Isolation and characterization of Emphasis Type="

Authors: YeongTae Kim Jonghee Oh KyungHwan Kim JaeYoul Uhm ByoungMoo Lee

Publish Date: 2009/05/16

Volume: 37, Issue: 2, Pages: 717-

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Abstract

Elicitins extracellular proteins from Phytophthora fungi elicit a hypersensitivity response HR including systemic acquired resistance in some plants The elicitin capsicein ~10 kDa was purified by FPLC from culture filtrates of P capsici Purified native and recombinant capsicein induced a hypersensitive response in leaves of the nonhost plants Nicotiana glutinosa and Brassica rapa subsp pekinensis To search for candidate capsiceininteracting proteins from N glutinosa a yeast twohybrid assay was used We identified a protein interactor that is homologous to a serine/threonine kinase of the plant receptorlike kinase RLK group and designated it NgRLK1 The ORF of NgRLK1 encodes a polypeptide of 832 amino acids 93490 Da A conserved domain analysis revealed that NgRLK1 has structural features typical of a plant RLK NgRLK1 was autophosphorylated with higher activity in the presence of Mn2+ than Mg2+Fungi of the genus Phytophthora are a major cause of crop destruction Root rot caused by Phytophthora capsici soil infection is a serious disease affecting the cultivation of red pepper 1 and Phytophthora blight of pepper caused by this pathogen is one of the most destructive soilborne diseases in Korea 2 Many Phytophthora produce elicitins members of a family of small extracellular proteins that cause a hypersensitive response associated with the defense response of Nicotiana and Brassica species 3 Elicitins are conserved 98amino acid ~10 kDa holoproteins that are abundantly secreted into the medium 4 5 6 7Elicitin genes and proteins have been cloned and purified from many species including P parasitica 8 P cryptogea 9 P sojae 10 11 P infestans 12 and P cinnamomi 13 although the elicitin gene from P capsici has not yet been cloned All known Phytophthora elicitins show 60 sequence identity 4 Elicitins from different fungal species cause necrotic lesions on tobacco but plant defense reactions are induced at the same level 5 The major 98amino acid elicitins of Phytophthora have been divided into acidic α and basic βelicitins 14 15 The βelicitins produce greater necrosis than the αelicitins and the difference in biological activity is correlated with differences in sequence and physicochemical features among them 4 16 17 Kamoun et al have proposed dividing the elicitins and elicitinlike proteins of Phytophthora and Pythium species into five classes based on phylogenic analysis and the overall structures of the proteins 12 Jiang et al searched for new elicitin and elicitinlike gene family members by making use of a Phytophthora expressed sequence tag EST database and classified all family members of elicitin based on sequence and protein motifs 18Although elicitins are thought to be major determinants of nonhost resistance against Phytophthora infection in Nicotiana species 5 19 the role of elicitins in the biology of Phytophthora are not clear and little is known about the molecular mechanisms involved in elicitin activity Specific highaffinity binding sites for βcryptogein of P cryptogea were observed in tobacco plasma membranes 20 but these proteins did not exhibit any protease βglucanase or phospholipase activity and no other enzymatic activity has been reported for them 3 21 One likely biological role for elicitins is as sterol scavengers because they are known to be sterol carrier proteins and Phytophthora species do not synthesize sterols 22Elicitins are known to be secreted by Phytophthora species and have been assumed to localize in the extracellular space of plant tissue Consequently it is conceivable that elicitins interact with a host molecule in the plant plasma membrane to initiate plant cell responses following elicitin recognition Despite the identification of many elicitin proteins membrane receptors for elicitins have not been clearly identified to date In a yeast twohybrid assay Tyler unpublished data found a tobacco cDNA encoding an intracellular elicitinbinding protein which contained a nucleotidebinding site and leucinerich repeat motifs 3 The protein expressed from this cDNA was required for the elicitin response in tobacco but its expression in tomato or petunia did not result in gain of an elicitin response in those plants Tyler suggested that additional membrane receptor proteins may be required for the elicitin response Kanzaki et al isolated and characterized NbLRK1 which is a P infestans INF1 elictininteractor from N benthamiana 23 In their study INF1 was found to bind to the intracellular domain kinase domain of NbLRK1 Therefore they speculated that plant recognition of INF1 takes place inside the plant cellsIn this paper we report the molecular cloning and protein purification of the elicitin from P capsici In addition we used a yeast twohybrid assay to identify a capsiceininteracting protein from tobacco N glutinosa Sequence analysis revealed that the capsiceininteracting protein designated NgRLK1 has domains typical of plant receptorlike kinases RLKs The kinase domain of NgRLK1 was expressed in Escherichia coli and this domain was autophosphorylated in the presence of Mn2+ or Mg2+

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