Investigate On The Non-host Resistance Of Oryza Sativa To The Puccinia Triticina And Proteomic Analysis Of Wheat Leaf Infection By Stripe Rust Fungus

The ability of an entire plant species to resist infection by all isolates of a pathogen species is termed non-host resistance (NHR). NHR can involve preformed physical or chemical barriers present on the plant surface or in epidermal cells that can stop establishment of infection structures as well as induced physical or chemical defenses (Heath 2000; Mysore and Ryu 2004). NHR is a universal plant resistence, and characterized as broad and durable. Study NHR can help us to understand the molecular mechanism of general and broad reistance better. so it has very important application value in the breed improvement of crop。Although most cereals are susceptible to at least one species of rust fungi, rice (Oryza sativa) is unusual in being a non-host species to all known rust diseases. The immunity of rice to rust disease is mediated by NHR. An obviously attractive proposition is to transfer this NHR from rice into wheat and explores the possibility of successfully transferring this resistance to other cereals agricultural cereals. So, this study, we screend non-host plant rice c.v Nipponbare, host plant wheat cv Mingxian 169 and Puccinia triticina f.sp. tritici (Ptt) as materials, employed histology, Microarray and Proteomics technique to dissect the NHR of rice to Ptt, respectively. Hopr to identify some genes or proteins could be related to possible mechanisms of NHR.Simultaneously, In this study, expression profiling of wheat cultivar‘Suwon11’infected by Pst pathotypes CY31 and CY23, representing compatible and incompatible reactions, respectively, were analyzed using the proteomics technique, followed by isolation of the differentially expressed proteins,and differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), together with the bioinformatics characterization to identify candidate genes of protein; qRT-PCR analyses were used to verify the transcription patterns of selected important candidate protein genes.The follow most important results were obtained:1. The wheat leaf rust fungus Puccinia triticina f.sp. tritici (Ptt) is not pathogenic on rice (Oryza sativa L.). Rice (Oryza sativa) cv. Nipponbare expresses non-host resistance (NHR) to Ptt. Upon inoculation with Ptt spores, approx 95% of the urediniospores germinated on the leaf surface, but only 10% of the germinated spores formed appressoria over the stomata at one day post inoculation (1 dpi). Hydrogen peroxide (H2O2) accumulated in host cells around the appressoria at 3 dpi. Approx. 3% of the appressoria produced short hyphae inside the leaf, and fluorescence was observed in tissue invaded by the hyphae by 7 dpi. At 22 dpi, 0.2% of the sites with appressoria formed branching infection hypha in mesophyll cells, but no substomatal vesicles, haustorial mother cells or haustoria were observed. The cultivar responded to the inoculation by brown discoloration of the local tissue and surrounding areas, followed by fragmentation of rust hyphi and appressoria.2. Microarray hybridization analysis of the host transcriptional response was performed 24 h after inoculation with Ptt strain PNMQ, revealing a set of genes that are up- or down-regulated following infection. In particular, the loci represented by five probe sets Os.55776.1.S1_x_at, Os.55647.1.A1_at, Os.55776.1.S1_at, OsAffx.10944.1.S1_x_at and OsAffx.10944.1.S1_at displayed the highest increase in expression compared to control inoculation. The probe sets represent at least one member of the receptor-like kinase family (RLK) that occurs within a cluster of RLK genes on chromosome 1. Other RLK genes, within the RLK gene cluster and at another location, also showed increases in expression after Ptt inoculation. These RLK genes responded differently when challenged with different rust strains, or challenged with different non-rust wheat pathogens that are non-pathogenic to rice indicating different sets of genes respond to non-host pathogens.3. Proteins were extracted from leaves inoculated with Ptt at 3 dpi and analyzed by two-dimensional gel electrophoresis (2-DE). A total 33 spots were reproducibly up-regulated and 9 were down-regulated by infection compared to the water inoculated control. Of these, 30 were identified by MALDI TOF Mass Spectrometry. The identified proteins participate in defense/stress responses, energy/carbohydrate metabolism, oxidation-reduction processes, protein folding/turnover/cleavage/degradation, signal transduction and cell death regulation. The results indicates that NHR of rice to Ptt is consistent with a shift in protein and energy metabolism, increased antimicrobial activities, possibly including phytoalexin accumulation and cell wall reinforcement, increased cell repair, antioxidive and detoxification reactions, and enhanced prevention of plant cell death. Nearly half of the up-regulated identified proteins were associated with chloroplast and mitochondrial physiology suggesting important roles for these organelles during NHR.4. In this study, a proteomics approach was used to study compatible and incompatible interactions between wheat and Puccinia striiformis f. sp. tritici (Pst). Seedlings of wheat cv. Suwon were inoculated with isolates of compatible race CYR31 and incompatible race CYR23. Total proteins isolated from wheat leaves were separated by two-dimensional gel electrophoresis. Compared to the mock inoculation, 41 differentially expressed proteins at 24 and 72 h post-inoculation were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), 23 of these proteins were positively identified.They were categorized into classes related to photosynthesis, antioxidant defense, signal transduction, and metabolism. Annotation of proteins induced in incompatible interactions revealed a rapid resistance response that included the induction of protein kinase signaling and the production of reactive oxygen species, which usually occur during R-gene-mediated responses. Proteins with antioxidant functions, e.g., ascorbate peroxidase, dehydroascorbate reductase, and glutathione S-transferase, were differentially expressed between compatible and incompatible interactions, indicating differential accumulation of reactive oxygen species in infected tissues. In addition, fragments of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) were identified in proteins isolated from Pst-infected leaves. Rubisco fragmentation in response to Pst inoculation was confirmed by western blot analysis, indicating that chloroplasts and photosynthesis in both the incompatible and compatible situations were affected by Pst infection.