CDNA-AFLP combined with functional analysis reveals novel genes involved in the hypersensitive response.
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.
Gabriëls
Suzan H E J
SH
Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands.
Takken
Frank L W
FL
Vossen
Jack H
JH
de Jong
Camiel F
CF
Liu
Qing
Q
Turk
Stefan C H J
SC
Wachowski
Ludvik K
LK
Peters
Jenny
J
Witsenboer
Hanneke M A
HM
de Wit
Pierre J G M
PJ
Joosten
Matthieu H A J
MH
D020418:Algal Proteins; D002963:Cladosporium / Q000235:genetics; D018076:DNA, Complementary; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000502:physiology; D020869:Gene Expression Profiling; D020868:Gene Silencing; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000502:physiology; D000090263:Leucine-Rich Repeat Proteins; D018551:Solanum lycopersicum / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology; D010838:Phytophthora / Q000235:genetics; D018515:Plant Leaves / Q000033:anatomy & histology / Q000378:metabolism / Q000382:microbiology; D010940:Plant Proteins / Q000235:genetics* / Q000502:physiology; D030821:Plants, Genetically Modified / Q000033:anatomy & histology / Q000378:metabolism / Q000382:microbiology; D011110:Polymorphism, Genetic; D011506:Proteins / Q000235:genetics / Q000502:physiology; D012269:Ribosomal Proteins / Q000235:genetics / Q000502:physiology; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000235:genetics
C085987:AVR4 protein, Cladosporium fulvum; D020418:Algal Proteins; D018076:DNA, Complementary; D005656:Fungal Proteins; D018841:HSP90 Heat-Shock Proteins; D000090263:Leucine-Rich Repeat Proteins; D010940:Plant Proteins; D011506:Proteins; D012269:Ribosomal Proteins; C085101:elicitin, Phytophthora; D020558:GTP Phosphohydrolases