Characterization by suppression subtractive hybridization of transcripts that are differentially expressed in leaves of apple scab-resistant and susceptible cultivars of Malus domestica

Abstract

In order to compare transcription profiles in cultivars of Malus domestica that are differentially sensitive to apple scab (Venturia inaequalis), two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) method. Subtraction hybridization was performed between cDNAs from uninfected young leaves of the resistant cultivar Remo and the susceptible Elstar. In total, 480 EST clones were obtained: 218 (ELSTAR) clones represent transcripts that are preferentially expressed in Elstar, while the other 262 (REMO) are derived from RNAs that are more highly expressed in Remo. The putative functions of about 50% of the cloned sequences could be identified by sequencing and subsequent homology searches in databases or by dot-blot hybridization to known targets. In the resistant cv. Remo the levels of transcripts encoding a number of proteins related to plant defense (such as beta-1,3-glucanase, ribonuclease-like PR10, cysteine protease inhibitor, endochitinase, ferrochelatase, and ADP-ribosylation factor) or detoxification of reactive oxygen species (such as superoxide dismutase) were highly up-regulated relative to the amounts present in cv. Elstar. Most surprising was the large number of clones derived from mRNAs for metallothioneins of type 3 (91 out of 262) found in the REMO population. The corresponding transcripts were only present in small amounts in young uninfected leaves of the cv. Elstar, but were up-regulated in the susceptible cultivar after inoculation with V. inaequalis. These results indicate that constitutively high-level expression of PR proteins may protect cv. Remo from infection by different plant pathogens.

Identification of expression profiles of tapping panel dryness (TPD) associated genes from the latex of rubber tree (Hevea brasiliensis Muell. Arg.)

“…The normalization step equalizes the abundance of cDNAs with the tester population and the subtraction step excludes the common molecules between the tester and driver populations and achieves a greater than 1,000-fold enrichment of diVerentially expressed cDNAs (Diantchenko et al 1999). The technique has already been applied in many studies to isolate genes involved in responses to biotic and abiotic stresses (Mahalingam et al 2003;Degenhardt et al 2005;Zhang et al 2005).…”

Powdery Mildew Induces Defense-Oriented Reprogramming of the Transcriptome in a Susceptible But Not in a Resistant Grapevine

“…In apple (Malus domestica), the association of elevated defense gene expression with resistance to the apple scab fungal pathogen (Venturia ineaqualis) was recently reported (Degenhardt et al, 2005). In the scab-resistant variety Remo genes encoding PR-10, PR-2, PR-3, Cys protease inhibitor, and metallothioneins were constitutively expressed at higher levels than in the scabsusceptible variety Elstar in the absence of the pathogen.…”

Differentially expressed cDNAs at the early stage of banana ripening identified by suppression subtractive hybridization and cDNA microarray

“…SSH combines SSH with PCR to generate a population of PCR fragments enriched for sequences that are diVerentially expressed. This technique, in combination with cDNA arrays, has been successfully used to isolate diVerentially expressed genes in diVerent plant systems including potato (Faivre-Rampant et al 2004), Arabidopsis (Hinderhofer and Zentgraf 2001), alfalfa (Hays and Skinner 2001), rice (Chen et al 2002), wheat (Luo et al 2002), apple (Degenhardt et al 2005), maize (Zheng et al 2004), soybean (Liao et al 2003), poplar (Rishi et al 2004), Eucalyptus (Paux et al 2004), cotton (Ji et al 2003), barley (Jang et al 2003), and sugarcane (Watt 2003). However, to our knowledge, there is only one report identifying diVerentially expressed genes at the late stage of postharvest banana ripening using SSH, followed by diVerential screening of cDNA (Manrique-Trujillo et al 2006).…”