Cloning And Expression Of Defence-related Genes Induced By Ralstonia Solanacearum In Potato

On a global scale, potato (Solanum tuberosum) is an important crop species for the purpose of grain and vegetable consumption. Potato has wide adaptability, well fertility, high economic efficiency and rich nutritional value to be suitable for industrial processes. It plays a significant role in the food safety architectures. The plantation of potato and the production of potato are seriously threatened by bacterial wilt disease (Ralstonia solanacearum). It's very important to improve the genetic resistance of potato cultivars for disease control. This presses for clarification of the molecular mechanism of potato-Ralstonia interactions and for discovery and utilization of the good resistance genes to improve the hereditary constitution for disease resistance. At the present time, very little is known about the amount and type of defense related genes or resistance genes expressed during the stages of potato-Ralstonia interaction, without mentioning the regulations. In order to acquire some useful information about the molecular mechanism, here we presented the screening and identification of potato defense related genes and their expression patterns.The SSH (suppression subtractive hybridization) technique was used to construct an EST library. SMART (switching mechanism at 5' end of RNA transcript) and LD-PCR (long distance PCR) techniques were used to construct a full length cDNA library. Gene ontology method was used for classification and functional annotation of the related genes. For validation of the expression patterns of the defense related genes, qRT-PCR and Northern blotting were used. The main results obtained are listed bellow:To identify potato genes differentially expressed during the early stages of infection, two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) approach. Subtraction hybridization was conducted between cDNAs from stems and leaves of the susceptible genotype'zhongshu 3'inoculated with R. solanacearum and the mock inoculated with water, in both directions. A total of 189 unique sequences were obtained from the library enriched for the inoculated susceptible'zhongshu 3'sequences. By homology search and gene ontology analyses, the identified ESTs were mainly putatively categorized as belonging to: biology process (25.42%), cellular component (15.57%), and molecular function (40.98%). 22 ESTs (18.03%) were distributed among the unknown GO principles. RT-PCR analyses of 5 transcripts identified as differentially expressed between the inoculated and mock-inoculated plants revealed distinct kinetics of gene up-regulation at the early stage of the disease.Using gene ontology hierarchy and prior known genes, this is the first study to display the extent of EST transcript diversity in susceptible genotype of potato after infection by R. solanacearum which were poured into soil surrounding wounded roots. By the comparative analyses with other two similar research works including ESTs from potato inoculated with R. solanacearum by so called'stem culture in Bacterial solution method'and ESTs from grapes inoculated with Xylella fastidiosa, some similarities and differences were identified among the defense-related genes. This study has identified likely molecular targets for developing bacterial wilt resistant varieties and for characterizing their key defense genes.A full length cDNA library was constructed, with which RACE (rapid amplification of cDNA ends) method was combined to clone a dozen of key defense genes involved in the early stages of potato-Ralstonia interaction. These genes included non-specific lipid transfer protein (StLTPa1, StLTPa7, StLTPb1, StLTPb3, and StLTPf10), remorin (StREMa4), plant ferritin (StFb8), yippee-like protein (StYPa5), proteinase inhibitor (StPIa3), multiprotein bridging factor (StMBFb7), NAC transcription factor (StNACb4) and so on.The non-specific lipid transfer proteins (nsLTPs) gene StLTPa7's expression pattern was investigated by using combined methods of inoculation with the pathogen, chemical treatments with abscisic acid, salicylate and methyl jasmonate, and ESEM-EDAX (environmental scanning electron microscopy-electron dispersive x-ray spectrometer) analysis. The results showed that StLTPa7 gene displayed a complex Ca2+-associated pattern of expression during the early stage of potato-Ralstonia interaction. Transcripts of the StLTPa7 gene were systemically up-regulated by infection with R. solanacearum. It was stimulated by salicylic acid, methyl-jasmonate, abscisic acid and CaCl2. The Ca2+ channel blocker, ruthenium red, partially blocked pathogen-induced expression of StLTPa7. In situ hybridization results showed that StLTPb7 mRNA was localized in inner phloem cells of vascular tissues of potato leaves and stems. These results suggested that the gene was involved in long distance signaling and played a key role in plant defense mechanisms. A putative working model on the Ca2+-associated expression pattern of StLTPa7 was proposed to explain the mechanism involved in the defense response to bacterial wilt.The transcripts of another nsLTP gene StLTPa1 accumulated in potato leave and stem tissues infected by R. solanacearum and showed significant differences between resistant and susceptible genotypes of potato. StLTPa1 transcription was induced faster, higher and stronger in resistant genotypes compared with susceptible genotypes by the pathogen. Dominant differences in the pathogen-induced gene expression pattern between the upper and lower leaves and stems were observed within the same genotypes. In situ hybridization localized the StLTPa1 mRNA in inner phloem cells of vascular tissues. Salicylic acid, methyl jasmonate and abscisic acid induced StLTPa1 gene expression in different manners. These three signal molecules could produce long-lasting effects on the expression of StLTPa1. These results showed that StLTPa1 be pathogen-responsive gene in potato defenses and involved in a complex gene regulation network. These results suggested the causality between the gene expression and genotypes with different resistance.Two R. solanacearum cold-adapted strains isolated from the potato rhizosphere soil and potato tuber were identified as race 3 biovar 2 by microbiological, pathological and molecular methods. The strains had the classical characters of small colony variants (SCVs): pinpoint colonies that were 1/10 smaller than those of the wild-type strain, smaller cells with thicker extracellular substance, rough and strongly cohesive colony on solid media, cell-cell aggregating behavior, reduced growth rate and symptomless latent infection. To our knowledge this is the first isolation of SCVs in plant pathogen of R. solanacearum. The SCVs could not induce the up-regulated expression of StLTPa1 or StLTPa7 gene, showing that the expressions of these genes were related to the pathogenity of R. solanacearum.By construction of SSH-EST and cDNA library, gene cloning and expression analysis, the content of R.solanacearum-induced ESTs and the amount and type of defense related genes expressed during the early stages of potato-Ralstonia interaction was preliminarily determined and the biological processes in which the defense related genes involved in came to understand. This study has identified some key defence gene as likely molecular targets for developing bacterial wilt resistant varieties and for characterizing their resistance genes. Based on the comparative EST analyses, the similarities and differences were of defense-related genes involved in two types of vascular disease, and it is clear that different inoculation methods could induce different defence-related genes. The expression patterns of two defense genes of StLTPa1 and StLTPa7 were displayed. It was deduced that there must exist a complicated network composed with multiple genes, multiple pathways and multiple biological proceeds in the early stages of potato-Ralstonia interaction.Therefore the SSH library that we have created provides a basis for the identification of potato defense genes that are up-regulated during the early interaction with the pathogen, which could be important for next intensive study on gene expressions and for the molecular mechanism of potato defense to R. solanacearum.