| Wheat leaf rust caused by Puccinia triticina infection is a common, widespread wheat diseases, epidemic year caused serious damage to wheat production. Study on the mechanism of host resistance is the most economical, effective, sustainable and environmentally friendly method to control the disease. Our group has committed to the research on the molecular mechanism of wheat leaf rust resistance in Triticum aestivum.Puccinia triticina is the highly specialized living parasitic fungus. In the incompatible combination, the wheat starts hypersensitive reaction(HR) to resist the infection of Puccinia triticina. HR is a rapid form of cell death, which locates at P. triticina infection site contacting haustorrium mother cell, thereby limiting the extension of the pathogen. Our research has focused on the hypersensitive reaction mechanism of signal transduction caused by P. triticina infection. On the basis of our previous work, we have used elicitor-suspension cells and P. triticina- wheat combinations to explored the function of Ca2+ and NO in the process of hypersensitive reaction. Furthermore, we used the high- throughput transcriptome sequencing technology to analyze the gene expression pattern mediated by calcium. The main results are as follows:1. Based on wheat and P. triticina interaction system, we used wheat suspension cells-elicitors interaction system. Suspension cells of wheat varieties ’Lovrin 10’ and ’Zhengzhou 5389’ were stimulated with IWF-260, a leaf intercellular washing fluid induced by leaf rust. The dynamics and interaction of Ca2+ and nitric oxide(NO) in the hypersensitive response induced by IWF-260 were analysed. The fluorescence molecular probe Fluo-3AM and DAF-FM DA were used to mark Ca2+ and NO, respectively. The two kinds of suspension cells showed differences in the concentration of Ca2+. ’Lovrin 10’, a disease-resistant variety, showed two peaks of [Ca2+]cyt, at 330 and 700 s, on stimulation, whereas ’Zhengzhou 5389’, a susceptible variety, showed no obvious change in [Ca2+]cyt with stimulation. The increased [Ca2+]cyt depended on Ca2+ flowing into cells, which suggests that Ca2+ may be involved in the hypersensitive response. Like Ca2+, NO showed a similar pattern after elicitor stimulation. For ’Lovrin 10’, NO showed a peak, with no change for ’Zhengzhou 5389’. Thus, NO production and extracellular calcium influx are closely related to wheat suspension cells’ response to stimulation; NO may play a role in downstream of calcium.2. Secondly, in vivo wheat- P. triticina interaction system, the wheat cultivar ’Lovrin 10’ and races 165 and 260 were used to constitute compatible and incompatible combinations to investigate the relationship between NO and H2O2 and between NO and calcium(Ca2+) signaling in the cell defense process by pharmacological means. The specific fluorescent probe DAF-FM DA was coupled with confocal laser scanning microscopy and used to label intracellular nitric oxide(NO) and monitor the real-time NO dynamics during the processes of wheat defense response triggered by P. triticina infection. The results showed that at 4 h after inoculation, weak green fluorescence was observed in the stomatal guard cells at the P. triticina infection site in the incompatible combination, which indicates a small amount of NO production. Twelve hours after inoculation, the fluorescence of NO in cells adjacent to the stomata gradually intensified, and the NO fluorescent area also expanded continuously; the green fluorescence primarily occurred in the cells undergoing a hypersensitive response(HR) at 24-72 h after inoculation. For the compatible combination, however, a small amount of green fluorescence was observed in stomata where the pathogenic contact occurred 4 h after inoculation, and fluorescence was not observed thereafter. The enzyme activity detection results showed that the activity level of the NOS or the NR is higher in the incompatible combination compared to that of the compatible combination, which indicated that NO in the incompatible combination came from the NOS or NR enzyme catalytic pathway. The enzyme inhibitor experiment showed that, no matter the NOS inhibitor L-NAME or the NR inhibitor Na2WO4 was injected, both inhibitors delayed the occurrence of the HR reaction, which indicated that both NOS and the NR is involved in the NO mediated HR reaction. Injections of the NO scavenger c-PTIO prior to inoculation postponed the onset of NO production to 48 h after inoculation and suppressed HR advancement. The injection of imidazole, a NADPH oxidase inhibitor, or EGTA, an extracellular calcium chelator, in the leaves prior to inoculation, delayed the onset of NO production in the incompatible combination and suppressed HR advancement. Combined with the results of previous work in our laboratory, it can be concluded that, Ca2+ and hydrogen peroxide(H2O2) are involved in upstream of NO production to induce the HR cell death during P. triticina infection, and Ca2+, NO and H2O2 are jointly involved in the signal transduction process of HR in the interaction system.3. According to the previous two results, we found in the process of HR induced by wheat’s resistance to the P. triticina infection, calcium played an important role upstream in the signal transduction pathways. Then we used the high-throughput transcriptome technology to analyze the gene expression pattern mediated by the calcium during the wheat and P. triticina interaction. Wheat Tc Lr26 was selected as the experiment material, Tc Lr26 and L10 contain the same leaf rust resistance gene Lr26. Tc Lr26 leaf were pre-injected with EGTA and then inoculated with P. triticina, leaf without drug treatment were used as the control. Leaf sample were collected at different time after inoculation and its RNA were extracted to conduct transcriptome sequencing(RNA-seq) analysis. Data analysis was performed including de novo assembly, differential expression analysis, database annotation. The recombinant contigs obtained 80975 unigenes database annotation, annotation analysis of COG, GO functional classification and KEGG pathway mapping. Compared with the non-drug injection samples, the injection of the calcium chelator EGTA significantly differentially expressed genes, these genes at early period after inoculation, a total of 4311, of which 1673 were up-regulated, 2638 down regulated in drug treatment, at later period these genes had a total of 1416, including 829 up-regulated expression and 587 down regulated, the genes functional annotation were analysed in this paper. In the meantime, we have analyzed the expression level of 10 GOI in the early and late stage of the interaction using RT-q PCR, and the result was accordance with those in the transcriptome sequencing. By analyzing the genes related to NO and H2O2 metabolism, we have came to the result that the expression of NOS, NR, NR-1, CAT-1, SOD1.1 are affected by the calcium concentration adjusted by EDTA. The results have provided molecular evidence in the level of transcriptome sequencing for previous chapters, which used pharmacology to prove that calcium is acting in the upstream of H2O2 and NO.In summary, we believed in the process of wheat and P. triticina interaction, Ca2+, H2O2 and NO played as important signaling molecules involved in the process of HR expression induced by the P. triticina infection, and calcium ions functioned upstream in the signaling pathway. Calcium mediates many physiological processes related to the resistance of wheat to the P. triticina. |
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