Isolation Of Resistance Related Genes To Ralstonia Solanacearum And Their Function Analysis In Diploid Potato

Potato (Solanum tuberosum L.) as one of top four important crops in the world after wheat, corn and rice, plays more and more important role in food security. Bacterial wilt caused by Ralstonia solanacearum (Rs) is one of devastating diseases of potato. As a soil-borne disease, there are no any efficient chemical methods to control it. Probably, the most promising way is to develop resistant cultivars. During the past years, research mainly focused on pathology, taxonomy and epidemiology of the pathogen. The genetics of host resistance and the mechanism of potato-Rs interaction along with regulation of gene expression are still poorly understood. In this study, gene expression of the incompatible interaction of potato-Rs was surveyed. The main results of this investigation are as follows:1. A new inoculation method, named as "Stem Culture in Bacterial Solution Method" was established. Two conventional methods and the new method were used to evaluate the resistance against Rs in two potato populations as well as in seven tomato lines. The results showed that the new method is more economic and reproducible, simpler and easier to operate than the two conventional methods. Six Rs-resistant and seven Rs-susceptible potato genotypes were obtained. Six genotypes (e.g. the most resistant clone ED13 and susceptible clone ED25) have been used as parents to construct mapping populations, which are useful to study the inheritance of Rs-resistance in future.2. A subtractive cDNA library of Rs-resistant potato at early stage was constructed using suppression subtractive hybridization (SSH) and macroarrays. Totally 123 differentially expressed uni-gene fragments were identified. Ninty-nine ESTs (80%) have high similarity with genes (fragments) in the NCBI database (0< E-value< 10¹⁰), sixteen (13%) have low identity (E-value>10"10), eight (8%) have no match. The genes with low identity and no match to known genes may represent novel genes. Seventy percent of 123 ESTs were related to the primary metabolism, energy, cell structure, gene regulation, protein synthesis, protein modification and processing, transporters, disease defence, transcription and signal transduction, etc. while the remaining 30% were function unknown or unclassified.Fourty-four ESTs out of the 123 differentially expressed fragments were considered as Rs-resistance related ESTs and were classified into six groups, which are involved in basal disease resistance process, including signal recognation, signal transudation, hypersensitive response, systemic acquired resistance, cell rescue and protection, and resistance or defense, etc. Most of these ESTs shared high similarity with known genes, indicating that these genes may be conservative in fuction and play important roles in disease resistance in different plant species.3. A full-length cDNA library of Rs-resistant potato was constructed based on SMART (switching mechanism at 5' end of RNA transcript) and LD-PCR (long distance PCR) techniques. The titers of unamplified and amplified cDNA library were 4.4×10⁶pfu/ml and 1.8×10¹⁰pfu/ml respectively. The rate of recombination was 96%. The length of inserted fragments was 400-1800 bp and the average size was 700-1000 bp.4. Three full-length cDNAs (StPI, StPMEI and StDnaJ) with completed open reading frame (ORF) were cloned from Rs-resistant potato leaves using RACE method combined with LD-PCR and cDNA library as well as four other genes' RACE PCR products. Expression patterns of these genes were also confirmed using Northern blot and semi-quantitative RT-PCR.StPI gene encodes a protein of 116 amino acids, and shares 89% identity with potato proteinase inhibitor I precursor in nucleotide and 74% in amino acid. StPI gene was induced by Rs and also regulated by JA. StPI gene is one of the members in potato proteinase inhibitor gene family, and may play an important role in potato's resistance to Rs. This gene has been registered in GenBank and the accession number is DQ822994.StPMEI gene encodes a protein of 198 amino acids, and shares 85% identity with tobacco invertase/pectin methylesterase inhibitor in nucleotide and 78% in amino acid. The expression of StPMEI was inhibited by Rs, which might result in improving the activity of pectin methylesterase (PME) and stiffening the cell wall in early stage post Rs-invasion. Whereas StPMEI gene was up-regulated by JA, which may be related to cell wall extension and building up in later stage. This gene has been registered in GenBank and the accession number is DQ822993.StDnaJ gene encodes a protein of 177 amino acids, and shares 84% identity with DnaJ-like 20 of Arabidopsis thaliana in nucleotide and 59% in amino acid. StDnaJ gene was induced by Rs and also regulated by JA. This gene was induced earlier and its high expression level persisting time was obviously shorter treated with JA than that infected by Rs. StDnaJ gene has been registered in GenBank and the accession number is DQ885360.L51 (methyl-CpG-binding domain-containing protein) and L77 (encoding receptor-like kinase RHG1) have different Rs-induced expression patterns. L51 reached the highest expression level in 6-12h p.i. while L77 did so in 48h p.i. These two genes were not regulated by JA.L181 (Phosphatase 2C) exists both in resistant and susceptible genotypes. This gene was down-regulated by Rs in resistant genotype, whereas up-regulated by JA, but was not regulated by JA in susceptible genotype, indicating that Rs invasion may have different signal transduction pathway with JA-treatment for this gene and this gene show its function due to stress induction.L362 (Protein kinase Pti1) was up-regulated by Rs and JA, and its expression pattern was similar to that of StDnaJ. The mRNA accumulation of L362 reached the highest level before 24h post treated by Rs and JA. Also this gene was induced earlier treated with JA than that infected by Rs. Pti1 gene is located downstream of Pto and involved in disease resistance in tomato, suggesting that L362 may also play an important role in Rs-resistnce reaction in potato.This investigation, although far from being exhaustive, has provided new molecular insights into the nature of the potato-Rs interaction in potato leaves. The ESTs reported here may provide useful data for improving our knowledge of potato resistance to pathogens, and may also be used as candidate genes for developing molecular markers to assist potato genetic breeding in future.