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In Planta Effector Competition Assays Detect Hyaloperonospora arabidopsidis Effectors That Contribute to Virulence and Localize to Different Plant Subcellular Compartments

July 2013 , Volume 26 , Number  7
Pages  745 - 757

Jorge Luis Badel , Sophie J. M. Piquerez , David Greenshields , Ghanasyam Rallapalli , Georgina Fabro , Naveed Ishaque , and Jonathan D. G. Jones

The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, U.K.

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Accepted 14 March 2013.

The genome of the pathogenic oomycete Hyaloperonospora arabidopsidis is predicted to encode at least 134 high-confidence effectors (HaRxL) carrying the RxLR motif implicated in their translocation into plant cells. However, only four avirulence genes (ATR1, ATR13, ATR5, and ATR39) have been isolated. This indicates that identification of HaRxL effectors based on avirulence is low throughput. We aimed at rapidly identifying H. arabidopsidis effectors that contribute to virulence by developing methods to detect and quantify multiple candidates in bacterial mixed infections using either Illumina sequencing or capillary electrophoresis. In these assays, referred to here as in planta effector competition assays, we estimate the contribution to virulence of individual effectors by calculating the abundance of each HaRxL in the bacterial population recovered from leaves 3 days after inoculation relative to abundance in the initial mixed inoculum. We identified HaRxL that enhance Pseudomonas syringae pv. tomato DC3000 growth in some but not all Arabidopsis accessions. Further analysis showed that HaRxLL464, HaRxL75, HaRxL22, HaRxLL441, and HaRxL89 suppress pathogen-associated molecular pattern-triggered immunity (PTI) and localize to different subcellular compartments in Nicotiana benthamiana, providing evidence for a multilayered suppression of PTI by pathogenic oomycetes and molecular probes for the dissection of PTI.

© 2013 The American Phytopathological Society