APETALA2/ethylene‐responsive factor (AP2/ERF) family transcription factors are well‐documented in plant responses to a wide range of biotic and abiotic stresses, but their roles in mediating elicitor‐induced disease resistance remains largely unexplored. PevD1 is a Verticillium dahliae secretory effector that can induce disease resistance in cotton and tobacco plants. In our previous work, Nicotiana benthamiana ERF114 (NbERF114) was identified in a screen of genes differentially expressed in response to PevD1 infiltration. Here, we found that the ortholog of NbERF114 in Arabidopsis thaliana (ERF114) also strongly responded to PevD1 treatment and transcripts were induced by Pseudomonas syringae pv. tomato (Pst) DC3000 infection. Loss of ERF114 function caused impaired disease resistance, while overexpressing ERF114 (OE‐ERF114) enhanced resistance to Pst DC3000. Moreover, ERF114 mediated PevD1‐induced disease resistance. RNA‐sequencing analysis revealed that the transcript level of phenylalanine ammonia‐lyase1 (PAL1) and its downstream genes were significantly suppressed in erf114 mutants compared with A. thaliana Col‐0. Reverse transcription‐quantitative PCR (RT‐qPCR) analysis further confirmed that the PAL1 mRNA level was significantly elevated in overexpressing OE‐ERF114 plants but reduced in erf114 mutants compared with Col‐0. Chromatin immunoprecipitation‐qPCR (ChIP‐qPCR) and electrophoretic mobility shift assay verified that ERF114 directly bound to the promoter of PAL1. The gene expression profiles of ERF114 and PAL1 in oestradiol‐inducible transgenic plants confirmed ERF114 could activate PAL1 transcriptional expression. Further investigation revealed that ERF114 positively modulated PevD1‐induced lignin and salicylic acid accumulation, probably by activating PAL1 transcription.

Xenocoumacin 1 (Xcn1), which is produced by Xenorhabdus nematophila CB6, exhibits strong inhibition activity against plant pathogens, especially fungi and oomycetes. Therefore, it has attracted interest in developing it into a novel biofungicide applicable for plant protection. However, its low yield with concomitant high cost during the fermentation process limits its widespread application. In this study, we replaced the native promoter of xcnA with the arabinose-inducible araBAD promoter (PBAD), a well-known and widely used promoter for expressing heterologous genes, to evaluate its effects on Xcn1 yield and antimicrobial activity. Compared with wildtype strain, the fermentation yield of Xcn1 was improved from 68.5 mg/L to 249.7 mg/L (3.6-fold) and 234.9 mg/L (3.4-fold) at 0.5% and 1.0% L-arabinose concentration, respectively. We further explored the transcription level of the biosynthesis related genes of Xcn1 and found that their upregulation resulted in the yield improvement of Xcn1. Moreover, the antimicrobial activity of Xcn1 against Bacillus subtilis and Phytophthora capsici was determined by agar diffusion plate and growth inhibition assay, as expected, it was also found to be enhanced. The promoter-replacement strategy utilized here improves the yield of Xcn1 efficiently, which provides a basis for the industrial production of Xcn1.

Xenocoumacin 1 (Xcn1) is an excellent antimicrobial natural product against Phytophthora capsici. However, the commercial development of Xcn1 is hindered by the low yield, which results in high application costs. In this study, multiple metabolic strategies, including blocking the degradation pathway, promoter engineering, and deletion of competing biosynthetic gene clusters, were employed to improve the production of Xcn1, which was increased from 0.07 to 0.91 g/L. The formation of Xcn1 reached 1.94 g/L in the TB medium with the final strain T3 in a shake flask and further reached 3.52 g/L in a 5 L bioreactor, which is the highest yield ever reported. The engineered strain provides a valuable platform for production of Xcn1, and the possible commercial development of the biofungicide. We anticipate that the metabolic engineering strategies utilized in this study and the constructed constitutive promoter library can be widely applied to other bacteria of the genera Xenorhabdus and Photorhabdus.