Defense Responses Including Oxidative Burst And Defense Gene Expression In The Interaction Between Wheat And Stripe Rust And Identification And Functional Characterization Of Resistance-Related Genes

Stripe rust, caused by Puccinia striiformis Westend f. sp. tritici Eriks. & Henn. (Pst), is one of the most widely destructive leaf diseases of wheat (Triticum aestivum L.) in the world. Wheat yield can be greatly reduced, even completely destroyed depending upon the level of the disease epidemic. It has been proven that breeding and rational utilization of disease-resistant varieties is the safest, most economical and effective method to control wheat stripe rust. Investigations to the resistant mechanism of the host plant and the interactions between wheat and the stripe rust pathogen from histological, histochemical and molecular biological aspects are important to give further information for the rational use of resistant genes in the improvement of resistance in cultivars.This study can be divided into two parts, one part is employed light and fluorescent microscopy, H2O2 staining, and defense gene profiling to compare the incompatible interaction involving resistance gene Yr10 and Pst in Moro wheat with a compatible interaction involving Fielder in order to elucidate the relationships among morphological aspects of pathogen development, the HR and ROS. The expression profiles of a series of defense-related genes which were studied by qRT-PCR permitted development of an integrated, time-course portrait of the host-parasite interaction of Pst in susceptible Fielder and Yr10-resistant Moro linking morphological responses to regulation of gene expression.The other part, on the basis of previous constructed suppression subtractive hybridization (SSH) the incompatible cDNA library of wheat (cv. Suwon 11) leaves infected by Pst CYR23, or from the NCBI GenBank wheat EST database, we isolated and characterized 3 full length cDNA sequences which were the CBS domain containing protein gene, the calcium binding EF-hand protein gene and translationally controlled tumor protein gene from wheat leaves infected with Pst through in silico cloning and reverse transcription PCR (RT-PCR) approaches; Characterized the molecular features and transcription profiles of these genes in the wheat defense responses to Pst, phytohormones and abiotic stress stimuli by qRT-PCR analysis; Subcellular localization of the calcium binding EF-hand protein gene and the translationally controlled tumor protein gene by introducing the GFP fusion protein constructs via particle bombardment in onion epidermal cells; Functional anlysis of the translationally controlled tumor protein gene was carried out thought BSMV-VIGS (Barley Stripe Mosaic Virus-Virus Induced Gene Silencing) method. The main studies contents and results are as follows:1. This study integrated defense-related genes profiling with histological and histochemical studies to develop a detailed time-course description of Pst penetration and infection of susceptible Fielder wheat and resistant Moro that contains the Yr10 gene for stripe rust resistance. Penetration and establishment events of the fungus within the leaf are very similar in both Fielder and Moro until approximately 6 to 12 days after inoculation (dai) after which phenomena associated with oxidative burst and hypersensitive response (HR) were observed in Moro that terminated further growth of the pathogen. Differential upregulation of transcripts of defense signaling genes in Moro compared to Fielder were observed as early as 2-6 dai and from 4-14 dai among defense-related PR-proteins. We demonstrated that recognition responses in hosts occur early in the host-parasite interaction but that key defense responses that terminate pathogen development and response occurred later.These results are the first among cereal rusts to integrate detailed morphological aspects of defense reactions including oxidative burst and HR with transcript profiling of annotated genes and permitted interpretation of the Yr10-stripe rust interaction in the context of current models of host-parasite interactions. These results will also permit identification of unannotated genes based on profile expression in the near future and thus uncovering additional key factors involved in the Yr10 mediated resistance response to P. striiformis in wheat.2. The CBS domain containing protein gene, tentatively designated as TaCDCP1 (Triticum aestivum CBS domain containing protein 1), was predicted to encode 217 amino acids protein which contained two conserved cystathionine beta-synthase (CBS) domains and was without transmembrane domain or signal peptide sequence. The deduced protein was predicted existing in chloroplast stroma. The amino acid sequence of TaCDCP1 shares 92%, 72% and 63% identify with the homologs in barley (Hordeum vulgare) , rice (Oryza sativa) and maize (Zea mays), respectively. The TaCDCP1 gene was highly expressed in leaves than in roots and stems. Challenged by Pst, TaCDCP1 was induced by this fungus in both incompatible and compatible interactions, with the maximal expression at 18 h post inoculation (hpi) and 96 hpi, respectively. Its transcript accumulation was much higher in the incompatible interaction than in the compatible interaction at the early stage of infection (18-48 hpi), but much lower at the late stage (96-120 hpi). The expression of TaCDCP1 was also up-regulated after treated by phytohormones such as abscisic acid (ABA), and down-regulated by benzyladenine, ethylene, gibberellins, methyl jasmonate and salicylic acid to a certain degree. And it was obviously up-regulated by various abiotic stresses, such as low temperature and drought. However, mechanical wound and high salinity stress could not induce the expression of TaCDCP1. These results suggest that TaCDCP1 is probably involved in the disease resistance and defense response in wheat to Pst through ABA pathways, and also participate in the signal transmission pathways under low temperature, and drought conditions.3. The calcium binding EF-hand protein gene, designated as TaCab1 (Triticum aestivum calcium binding EF-hand protein 1), did not have an intron and was predicted to encode a 216 amino acid protein which possesses an N-terminal region with a signal peptide, a transmembrane domain, an EF-hand motif and a caleosin domain. TaCab1 The results of transient assays with constructs of TaCab1 with green fluorescent protein (GFP) gene indicated that TaCab1 encodes a transmembrane protein. Quantitative real-time PCR (qRT-PCR) analyses revealed that TaCab1 was highly expressed in leaves than roots and stems. Although up-regulated expression profiles of TaCab1 were quite similar in both incompatible and compatible interactions, its transcript accumulation in the compatible interaction was much higher than in the incompatible interaction. The transcription of TaCab1 was consistently up-regulated after treated with salicylic acid (SA), and it also up-regulated at different degrees after treated by other phytohormones and stress stimuli. These results suggest that TaCab1 is involved in the plant-pathogen recognition, symptom development, and the basal tolerance to biotic and abiotic stresses through the SA signaling pathway.4. The wheat translationally controlled tumor protein designated as TaTCTP1 (Triticum aestivum translationally controlled tumor protein 1). It had 1647 bp DNA sequence with 4 introns and 5 extrons. TaTCTP1 was predicted to encode a 168 amino acid protein, which possesses an Mss4-like and Mss4/TCTP-associated superfamily conserved domains and 2 TCTP conserved sites, TCTP₁ and TCTP₂, and was without transmembrane domain or signal peptide sequence. The results of transient assays indicated that TaTCTP1 encodes a cytoplasmic protein. qRT-PCR analyses revealed that TaTCTP1 was constitutively expressed in leaves, roots and stems. The transcription level of TaTCTP1 was much higher in the incompatible interaction than in the compatible interaction at the early stage of infection (12-48 hpi), a little lower at the late stage (96-120 hpi), but was still at a high level. The transcription of TaTCTP1 was up-regulated after treated by ethylene, low temperature and high salinity. Functional analysis system mediated by BSMV-VIGS revealed that wheat plants with silenced gene of TaTCTP1 produced considerable amount of sporulation after inoculated with Pst race CYR23, and the leaf symptom was not changed after inoculated with Pst race CYR31. These results suggested TaTCTP1 could play an very important role in wheat resistant response against Pst and defense response to low temperature and high salinity through ethylene pathway.