| Calcium signaling was validated to participate in the early process of signal transduction during the interaction of wheat and Puccinia triticina by subcellular localization of calcium ion and our previous study of changes of [Ca2+]cyt concentration after elicitors stimulated wheat suspension cells. Bait vector of Ta Ca M4-1 was constructed and positive clone was screened out via yeast two-hybrid from c DNA library of Puccinia triticina infected wheat leaves. Ta CAMTA4 is one of the positive clone which interacting with Ta Ca M4-1. From there, the overall length of Ta CAMTA4 was obtained by 5’ RACE PCR and fragment assembly. CAMTA(calmodulin-binding transcription activator)is also named SR, they are a kind of highly conserved transcription family during evolution. So far, six members of CAMTA family were found in Arabidopsis thaliana, and they regulate the genes related to biotic stress and abiotic stress together. In our previous research, we have analysed the expression level of Ta CAMTA4 on Puccinia triticina infected wheat leaves, and found that in the incompatible combination, the expression level of Ta CAMTA4 was down regulated in the early period of 4-8 h. This result indicated that Ta CAMTA4 may regulates the resistance of wheat to Puccinia triticina negatively.To illuminate the function of Ta CAMTA4 during the resistance of wheat to Puccinia triticina, BSMV-VIGS system was adopt in this study. First, the condition of experiment was optimized. Then the two different vectors of Ta CAMTA4 were constructed. And the efficiency of Ta CAMTA4 gene silencing was compared by inoculating a single virus or their mixture. And the development of Puccinia triticina on Ta CAMTA4 gene silenced wheat leaves was observed. From there, de novo transcriptome analysis was performed on Ta CAMTA4 silenced leaves at different time points(early period: 8 h, 12 h; late period: 20 h, 36 h) to explore differently expressed genes and get their annotation information. And the genes regulated by Ta CAMTA4 were analyzed.The results we obtained in this study were as follows:1. The condition for BSMV-VIGS system on wheat lovrin10 was optimized. The two VIGS vectors of Ta CAMTA4 were constructed and their mixture was used to infect wheat to compare their silencing efficiency. The vector BSMV: Ta CAMTA4-2 was confirmed to be the best virus to silence target gene.2. The development of Puccinia triticina race 165 on Ta CAMTA4 silenced wheat leaves was depressed, and the mycelia area was observed to be diminished by fluorescence microscope; The sorus on silenced leaves was diminished compared with the control leaves.3. The resistance of Ta CAMTA4 silenced wheat leaves to Puccinia triticina race 260 was enhanced; The area of cells which had hypersensitive reaction was diminished compared with the control leaves.4. Ta CAMTA4 silenced and control wheat leaves were inoculated with Puccinia triticina race 260, then the third leaves were collected at different time points and subjected to transcriptome sequencing; Total output is 24185102400 nt, and 72957 Unigene via de novo assembly.5. Of the 72957 Unigenes, 60948 of them were annotated with NR,NT,Swiss-Prot,KEGG,COG,GO database.6. Analysis of different expressed genes was performed, and 3456 genes were up-regulated, 576 genes were down-regulated at early period(8 h, 12 h); 4180 genes were up-regulated, 633 genes were down-regulated at late period(20 h, 36 h).7. Relative gene expression of five candidate genes was analysed by RT-q PCR. The results of RNA-seq was validated to be reliable.8. Different expressed genes were classified and annotated by GO and KEGG database, and Ta CAMTA4 was proved to participate in many physiological processes, such as metabolic process, response to stimulus, biological regulation and so on. In pathway of regulation of autophagy, the expression of key gene Ta ATG8 was up-regulated because of the silencing of Ta CAMTA4. In plant-pathogen interaction pathway, expression of CDPK, HSP90, PR1, WRKY were also up-regulated. |
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