Identification of Gene Involved in Cypress Canker by PCR-Select Subtractive Hybridisation Approach

Abstract

Cypress canker is the most serious biological threat faced by cypress in Europe and North America. Tree breeding strategies retain canker resistance the primary selection criterion. Identification of genes activated or inhibited during the infection process is the basis to better understand the canker resistance. PCR-select (suppression subtraction hybridization) technique of isolation of genes specific for an infection process, was applied for analysis of host-pathogen interactions in the pathosystem Cupressus sempervirens / Seiridium cardinale. The subtraction, with RNA from the early stages of infection of S. cardinale, as a tester, and RNA from uninfected C. sempervirens, as a driver, enriched the pool of cDNA molecules with the ones specific for infection. The first step, was to develop a critical protocol for RNA isolation from cypress bark to provide a good quality of RNA for the further analysis. In a second step, 5 years-old seedlings of C. sempervirens were artificially infected by virulent strain of S. cardinale. Particular attention was paid in the experimental design to avoid to select genes that were activated only by wounding. A third step, was the isolation of pathogen DNA to monitor, by Real-time PCR, the pathogen spatial colonization in the bark along the stem. In the fourth step, a subtractive procedure to obtain an enriched library of cDNA, by PCR-Select, was carried out to select putative genes. To this purpose databank similarity searches were performed with the Blastx. program maintained at NCBI. In this study we succeeded in identifying about 100 cDNA clones significantly expressed in infected hosts but not in the uninfected control. The expression of several of these genes showing sequence similarity with resistance- or stress-related genes from other plant species were identified.