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

Abstract

Background: As 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.

Results: In 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.

Conclusions: This 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.

Keywords: Agrobacterium-mediated transformation; Copy number; GFP; Oomycete; Phytophthora palmivora.

Fig. 1

Schematic representation of pCB301TOR. RB, T-DNA right border; LB, T-DNA left border. Pham34 and Phsp70, promoters of ham34 and hsp70. Tham34 and Thsp70, terminators of ham34 and hsp70. MCS, multiple cloning site. NPTII, neomycin phosphotransferase II

Fig. 2

An outline of the method developed for Agrobacterium-mediated transformation of P. palmivora. Three representative transformants from two selection plates are shown in the picture at the bottom

Fig. 3

Expression of GFP in a representative transformant transformed with pCB301TOR-GFP. Photographs of mycelia (a), sporangia (b), sporangia and immobile zoospores (c) of the wild type strain (WT) and a representative GFP transformant (Tfm) under bright field and GFP fluorescence channel (GFP). Note that empty sporangia (indicated by arrows) did not show GFP fluorescence; Sporangia of the wild type strain showed some auto-fluorescence under GFP channel when 20× objective was used. Scale bars are shown at the bottom of each panel

Fig. 4

Southern blot analyses of P. palmivora transformants. Total DNA was digested using EcoRI and the blots were probed with a biotin-labeled DNA fragment encoding the open reading frame of NPTII. WT, wild-type P. palmivora strain. P, EcoRI-digested plasmid pCB301TOR-GFP. The numbers on the left of the blots represent the sizes (Kb) of DNA bands from NEB 1Kb DNA ladder