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A functional cloning strategy, based on a binary PVX‐expression vector, to isolate HR‐inducing cDNAs of plant pathogens
Author(s) -
Takken Frank L.W.,
Luderer Rianne,
Gabriëls Suzan H.E.J.,
Westerink Nienke,
Lu Riu,
De Wit Pierre J.G.M.,
Joosten Matthieu H.A.J.
Publication year - 2000
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2000.00866.x
Subject(s) - cloning (programming) , computational biology , vector (molecular biology) , expression vector , biology , cloning vector , virology , potato virus x , molecular cloning , genetics , complementary dna , computer science , gene , recombinant dna , plant virus , virus , programming language
Summary We have devised a novel, high‐throughput functional cloning method to isolate cDNAs from plant pathogens of which the products elicit a hypersensitive response (HR) in plants. Copy DNA, made from RNA isolated from the tomato pathogen Cladosporium fulvum grown under nutrient‐limiting conditions in vitro , was cloned into a binary, potato virus X (PVX)‐based expression vector and transformed to Agrobacterium tumefaciens . 9600 colonies were individually toothpick‐inoculated onto leaflets of tomato plants resistant to C. fulvum . Four cDNAs were identified whose expression induced formation of a necrotic lesion around the inoculation site. One of these clones, specifically inducing HR on tomato plants carrying the Cf‐4 resistance gene, encodes race‐specific elicitor AVR4. The other three cDNAs, inducing a non‐genotype‐specific HR, encode a protein highly homologous to bZIP, basic transcription factors. To determine whether this approach has general applicability, part of the library was also inoculated onto Nicotiana tabacum var. Samsun NN, which is not a host for C. fulvum . Four independent HR‐inducing cDNAs were identified which all encode ECP2, an extracellular protein of C. fulvum known to induce necrosis in certain Nicotiana species. These observations confirm that this functional screening method is a versatile strategy to identify cDNAs of pathogens that encode (race‐specific) elicitors and other HR‐inducing proteins.