Pseudomonas syringae, a major hemibiotrophic bacterial pathogen, causes many devastating plant diseases. However, the transcriptional regulation of plant defense responses to P. syringae remains largely unknown. Here, we found that gain-offunction of BTB AND TAZ DOMAIN PROTEIN 4 (BT4) enhanced the resistance of Arabidopsis (Arabidopsis thaliana) to Pst DC3000 (Pseudomonas syringae pv. tomato DC3000). Disruption of BT4 also weakened the salicylic acid (SA)-induced defense response to Pst DC3000 in bt4 mutants. Further investigation indicated that, under Pst infection, transcription of BT4 is modulated by components of both the SA and ethylene (ET) signaling pathways. Intriguingly, the specific binding elements of ETHYLENE RESPONSE FACTOR (ERF) proteins, including dehydration responsive/C-repeat elements and the GCC box, were found in the putative promoter of BT4. Based on publicly available microarray data and transcriptional confirmation, we determined that ERF11 is inducible by salicylic acid and Pst DC3000 and is modulated by the SA and ET signaling pathways. Consistent with the function of BT4, loss-of-function of ERF11 weakened Arabidopsis resistance to Pst DC3000 and the SAinduced defense response. Biochemical and molecular assays revealed that ERF11 binds specifically to the GCC box of the BT4 promoter to activate its transcription. Genetic studies further revealed that the BT4-regulated Arabidopsis defense response to Pst DC3000 functions directly downstream of ERF11. Our findings indicate that transcriptional activation of BT4 by ERF11 is a key step in SA/ET-regulated plant resistance against Pst DC3000, enhancing our understanding of plant defense responses to hemibiotrophic bacterial pathogens.

BackgroundRecent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts.MethodsThe AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids.ResultsWe characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%.ConclusionsAFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist’s toolbox.Electronic supplementary materialThe online version of this article (doi: 10.1186/s12896-017-0394-x) contains supplementary material, which is available to authorized users.

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Laccases are multicopper oxidases (E.C. 1.10.3.2) that catalyze the oxidation of many phenolic compounds. In this study, a novel laccase, Stlac4, from Setosphaeria turcica was cloned and expressed in Escherichia coli by insertion into the pET-30a expression plasmid. The recombinant laccase was purified and visualized on SDS-PAGE as a single band with an apparent molecular weight of 71.5 KDa, and confirmed by Western blot. The maximum activity of the purified laccase was 127.78 U · mg −1 , the optimum temperature and pH value were 60°C and 4. Laccases are wide-spread in nature and over 220 species of laccases have been isolated from many sources,including plants, fungi, bacteria, and insects [3][4][5][6][7]. Fungal laccases are the most extensively studied multicopper oxidases, and while primarily considered to be involved in lignin degradation [8], recent studies have shown that fungal laccases participate in fruiting body formation [9], pigment biosynthesis [10], pathogenesis, and stress defense [11]. Laccases are also very important and valuable enzymes for various biotechnological and industrial applications, such as biodegradation of lignin without polluting the environment, degradation of recalcitrant compounds, environmental protection and bioremediation, biological bleaching in the paper industry, and textile dye decolorization [12,13]. Several laccase genes have been cloned from different fungal sources and heterologously expressed with the specific purpose of using laccases more efficiently in biotechnology. Like other enzymes, laccases often face harsh conditions in industrial processes, such as high temperature, high salt concentration, and extremely acidic or alkaline pH [14,15]. Identification of laccase enzymes robust to harsh conditions could improve economic viability of this process. The majority of fungal laccases operate in the range of 30-55°C, and their optimal pH range is limited to mildly acidic conditions [16]. Many strategies Shuangxin Ma and Ning Liu contributed equally to this work.68 |

A pathogenic mutant, BCG183, was obtained by screening the T-DNA insertion library of Botrytis cinerea. A novel pathogenicity-related gene BcKMO, which encodes kynurenine 3-monooxygenase (KMO), was isolated and identified via thermal asymmetric interlaced PCR, bioinformatics analyses, and KMO activity measurement. The mutant BCG183 grew slowly, did not produce conidia and sclerotia, had slender hyphae, and presented enhanced pathogenicity. The phenotype and pathogenicity of the BcKMO-complementing mutant (BCG183/BcKMO) were similar to those of the wild-type (WT) strain. The activities of polymethylgalacturonase, polygalacturonase, and toxins were significantly higher, whereas acid production was significantly decreased in the mutant BCG183, when compared with those in the WT and BCG183/BcKMO. Moreover, the sensitivity of mutant BCG183 to NaCl and KCl was remarkably increased, whereas that to fluconazole, Congo Red, menadione, H2O2, and SQ22536 and U0126 [cAMP-dependent protein kinase (cAMP) and mitogen-activated protein kinase (MAPK) signaling pathways inhibitors, respectively] were significantly decreased compared with the other strains. Furthermore, the key genes involved in the cAMP and MAPK signaling pathways, Pka1, Pka2, PkaR, Bcg2, Bcg3, bmp1, and bmp3, were significantly upregulated or downregulated in the mutant BCG183. BcKMO expression levels were also upregulated or downregulated in the RNAi mutants of the key genes involved in the cAMP and MAPK signaling pathways. These findings indicated that BcKMO positively regulates growth and development, but negatively regulates pathogenicity of B. cinerea. Furthermore, BcKMO was found to be involved in controlling cell wall degrading enzymes activity, toxins activity, acid production, and cell wall integrity, and participate in cAMP and MAPK signaling pathways of B. cinerea.