Identification Of Genes Involved In Resistance Of Nicotiana Benthamiana To Xanthomonas Oryzae Pv. Oryzae And Tomato To Cladosporium Fulvum

Gene-for-gene resistance and non-host resistance are two important types of plant disease resistance. Plant gene-for-gene resistance is conferred by plant disease resistance (R) genes to pathogen strains carrying complementary avirulence (Avr) genes; while non-host resistance, manifesting at species level, is the most widely-distributed and longlasting type of plant disease resistance in the nature. This thesis research concerns two parts of contents. Part I focuses on cloning and bioinformatic analysis of 89 ACE (Avr/Cf-elicted) cDNA fragments, while part II is about functional analysis of genes corresponded to 62 fragments in rice bacterial leaf blight pathogen Xanthomons oryzae pv. oryzae (Xoo) and Cf/Avr-mediated hypersensitive response (HR) employing the virus-induced gene silencing (VIGS) technique, thereby screening for the candidate genes which are required for the non-host resistance of Nicotiana benthamian to Xoo and gene-for-gene resistance of tomato to the leaf mould fungus Cladosporium fulvum. The main results are as follows:1. 89 ACE fragments displaying differential expression between seedlings showing and without showing Cf-4- and Cf-9-dependent HR were cloned. The sequences were deposited at GenBank under the accession numbers EL645744-EL645832. Among the 89 ACE fragments, 79 showed homology to sequences with known functions, such as photosynthesis (20.3%), metabolism (14.7%), defence response (10.1%), signal transduction (10.1%), HR and cell death (1.1%), transcriptional regulation (10.1%), protein synthesis (9%), stress response (1.1%), cell membrane fusion and secretion, and membrane trafficking (2.2%), and miscellaneous biological processes (10.1%). Additionally, 9 ACE fragments corresponded to genes encoding proteins with an unknown function, and 1 had no significant similarity to known sequences.Analysis for the 89 ACE sequences in combination with 189 ACE fragments cloned previously by our laboratory revealed that 38% (106/278) ACE fragments showed significantly different expression (either induced or repressed) upon development of the Avr4/Cf-4- and .Avr9/Cf-9-dependent HR. The majority of these (90/106, 85%) displayed significantly greater differential expression upon development of ,Avr4/Cf-4-dependent HR than that of Avr9/Cf-9-dependent HR, indicating that HR and defence responses induced by Avr4/Cf-4 are stronger than that by Avr9/Cf-9. Expression of most photosynthesis-related ACE genes were down-regulated, while that of most ACE genes related to metabolism, defence response, signal transduction, and transcriptional regulation were up-regulated, demonstrating that development of Cf/Avr-dependent HR induces defense response, signal transduction and transcriptional regulation, while represses photosynthesis.Among the 89 ACE fragments, 43 (encoding 36 types of proteins) were unique in comparison with all known sequences that are responsive to Avr/Cf interaction, including the 189 ACE fragments cloned previously by our laboratory and the ACRE and ART sequences cloned by other groups, thus providing novel gene resources and information potentially useful in elucidating the mechanism of Cf/Avr-dependent HR and resistance.2. The role of 62 genes in Cf-4/Avr4- and Xoo-induced HR was analyzed using TRV-mediated VIGS. The 62 genes comprise 51 ACE and 11 genes related to ubiquitin-mediated protein degradation pathway and Ca²⁺ signal transduction. Silencing of 12 fragments significantly compromised the two types of HR, thus the 12 genes might play an important role in the two types of HR. The 12 genes included ACE175, which encodes a suberization-associated anionic peroxidase; ACE150, which encodes a subtilisin-like Proteinase; ACE43, which encodes a member of the ERF/AP2 transcription factor family; ACE80, which encodes a pathogenesis-related protein; ACE117, which encodes a lipase/esterase family protein; ACE genes 35, 95 and 112 encoding proteins with an unknown function, and UBH encoding an ubiquitin carboxyl-terminal hydrolase, two UBC genes under accession numbers L23762 and AF332958, encoding an ubiquitin-conjugating enzyme, and UBD encoding an ubiquitin fusion degradation protein. Silencing of 8 out of the 12 genes, including ACE genes 35, 95, 150 and 175, and UBH, UBC (L23762), UBC (AF332958) and UBD, significantly reduced Cf-4/Avr4-dependent HR, therefore these 8 genes may be involved in Cf/Avr-mediated gene-foe-gene resistance; while silencing of 10 out of the 12 genes, including ACE genes 35, 43, 80, 95, 112, 117 and 175, and UBH, UBC (L23762) and UBD, significantly decreased Xoo-induced HR, therefore these 10 genes may play a role in regulation of non-host resistance to Xoo. Among the 12 genes, 6, including ACE genes 35, 95, and 175, and UBH, UBC (L23762) and UBD, were potentially involved in regulation of both type resistance, while ACE150 and UBC (AF332958), and 4 ACE genes 43, 80, 112 and 117 possibly functioned specifically in regulation of Cf/Avr dependent resistance and non-host resistance to Xoo, respectively. Results obtained in this study suggest that uniquitin-miediated protein degradation pathway may play an important role in regulation of both gene-for-gene resistance and non-host resistance.