Functional Analyses Of The Cladosporium Fulvum Gene CfHNNI1 In Plant Nonhost Disease Resistance

Nonhost resistance, manifesting at species level, is an important type of plant disease resistacne. It is strong, longlasting and effective against a broad spectrum of plant pathgens, protecting plants from infection of most potential pathogens. Understanding and furthur exploitation of nonhost resistance will provide a new strategy to control plant diseases. Function of CfHNNI1 (C. fulvum host and nonhost plant necrosis inducer I), a gene isolated from tomato leaf mould pathogen Cladosporium fulvum, on induction of nonhost resistance, was studied in this thesis research, and the main results are as follows:1. Function of CfHNNI1 on induction of plant necrosis was investigated. Eleven plant species belonging to 6 families were subjected to examination of necrosis-inducing function of CfHNNI1. Transient expression of CfHNNI1 induced necrosis in species of at least 3 families, including Solanaceous species tomato and pepper, Cruciferous species Brassica napus and B. pekinensis, and Cucurbitaceous species cucumber;but not in Solanaceous species eggplant;Leguminous species pea and broad bean;and Chenopodiaceous species spinach. CfHNNI1-induced necrosis varied among species of different families or even among different species within the same family.2. Function of CfHNNI1 on induction of plant nonhost resistance was studied using transgenic plants. CfHNNI1 transgenic tobacco plants were constructed using Agrobacterium-mediated leaf disc approach. In these transgenic plants expression of CfHNNIl is under the control of the pathogen-inducible promoter of the gene HSR203J, and thus it did not occur under normal conditions, but strongly induced upon infection of tobacco black shank pathogen Phytophthora parasitica var. nicotianae. The fungal infection also induced expression of the hypersensitive response marker gene HSR203J, and PR genes, and inceased the resistance to this pathogen, indicating a role of CfHNNI1in. induction of plant nonhost resistance.3. Role of the open reading frames (ORFs) of the CfHNNIl cDNA in necrosis induction was analysed. Five constructs for transient plant expression of ORF (ATG273-TAG780), whose product contains domains conserved in bZIP transcription factors (TFs), and sequences starting from ATG3, ATG101, ATG155 and ATG262 and ending at TAG780, were constructed. Activity of the 5 cDNAs to induce necrotic response* in tobacco leaves was comparative analysed. In the sectors infiltrated with Agrobactrium suspensions expressing the sequences modified from CfHNNIl, the cDNA (ATG3-TAG780) induced rapid and strong necrosis;the cDNA (ATG273-TAG780) induced slightly less strong necrosis;while the sequences started from other ATGs did not induce any necrosis, demonstrating the essential role of the ORF (ATG273-TAG780) with conserved bZIP TF domains in necrosis induction.4. Procedure of prokaryotic expression of CfHNNIl was optimised. Construct for prokaryotic expression of CfHNNIl was made using pET-30a as vector. CfHNNIl was expressed effectively in Escherichia coli cells. The purified CfHNNIl protein was obtained by affinity chromatography using Ni2+-NTA His-Bind resin, facilitating furthur study on functions and mechanisms of CfHNNIl.