Identification of differentially expressed fungal genes in planta by suppression subtraction hybridization

In host-pathogen interactions, identification of pathogen genes expressed during plant infection poses a challenge, even though these genes may be strongly induced by signals from the host. Here, we describe the application of a PCR-based differential screening method to plant-fungal interactions. Suppression subtraction hybridization (SSH) provides a sensitive method to isolate fungal genes expressed in planta. Total RNA is isolated from infected plants for comparison with the pathogen in axenic culture, or, in the application described here, plants infected with a wild type isolate are compared with plants infected with a mutant. Following library construction, clones are sequenced and screened for differential expression in the two starting populations.