Investigating the H2O2 sensitivity of LbAP1, a YAP1-like homolog in the ectomycorrhizal fungus Laccaria bicolor.

Ectomycorrhizal fungi (ECM) are an important group of organisms that ensure that trees can flourish in all types of environmental conditions. By providing nutrients, water and protection against stress conditions in exchange for sugars, they allow their host trees to grow. The stress response of plants has already been studied in detail. However, stress response signalling remains poorly understood in ECM fungi. In this study, we assess the potential of H2O2 to regulate a putative transcription factor, LbAP1, using a bio-informatic approach, and localisation of heterologously expressed eGFP-fused LbAP1 upon H2O2 treatment. LbAP1 is a homolog of YAP1, a key regulator of ROS responses in Saccharomyces cerevisiae. Oxidation of two cysteines in YAP1, located in the N- and C-terminal CRD, results in nuclear accumulation through masking of the NES by disulfide bond formation and transcriptional activation of target genes. In contrast to YAP1, results obtained by fluorescence microscopy showed that an LbAP1-eGFP fusion protein did not accumulate in the nucleus upon treatment of transformed Saccharomyces cerevisiae BY4741 with 0.4 mM H2O2. As C-terminal eGFP could be interfering with disulfide bond formation, the H2O2 sensitivity of eGFP-LbAP1 was examined and found to also be unresponsive to H2O2. Upon analysis of both protein sequence and structure of YAP1 and LbAP1, it was revealed that LbAP1 contains 2 Cys less than YAP1. Furthermore, the Cys responsible for nuclear localisation upon oxidation in YAP1 are not conserved in LbAP1. The NLS and NES, however, are partially conserved. Structural predictions of LbAP1 also showed that the helix structures containing these important cysteines are not conserved, possibly indicating that LbAP1 nuclear localisation is not H2O2 dependent. From these results, we can conclude that LbAP1 regulation might be H2O2 independent, and that the cellular mechanisms of ECM fungi should be investigated separately to other fungi.