Detection of in vivo interactions between Arabidopsis class A-HSFs, using a novel BiFC fragment, and identification of novel class B-HSF interacting proteins

Abstract

Class A heat shock factors (Hsfs) of Arabidopsis are known to function as transcriptional activators of stress genes. Genetic and functional analysis suggests that HsfA1a and HsfA1b are central regulators required in the early phase of the heat shock response, which have the capacity to functionally replace each other. In order to examine Hsf interaction in vivo, we conducted interaction assays using bimolecular fluorescence complementation (BiFC) on Arabidopsis protoplasts co-transformed with suitable Hsf-YFP fusion genes. BiFC assays were quantified with confocal laser scanning microscopy and flow cytometry, and confirmed with immunoprecipitation assays. For each Hsf we could not only demonstrate homomeric interactions but also detect heteromeric interaction between HsfA1a and HsfA1b. Truncated versions of these of Hsfs, containing deletions of the oligomerization domains (ODs), provided clear evidence that the ODs are required and sufficient for the HSF interaction in vivo. By contrast there was only homomeric but no heteromeric interaction detected between two different class B Hsf transcription factors (HsfB1 and HsfB2b) in a yeast two-hybrid assay. HsfB1/HsfB2b functions are not directly linked with the expression of conventional heat shock genes; class B Hsfs are devoid of the activation domain motif conserved in class A Hsfs. In order to identify other proteins interacting with HsfB1 and HsfB2b we performed yeast two-hybrid screenings of cDNA libraries. Three of the identified proteins were common to both screenings. This suggests that HsfB1 and HsfB2b may be involved in complex regulatory networks, which are linked to other stress responses and signaling processes.