Agrosuppression: a bioassay for the hypersensitive response suited to high-throughput screening.
We describe a novel method, agrosuppression, that addresses the need for an assay of the hypersensitive response (HR) in intact plants that is rapid and adapted to high-throughput functional screening of plant and pathogen genes. The agrosuppression assay is based on inoculation of intact plants with a mixture of Agrobacterium tumefaciens strains carrying (i) a binary plasmid with one or more candidate HR-inducing genes and (ii) a tumor-inducing (oncogenic) T-DNA. In the absence of HR induction, tumor formation is initiated, resulting in a typical crown gall phenotype. However, upon induction of the HR, tumor formation by the oncogenic T-DNA is suppressed, resulting in a phenotype that can be readily scored. We tested and optimized agrosuppression in Nicotiana benthamiana using the inf1 elicitin gene from the oomycete pathogen Phytophthora infestans, which specifically induces the HR in Nicotiana spp., and the gene-for-gene pair Avr9/Cf-9 from the fungal pathogen Cladosporium fulvum and Lycopersicon pimpinellifolium (currant tomato), respectively. Agrosuppression protocols that can be rapidly performed using simple mechanical wounding of petioles of intact N. benthamiana plants were developed and appeared particularly adapted to intensive high-throughput screening. This assay promises to greatly facilitate the cloning of novel plant R genes and pathogen Avr genes and to accelerate functional analyses and structure-function studies of these genes.
Kamoun
Sophien
S
Department of Plant Pathology, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA. kamoun.1@osu.edu
Hamada
Walid
W
Huitema
Edgar
E
D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000254:growth & development; D001681:Biological Assay / Q000379:methods*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics; D008855:Microscopy, Electron, Scanning; D010838:Phytophthora / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics / Q000382:microbiology / Q000648:ultrastructure; D010940:Plant Proteins / Q000235:genetics; D010941:Plant Tumors / Q000382:microbiology; D010957:Plasmids / Q000235:genetics; D013314:Stress, Mechanical; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development / Q000382:microbiology
C109609:Cf protein, Lycopersicon esculentum; D004269:DNA, Bacterial; D018744:DNA, Plant; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D010940:Plant Proteins; C036483:T-DNA; C073605:AVR9 protein, Cladosporium fulvum