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cDNA-AFLP Combined with Functional Analysis Reveals Novel Genes Involved in the Hypersensitive Response

June 2006 , Volume 19 , Number  6
Pages  567 - 576

Suzan H. E. J. Gabriëls , 1 Frank L. W. Takken , 1 Jack H. Vossen , 1 Camiel F. de Jong , 1 Qing Liu , 1 Stefan C. H. J. Turk , 2 Ludvik K. Wachowski , 2 Jenny Peters , 2 Hanneke M. A. Witsenboer , 2 Pierre J. G. M. de Wit , 1 and Matthieu H. A. J. Joosten 1

1Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands; 2Keygene N.V., P.O. Box 216, 6700 AE, Wageningen, The Netherlands

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Accepted 27 January 2006.

To identify genes required for the hypersensitive response (HR), we performed expression profiling of tomato plants mounting a synchronized HR, followed by functional analysis of differentially expressed genes. By cDNA-AFLP analysis, the expression profile of tomato plants containing both the Cf-4 resistance gene against Cladosporium fulvum and the matching Avr4 avirulence gene of this fungus was compared with that of control plants. About 1% of the transcript-derived fragments (442 out of 50,000) were derived from a differentially expressed gene. Based on their sequence and expression, 192 fragments, referred to as Avr4-responsive tomato (ART) fragments, were selected for VIGS (virus-induced gene silencing) in Cf-4-transgenic Nicotiana benthamiana. Inoculated plants were analyzed for compromised HR by agroinfiltration of either the C. fulvum Avr4 gene or the Inf1 gene of Phytophthora infestans, which invokes a HR in wild-type N. benthamiana. VIGS using 15 of the ART fragments resulted in a compromised HR, whereas VIGS with fragments of ART genes encoding HSP90, a nuclear GTPase, an L19 ribosomal protein, and most interestingly, a nucleotide binding-leucine rich repeat (NB-LRR)-type protein severely suppressed the HR induced both by Avr4 and Inf1. Requirement of an NB-LRR protein (designated NRC1, for NB-LRR protein required for HR-associated cell death 1) for Cf resistance protein function as well as Inf1-mediated HR suggests a convergence of signaling pathways and supports the recent observation that NB-LRR proteins play a role in signal transduction cascades downstream of resistance proteins.

Additional keywords: CC-NB-LRR, defense.

© 2006 The American Phytopathological Society