Molecular Mechanism Of Wheat-Puccinia Striiformis F. SP. Tritici Interactions Regulated By Host Mirnas

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive wheat diseases worldwide. Breeding and rational utilization of disease-resistant varieties is the safest, most effective, most economical and environmentally sound method of controlling wheat stripe rust. However, the resistance of wheat cultivar will be lost because of the constant variation of the Pst virulence. The study for molecular mechanism of wheat-pathogen interactions is of great significance to explore the new strategies for durably controlling the disease. At present, wheat resistance to Pst has been broadly categorized into all-stage resistance (seedling resistance) and adult plant resistance (APR). Compared with seedling resistance, APR is much more stable and durable. APR indicates that the plants are susceptible to the disease at seedling stage, but resistant at adult stage. The previous study of transcriptome indicated that there was no significant difference about the gene kind and quantity, but the intensity and lasting time of genes expression may cause the change of resistance. So, the regulation at post-transcriptional level may play important roles in APR. miRNAs are important small molecular regulators, and they play very important roles in plant organ development, phase change and defense responses by regulating the expression of the corresponding target genes at post-transcriptional level. Thus, to confirm whether miRNAs are involved in the APR to stripe rust, we firstly elucidated the expression features of miRNAs at two growth stages (seedling stage and adult stage) of Xingzi9104, and identified several miRNAs, which possessed the tissue expression specificity. Meanwhile, we also analyzed the expression profiles of some miRNAs during the interactions between wheat and Pst. Using the degradome sequencing, we identified the target genes of miRNAs. In addition, the function of target genes participating into the ROS metabolism and protein degradation were explored using VIGS, transient expression and other technologies. The function of two conserved miRNAs and their corresponding target genes were also analyzed using the interaction systems of Suwonll and Pst. The above research preliminary revealed the roles of miRNAs in wheat-Pst interactions. It laid the foundation of further illuminating the regulation mechanism of miRNAs during the wheat-Pst interactions. And it would be of greatly important for the development of genetically improving disease resistance and durably control of the disease. The main research results were listed as follows:Identification the miRNAs and corresponding target genes involved in the interactions between wheat and PstTo explore the function of miRNAs involved in the compatible interaction at seedling stage and incompatible interaction at adult stage, four miRNAs libraries (ST-M, ST-I, AT-M and AT-I) were constructed using high-throughout sequencing technology. In the libraries of seedling stage, a total of533miRNA candidates were obtained, including36wheat known miRNA candidates. The remaining497miRNA candidates included7conserved and490non-conserved wheat miRNAs. While,636miRNA candidates were obtained, including526wheat-specific miRNA candidates in the libraries of adult stage, the remaining110miRNA candidates included42conserved and68new wheat miRNAs that could be mapped to known miRNAs of other plants. Compared these libraries of distinct two growth stages, we found that most miRNAs were consistent, but the expression level of them showed different. Twelve miRNAs were demonstrated to possess the tissue expression specificity using northern blot, and most miRNAs showed a similar expression trend in second-top leaves and flag leaves. Using the microarray technology, we got several miRNAs responsive to Pst at different growth stages. The expression profiles of another15miRNAs from two stage libraries were analyzed by quantitative real time PCR (qRT-PCR), and results indicated that these miRNAs could be regulated by the infection of Pst, and the expression of them showed diverse profiles. These results indicated that not only one or one kind of miRNAs regulated the susceptibility/resistance to Pst at seedling/adult stage of Xingzi9104, but the different expression of many miRNAs.Additionally, the target genes of wheat miRNAs were also confirmed by using degradome sequencing technology. Most of the annotated target genes were related to signal transduction, energy metabolism, and other functions. According to the expression abundance, we selected15target genes for relative expression analysis by qRT-PCR, and found that the transcript accumulation of many genes also revealed a diverse expression profiles. In addition, most miRNAs and target gene formed a complicated regulation network. The one for one model was not much. This indicated the regulation miRNAs involved was very complex. We also predicted3target genes of miRNAs from seedling stage libraries using the genome information. These data provide a foundation for evaluating a new potential function of miRNAs in wheat-Pst compatible interaction. Meanwhile, there was still a large number of unigenes that were not annotated. The function of miRNAs will be explored with