primers were designed and the full-length cDNA of EST was cloned with the method of rapid amplification of cDNA ends (RACE). The expression levels of PnPGIP were analyzed by qRT-PCR. Results: The full-length cDNA of PnPGIP was 1 171 bp and contained an intact open reading frame (ORF) of 981 bp, a 13 bp 5'-untranslated region (UTR), and a 177 bp 3'-UTR. The deduced amino acid sequence of PnPGIP has 326 amino acid residues which form a 36 770 polypeptide with a calculated pI of 5.83. qRT-PCR analysis indicated that the expression of PnPGIP was quickly induced after inoculation with Fusarium solani and Alternaria panax, and the highest transcription level was achieved at 4 h and 2 h post inoculation, respectively. Moreover, the expression of PnPGIP was induced in different degrees by methyl jasmonate (MeJA), ethylene (ETH), H2O2, and salicylic acid (SA). Conclusion: PnPGIP responds to F. solani and A. panax infection in the transcription level, and it is induced by several kinds of adversity stresses related signaling molecules. Therefore, PnPGIP may be involved in defense response of P. notoginseng against F. solani and A. panax.

Cloning and expression analysis of a chitinase gene PnCHI1 from Panax notoginseng / 中国中药杂志

Chitinases(EC3.2.1.14), which are present in various organisms, catalyze the hydrolytic cleavage of chitin and play a vital role in plant defense mechanisms against fungal pathogens.In addition, the chitinases are well known to regulate plant growth and development and are involved in programmed cell death(PCD).A chitinase expressed sequence tag(EST) was isolated from Panax notoginseng, and the full-length cDNA of this EST was cloned with the method of rapid amplification of cDNA ends and named as PnCHI1. PnCHI1 was 1 022 bp in length and contained an intact open reading frame(ORF) of 822 bp, a 26 bp 5'-untranslated region(UTR), and a 174 bp 3'-UTR.The predicted protein of PnCHI1 with 273 amino acid residues belongs to glycoside hydrolase family 19 and fell into the class IV of chitinases through phylogenetic analysis.QRT-PCR analysis showed that the expression of PnCHI1 was induced by methyl jasmonate, ethylene, H2O2, and salicylic acid.PnCHI1 was quickly induced after inoculation with Alternaria panax.Moreover, the expression level of PnCHI1 was increased after pretreatment with methyl jasmonate, and then the transcription level of PnCHI1was sharp increased after inoculation with Fusarium solani,and the highest transcription level was achieved at 4 h post inoculation.But the expression level of PnCHI1 in the sterile water pretreated P.notoginseng was increased gradually after inoculation with F.solani, and the highest expression level was achieved at 48 h post inoculation.All the results of present study indicated that PnCHI1 was involved in defense response of P.notoginseng against the F.solani and A.panax.

New Assay Method for Allene Oxide Cyclase, an Important Enzyme in Jasmonic Acid Biosynthesis.

Aims: Allene oxide cyclase (AOC) (EC 5.3.99.6) is an important enzyme of jasmonates (JAs) biosynthesis. JAs are important signals that play a pivotal role in defense response of plants to environmental cues. Regulation JA biosynthesis is believed useful for elucidating the mechanism of plant defense system. Despite the high potential of AOC as a target for JA biosynthesis inhibitors, an efficient assay method suitable for screening AOC inhibitors is still not available. The aim of this work is to develop an efficient AOC assay method. Study Design: Using excess amount of purified recombinant allene oxide synthase (AOS) combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-HPOT), we established an efficient method to generate (12,13S)-epoxyoctadecatrienoic acid (EOT), the substrate of AOC. The AOS produced EOT was subsequently converted to (9S,13S)-12-oxo- (10,15Z)-phytodienoic acid (OPDA) by using purified recombinant AOC in a real time manner and the amount of OPDA was determined by HPLC. Place and Duration of Study: All the experiments were conducted from October 2009 to March 2013 at Akita Prefectural University, Japan. Methodology: The recombinant AOS and AOC were expressed in E. coli. The target proteins were purified using affinity chromatography, respectively. The unstable EOT was generated by using excess AOS combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid. The AOC synthesized OPDA was characterized by the comparison of HPLC retention time with the OPDA standard. AOC activity was calculated by determine the amount of OPDA in the assay system. Results: We found in the presence of 50 nmol of purified AOS together with 20 M 13-HPOT, the synthesis of OPDA was saturated when using 5 nmol of purified AOC in the enzyme reaction for 30 min. Our results indicated that the AOC activity can be determined by dual enzyme system. Conclusion: We established an efficient assay method for AOC which may be applied for screening of AOC inhibitors.

Observations of Infection Structures after Inoculation with Colletotrichum orbiculare on the Leaves of Cucumber Plants Pre-inoculated with Two Bacterial Strains Pseudomonas putida or Micrococcus luteus

Infection structures were observed at the penetration sites on the leaves of cucumber plants inoculated with Colletotrichum orbiculare using a fluorescence microscope. The cucumber plants were previously drenched with suspension of bacterial strains Pseudomonas putida or Micrococcus luteus. The plants pre-inoculated with both bacterial strains were resistant against anthracnose after inoculation with C. orbiculare. To investigate the resistance mechanism by both bacterial strains, the surface of infected leaves was observed at the different time after challenge inoculation. At 3 days after inoculation there were no differences in the germination and appressorium formation of conidia of C. orbiculare as well as in the callose formation of the plants between both bacteria pre-inoculated and non-treated. At 5 days, the germination and appressorium formation of the fungal conidia were, however, significantly decreased on the leaves of plants pre-inoculated with M. luteus at the concentration with 1.0 x 10(7) cfu/ml. Furthermore, callose formation of plants cells at the penetration sites was apparently increased. In contrast, there were no defense reactions of the plants at the concentration with 1.0 x 10(6) cfu/ml of M. luteus. Similarly, inoculation P. putida caused no plant resistance at the low concentration, whereas increase of callose formation was observed at the higher concentration. The results of this study suggest that the resistant mechanisms might be differently expressed by the concentration of pre-treatment with bacterial suspension.