Journal Article

An improved, versatile and efficient modular plasmid assembly system for expression analyses of genes in Xanthomonas oryzae

Yan, Yichao, Wang, Yanyan, Yang, Xiaofei, Chen, Gongyou, Zhang, Cuiping, Yang, Ching‐Hong, Li, Yilang, Zou, Lifang, Huang, Mengsang, Yang, Ruihuan, Haq, Fazal

2021

Overview

Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc) infect rice, causing bacterial blight and bacterial leaf streak, respectively, which are two economically important bacterial diseases in paddy fields. The interactions of Xoo and Xoc with rice can be used as models for studying fundamental aspects of bacterial pathogenesis and host tissue specificity. However, an improved vector system for gene expression analysis is desired for Xoo and Xoc because some broad host range vectors that can replicate stably in X. oryzae pathovars are low‐copy number plasmids. To overcome this limitation, we developed a modular plasmid assembly system to transfer the functional DNA modules from the entry vectors into the pHM1‐derived backbone vectors on a high‐copy number basis. We demonstrated the feasibility of our vector system for protein detection, and quantification of virulence gene expression under laboratory conditions and in association with host rice and nonhost tobacco cells. This system also allows execution of a mutant complementation equivalent to the single‐copy chromosomal integration system and tracing of pathogens in rice leaf. Based on this assembly system, we constructed a series of protein expression and promoter‐probe vectors suitable for classical double restriction enzyme cloning. These vector systems enable cloning of all genes or promoters of interest from Xoo and Xoc strains. Our modular assembly system represents a versatile and highly efficient toolkit for gene expression analysis that will accelerate studies on interactions of X. oryzae with rice. We developed a modular plasmid assembly system to transfer DNA modules from entry vectors into high‐copy number pHM1‐derived vectors and use it for protein detection, mutant complementation, and tracing pathogens in leaves.

• Publisher: John Wiley & Sons, Inc,John Wiley and Sons Inc
• Subject: Assembly / Bacteria / Bacterial diseases / Bacterial infections / bacterial leaf streak / Binding sites / Blight / Cloning / Cloning vectors / Complementation / Copy number / Deoxyribonucleic acid / DNA / E coli / Economic importance / Enzymes / Expression vectors / Gene expression / Genes / Genetic engineering / Genetic vectors / Host range / Leaf streak / Leaves / modular plasmid assembly / Modular systems / mutants / paddies / Pathogenesis / pathovars / pHM1 / plant pathology / Plasmids / promoter‐probe vector / Protein expression / protein expression vector / protein synthesis / Proteins / restriction endonucleases / Rice / Rice fields / Technical Advance / Tobacco / Virulence / Virulence (Microbiology) / Xanthomonas oryzae / Xanthomonas oryzae pv. oryzae