The root endophytic fungus Piriformospora indica plays an important role in increasing abiotic stress tolerance of its host plants. To explore the impact of P. indica on freezing tolerance, Arabidopsis seedlings were co-cultivated with P. indica exposed to −6°C for 6 h. Freezing stress decreased the survival rate, electrolyte leakage, leaf temperature, water potential and chlorophyll fluorescence of Arabidopsis plants in comparison to the controls. P. indica colonizion reduced the negative effects of freezing, and the plants contained also higher amounts of soluble proteins, proline and ascorbic acid during the post-thaw recovery period (4°C; 12 h). In contrast, the H2O2 and malondialdehyde levels were reduced in seedlings colonized by the fungus. The brassinolide (BR) and abscisic acid (ABA) levels dramatically increased and the transcript levels of several crucial freezing-stress related genes (CBFs, CORs, BZR1, SAG1 and PYL6) were higher in inoculated plants during the post-thaw recovery period. Finally, inocculated mutants impaired in the freezing tolerance response (such as ice1 for INDUCER OF CBF EXPRESSION1, a crucial basic helix-loop-helix transcription factor for the cold-response pathway in Arabidopsis, cbf1, −2, −3 for C-REPEAT-Binding Factor, cor47 and −15 for COLD-REGULATED and siz1 encoding the SUMO E3 LIGASE) showed better survival rates and higher expression levels of freezing-related target genes after freezing compared to the inocculated controls. Our results demonstrate that P. indica confers freezing tolerance and better post-thaw recovery in Arabidopsis, and stimulates the expression of several genes involved in the CBF-dependent pathway.