the function revelation of target genes.Functional analysis of miRNAs and their target genes involved in the wheat APR Through the degradome sequencing, the target gene of two miRNAs (1136-P3, PN-2013) was identified to be one sequence. And this sequence was demonstrated to could be cleaved by these two miRNAs in tobacco leaves experimentally. PN-2013was found to be regulated by the Pst infection, and it participated the interactions of Xingzi9104and Pst. The full-length cDNA of TaMDHAR were isolated from the wheat cultivarXingzi9104through in silico cloning, and the cleavage sites were located in the3’UTR. After bioinformatics analysis, TaMDHAR showed high evolutionary relationship with the MDHAR gene of Aegilops tauschii, and it was highest homologous to MDHAR2when it was balsted with all the six MDHAR genes of Arabidopsis. In additon, TaMDHAR and PN-2013, not1136-P3, showed a divergent expression patterns in wheat-Pst adult incompatible interaction. The TaMDHAR knockdown resulted in improved wheat resistance to Pst at the seedling stage. The TaMDHAR knockdown resulted in a much lower content of AsA, and it reduced the activities of APX and CAT. The reduced activities of APX and CAT resulted in a greater H2O2accumulation, which stimulated the occurrence of hypersensitive response (HR).Meanwhile, the candidate target gene of a novel miRNAs (PC-395) was isolated through the degradome sequencing. Using the co-transformation technology, we demonstrated the sequence could be cleaved by PC-395in tobacco leaves experimentally. PC-395was confirmed to be regulated by the Pst infection, so, the full-length cDNA of TaULP5were isolated from the wheat cultivarXingzi9104through in silico cloning. After bioinformatics analysis, TaULP5showed high evolutionary relationship with the ULP5gene of Brachypodium distachyon and barley. We confirmed the gene function of TaULP5through complementary in yeast mutant. Meanwhile, TaULP5was located in the cytoplasm of Arabidopsis protoplast. When TaULP5was over-expressed in yeast, the resistant of yeast to high salinity and H2O2was improved. In the interactions of wheat and Pst, PC-395and TaULPS showed contrasting divergent expression patterns, and knockdown of TaULP5resulted in the induction of several PR genes. In addition, the wheat resistance to Pst at the seedling stage was improved. Thus it can be seen, TaULP5possessed distinct function in plant response to biotic and abiotic stresses.Functional analysis of miRNAs and their target genes involved in wheat basal resistanceIn the previous study, the conserved miRNAs played important roles in the PTI interaction between plant and pathogen. Using qRT-PCR, we found the expression of tae-miR164and tae-miR408in the interactions of Xingzi9104(seedling stage and adult stage) to CYR32was similar with their expression in the Suwonll response to CYR23and CYR31. This indicated tae-miR164and tae-miR408may play a certain roles in the wheat basal resistance to Pst. Therefore, the function of tae-miR164, tae-miR408and their target genes were analyzed using the interaction systems of Suwonll to Pst. It laid the foundation of function exploration of conserved miRNAs during the plant-pathogen interactions.The full-length cDNA of the candidate target gene of tae-miR164, TaNAC21/22were isolated from the wheat cultivar Suwonll through in silico cloning, which possessed the resistance in the whole stages. It was a NAC transcription factor gene belonging to the NAM subfamily. After bioinformatics analysis, TaNAC21/22showed high homologous with the NAC gene of barley and NAC21/22of Brachypodium distachyon. The interaction between TaNAC21/22and tae-miR164was confirmed experimentally through co-transformation in tobacco leaves. Transcript accumulation of TaNAC21/22and tae-miR164showed contrasting divergent expression patterns in wheat response to Pst. TaNAC21/22was confirmed to be located in the nucleus and could function as a transcriptional activator. Silencing of individual gene showed that TaNAC21/22negatively regulates resistance to stripe rust. These results indicate that the target of tae-miR164, a novel NAC transcription factor from the NAM subfamily of wheat, plays an important role in regulating resistance of host plants to stripe rust.Meanwhile, the target gene of tae-miR408, designated TaCLP1, was isolated from wheat cultivar Suwonll through in silico cloning. There was a cleavage site of tae-miR408in the ORF of TaCLPl. TaCLPl could be cleaved effectively by tae-miR408through co-transformation technology in tobacco leaves. After bioinformatics analysis, TaCLP1showed the highest homologous with BCP gene of barley. Transcript accumulation of TaCLPl and tae-miR408showed contrasting divergent expression patterns in wheat response to Pst and high copper ion stress. Overexpression of TaCLPl in yeast (Schizosaccharomyces pombe) significantly increased cell growth under high salinity and Cu2+stresses. Silencing of individual cDNA clones in wheat challenged with Pst indicated that TaCLPl positively regulates resistance to stripe rust. The results indicate that the target of tae-miR408, TaCLPl, playe an important role in regulating resistance of host plants to abiotic stresses and stripe rust, and such interactions can be a valuable resource for investigating stress tolerance in wheat.