Transient transformation of Podosphaera xanthii by electroporation of conidia.

David Vela-Corcía,Alejandro Pérez-García,Juan Antonio Torés,Diego Romero,Antonio De Vicente

doi:10.1186/s12866-014-0338-8

Abstract

BackgroundPowdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew.ResultsTo introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B.ConclusionThis is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.

Highlights

Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops
Hygromycin B resistance is not suitable for in planta selection Hygromycin B resistance is one of the genetic markers most commonly used in the transformation of filamentous fungi
We initially tried to introduce into P. xanthii different plasmids containing the hygromycin B resistance cassette using electroporation (Table 1)

Summary

Introduction

Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. In Spain, as in many other countries around the world, Podosphaera xanthii (synonym Podosphaera fusca) is considered to be the main causal agent of powdery mildew in cucurbits and is one of the most important limiting factors in cucurbit production [7,8]. Its nature as an obligate biotrophic parasite has imposed severe limitations to experimentation in P. xanthii as in the rest of powdery mildew fungi. These limitations are currently being overcome by the use of novel physiological, genetic and molecular techniques [9,10,11,12]

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