We have devised a high-throughput functional cloning method to isolate cDNAs from Phytophthora boehmeriae of which the products elicit a hypersensitive response (HR) in tobacco. The cDNAs were cloned into a binary potato virus X (PVX)-based expression vector and transformed into Agrobacterium tumefeciens (Mog101). 4100 colonies were individually toothpick-inoculated onto leaflets of Nicotiana benthamiana. 12 cDNAs were identified whose expression induced formation of a necrotic lesion around the inoculation site. 7 of these clones have different sequences. One of these clones PBC43 encodes specific elicitin. Clone PBC163 encodes a protein highly homologous to Rab; PBC241 en-codes a prohibitin protein; PBN62 encodes a Heat Shock Protein 60 (HSP60). The other five cDNAs reveal no homology to known protein and are thus considered novel. These observations suggest that this functional screening method is a versatile strategy to identify cDNAs of pathogens that encode elicitors and other HR-inducing proteins.
LI JunZHANG HaiFengZHANG ZhengGuangWANG YuanChaoZHENG XiaoBo
