Identification Of Verticillium Wilt-Regulated Microrna In Eggplant And Functional Analysis Of MIR482 And STOL13A

Verticillium wilt is a notorious wilt disease affecting over many species of eudicot plants and mainly caused by a soil-borne fungal pathogen, Verticillium dahliae Kleb. Many economically important plants are susceptible to this disease, including cotton, eggplants and potato. But there remain at present no proven cures for this disease since the mechanisms of Verticillium wilt remain poorly understood. MicroRNAs (miRNAs), a recently discovered class of small (about 21nt in length), non-coding, endogenous, single-stranded RNAs in eukaryotes, regulate gene expression negatively at the post-transcriptional levels depending on the extent of complementation between miRNA and mRNA. Recent studies have shown that some ribosomal proteins may have additional functions apart from ribosome structure and protein biosynthesis, which could be released to regulate genes expression under stress. This study focuses on the possible role of miRNA and ribosomal proteins in Verticillium wilt. Our work will lay the foundation for further analysis and understanding of the possible molecular mechanism of Verticillium dahliae-caused defense responses in plants. The main results are as follows:In the first chapter, a computational homology search approach based on the conservation of miRNA sequences and the stem-loop hairpin secondary structures of miRNAs was adopted. The search was started with the known plant miRNAs compared to eggplant expressed sequence tags (EST) databases to find potential miRNAs. Following a filtering criteria, a total of 38 potential miRNAs belonging to 29 families were identified. Using online software psRNATarget, we further predicted target genes of these 38 miRNAs and got 137 potential targets genes. Most of these target genes were predicted to encode proteins that play a key role in eggplant growth, development, metabolism, and stress responses.To further identify miRNAs in eggplant and their response to Verticillium dahliae infection, we deep-sequenced two small RNA (sRNA) libraries prepared from mock-infected and infected seedlings of eggplants. Specifically,30,830,792 reads produced 7,716,328 unique miRNAs representing 99 known miRNA families that have been identified in other plant species. Two novel putative miRNAs were predicted with eggplant ESTs. The conservation of the identified 99 known miRNAs was investigated and the potential targets of the identified known and novel miRNAs were also predicted based on sequence homology search.By comparing the data of two sequenced sRNA libraries we found that the expression of 6 miRNA families was obviously different between the two libraries, which indicated that they may involved in plant response against Verticillium dahliae infection. We analyzed the expression pattern of partial differential expressed miRNA and their predicted target genes under Verticillium dahliae infection. The results showed similar changes to small RNA deep sequencing results. Moreover, the expression of miRNA showed a negative correlation with the changes of their target genes. By pharmacological experiment, we demonstrated that miR393 could regulate auxin pathways and play important roles in eggplant Verticillium wilt.Analysis of eggplant small RNA data sets revealed the presence of an unusually diverse superfamily, miR482, which responded to Verticillium dahliae infection. In this study, we generated several lines of transgenic potato which overexpressed miR482e to explore the role of miR482 in plant tolerance to Verticillium wilt. We found that overexpression of miR482e resulted in enhanced sensitivity to Verticillium dahliae infection in potato. By analyzing the published small RNA and degradome data set, we found that miR482e could target the coding sequence for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. MiR482e could cause its target mRNAs decay and production of secondary siRNAs. These secondary siRNAs targets other mRNAs of a defense-related protein, most of which are NBS-LRR proteins. Overexpressing miR482e in potato could result in decreasing of numerous resistant genes that related to plant pathogen resistance. Above results are consistent with the sensitive phenotype of miR482e overexpressed potato to Verticillium dahliae infection. Thus, we propose that miR482-mediated silencing cascade plays an important role in potato Verticillium wilt tolerance.In addition, we reported in previous study that L13a was responsive to Verticillium dahliae infection in a highly resistant eggplant species (Solanum torvum, SW). To elucidate the possible role of L13a in Verticillium dahliae infection, we cloned and characterized its cDNA (designated StoL13a) in this study. Expression level of StoL13a altered in response to Verticillium dahliae infection and phytohormone treatment. We expressed StoL13a in Verticillium dahliae sensitive potato and found that the transgenic potato plants were more resistant to Verticillium dahliae infection than the control plants. The transgenic plants showed a lower quantity of reactive oxygen species and attenuated oxidative injury. Also, six defense and antioxidant enzyme genes were up-regulated in the StoL13a ectopic expression plants. These results suggest that the StoL13a plays a role in plant defense to Verticillium dahliae infection.