Protocol

Phenotypic Analysis of Arabidopsis Mutants: Oomycete Pathogens

Adapted from Arabidopsis: A Laboratory Manual (eds. Weigel and Glazebrook). CSHL Press, Cold Spring Harbor, NY, USA, 2002.

INTRODUCTION

Various fungal pathogens are used in Arabidopsis pathogen studies, including Fusarium oxysporum, Alternaria brassicicola, Botrytis cinerea, and others. The oomycete pathogen Peronospora parasitica has been used by several groups and is described in this protocol. Working with Peronospora is complicated by the fact that it is an obligate biotroph, and consequently cultures must be maintained on living plants. There is no central repository for Peronospora stocks, but most investigators who work with them are willing to provide samples of infected tissue. These can be used to initiate new stock cultures, or they can be maintained as live cultures on seedlings. One of the most important factors in maintaining Peronospora is the humidity of the growth chamber, which must be kept at a minimum of 80%. Various Peronospora isolates are available. These vary with respect to which Arabidopsis ecotypes they can infect, because some combinations trigger gene-for-gene resistance. Thus, it is important that the appropriate ecotype is inoculated with the appropriate strain of pathogen. The extent of infections can be rated or quantitatively measured as the number of spores produced per plant, and frozen tissue stocks can be prepared from heavily infected tissue.