Establishment of a simple and efficient Agrobacterium-mediated transformation system for Phytophthora palmivora.

Dongliang Wu,Miaoying Tian,Janice Uchida,Yingchao Liu,Yingchao Liu,Natasha Navet

doi:10.1186/s12866-016-0825-1

Abstract

BackgroundAs an agriculturally important oomycete genus, Phytophthora contains a large number of destructive plant pathogens that severely threaten agricultural production and natural ecosystems. Among them is the broad host range pathogen P. palmivora, which infects many economically important plant species. An essential way to dissect their pathogenesis mechanisms is genetic modification of candidate genes, which requires effective transformation systems. Four methods were developed for transformation of Phytophthora spp., including PEG(polyethylene glycol)/CaCl2 mediated protoplast transformation, electroporation of zoospores, microprojectile bombardment and Agrobacterium-mediated transformation (AMT). Among them, AMT has many advantages over the other methods such as easy handling and mainly generating single-copy integration in the genome. An AMT method previously reported for P. infestans and P. palmivora has barely been used in oomycete research due to low success and low reproducibility.ResultsIn this study, we report a simple and efficient AMT system for P. palmivora. Using this system, we were able to reproducibly generate over 40 transformants using zoospores collected from culture grown in a single 100 mm-diameter petri dish. The generated GFP transformants constitutively expressed GFP readily detectable using a fluorescence microscope. All of the transformants tested using Southern blot analysis contained a single-copy T-DNA insertion.ConclusionsThis system is highly effective and reproducible for transformation of P. palmivora and expected to be adaptable for transformation of additional Phytophthora spp. and other oomycetes. Its establishment will greatly accelerate their functional genomic studies.

Highlights

As an agriculturally important oomycete genus, Phytophthora contains a large number of destructive plant pathogens that severely threaten agricultural production and natural ecosystems
The Polyethylene glycol (PEG)/CaCl2 protoplast transformation method was first established by Judelson et al [16] to transform P. infestans and it has been commonly used in transformation of several Phytophthora pathogens including P. infestans, P. parasitica [17], P. sojae [18,19,20], P. palmivora [21], P. cactorum [22], and P. capsici [23]
An Agrobacterium-mediated transformation (AMT) method was previously reported for P. infestans and P. palmivora [30], its further use in oomycete research has never been reported likely due to low success and low reproducibility of generating transformants

Summary

Introduction

As an agriculturally important oomycete genus, Phytophthora contains a large number of destructive plant pathogens that severely threaten agricultural production and natural ecosystems. The PEG/CaCl2 protoplast transformation method was first established by Judelson et al [16] to transform P. infestans and it has been commonly used in transformation of several Phytophthora pathogens including P. infestans, P. parasitica [17], P. sojae [18,19,20], P. palmivora [21], P. cactorum [22], and P. capsici [23] This method is labor intensive and requires large amounts of starting materials. AMT circumvents this issue as it usually generates the integration of one or two copies [30] It does not require protoplasting and specialized equipment such as gene gun (biolistic particle delivery system) or electroporator, and is easy in handling. An AMT method was previously reported for P. infestans and P. palmivora [30], its further use in oomycete research has never been reported likely due to low success and low reproducibility of generating transformants

Methods

Results

Discussion

Conclusion