Isolation And Analysis Of Resistence-related Genes During Periwinkle And Wheat Blue Dwarf Phytoplasma Interaction

Phytoplasmas are associated with diseases in over one thousand of plant species, especially are lethal in some woody plants. To date, Wheat blue dwarf?WBD? disease, caused by wheat blue dwarf phytoplasma, has only been reported in China. It is transmitted by leafhopper Psammotettix striatus L., and mostly distributes in arid and semiarid areas of northwestern China, including Shaanxi, Ningxia, Gansu and Inner Mongolia. The growth of infected wheat was depressed, and the wheat head sprouting and fructification were influenced. The disease could reduce the wheat yield, and threat wheat production. Recently, the researches involved in WBD phytoplasma genomes and pathogenicity have been carried out. However, the mechanisms of the interaction between host plant and phytoplasma were obscure. Consequently, understanding the expression profiling of plant-phytoplasma interaction at the transcriptome level, so as to elucidate the mechanisms involved in metabolism and defense after host plant infected by WBD phytoplasma, will be greatly important for WBD pathogencity research and resistant breeding. In the present study, c DNA-AFLP technique was used to analyze the differential expression profile of WBD phytoplasma-infected periwinkle?Catharanthus roseus?. Quantitative real-time PCR were carried out to verify the expression patterns of TDFs derived from c DNA-AFLP analysis. Pathogenesis-related protein?PRs? genes and some genes related with defense were full-length cloned using RACE method, following by bioinformatics characterization. Finally, the expression patterns of these genes were analyzed during the interaction between periwinkle and WBD phytoplasma. The main results are as follows:1. Using periwinkle as the material, the differential expression profile of WBD phytoplasma-infected flowers with virescence were analyzed by c DNA-AFLP technique. Two hundred and fifty-eight reliable TDFs were obtained after selective amplification and sequencing. Of the 258 transcripts, 201 TDFs were up-regulated during phytoplasma infection, while 57 were repressed. Meantime, 125 had relatively clear functions in various categories?eg. metabolism, energy, signal translocation and disease/defense and so on? when searching the non-redundant protein database. Therefore, these genes can be valuable resources for understanding molecular changes in the plant-phytoplasma interaction.2. Eight TDFs were selected for q RT-PCR assays. The same expression patterns of all TDFs were found as observed in the c DNA-AFLP analysis, except for one TDF which was appeared down-regulated on the gel, turned out to be up-regulated in q RT-PCR assays. The results revealed that the c DNA-AFLP technique was efficient and reliable in identifying expressed genes in host plant induced by phytoplasma infection.3. Among the isolated TDFs, two genes?W₁-23 and W₈-35? were speculated to be involved in flower virescence. The expression patterns of these two genes were detected in the flowers with discorloration, virescence, phyllody and healthy ones. The results showed that the expression levels of W₁-23 in different stages of flower malformation were higher than that in healthy control, while the expression of W₈-35 was up-regulated in flowers with discorloration, and down-regulated in flowers with virescence and phyllody. These indicated that W₁-23 and W₈-35 have a role during flower malformation.4. Ten candidated reference genes were selected, and their expression stability during WBD phytoplasma infection were evaluated using the algorithms ge Norm and Norm Finder program. The results showed that Cr L23 performed well when all samples were considered. Cr TUB and Cr TUA were the most stable reference genes for flower malformation during WBD phytoplasma infection. In addition, Cr ACT was the most stable reference gene for samples from different tissues of periwinkle.5. According to the sequences derived from c DNA-AFLP analysis, two PR genes were cloned with RACE method. The full length of Cr PR5 was 1049 bp,and the ORF was 750 bp which encoded 249 aa. While Cr PR6 was 1682 bp in length, and the ORF was 1170 bp, which encoded 389 aa. Cr PR5 was predicted to have a signal peptide within its N terminal residues and lack the transmembrane. However, Cr PR6 had no signal peptide or transmemberane region, and was predicted to be localized on peroxisome.6. q RT-PCR analysis was carried out for the expression of Cr PR1 a, Cr PR2, Cr PR4, Cr PR10 a, Cr PR5, Cr PR6, Cr PR13 and Cr PR14 during WBD and Jujube Witches' Broom?JWB? phytoplasma infection, respectively. The expression patterns were different for the same gene in two phytoplasma-infected periwinkles. Most of the genes were induced by WBD phytoplasma after grafted 5 days, while the time point of being induced by JWB phytoplasma was later. The results indicated that PRs have different roles in these two periwinkle-phytoplasma systems.7. The full length of Cr CBSX3 was cloned using the RACE method. The ORF of Cr CBSX3?1051bp? was 618 bp, and encoded 205 aa. Expression profiles analysis showed that Cr CBSX3 expressed with the highest level in the flower, and the lowest one in the root. In addition, Cr CBSX3 was strongly induced after WBD phytoplasma grafted 10 days during the periwinkle-WBD interactions. Subcellular localization in Nicotiana benthamiana leaves showed that Cr CBSX3 localized in the cytoplasm. Transient expression analysis indicated that Cr CBSX3 could suppress the cell death caused by BAX in tobacco cells, which indicated that Cr CBSX3 had a vital role in the early stage of this interaction system.