Heat stress-induced activation of a Trichoderma harzianum PIL superfamily gene

Various plasma membrane compartments in fungal cell wall are involved in a wide range of cellular functions. Eisosome protein Pil1 (phosphorylation is inhibited by long chain bases) in yeast cells are known to be involved genetic pathway that controls the cellular responses to heat stress. The PIL gene superfamily is well conserved among different organisms. In this study, a full-length ThPIL1 gene that encodes for a 357 amino acid protein was cloned from Trichoderma harzianum T35 strain. A comparative analysis of ThPIL1 gene sequence with other PIL1 genes of different organisms and Trichoderma spp. shows degree of sequence identity ranging from 78 to 95%. Southern blot analysis showed that there existed only one copy of the ThPIL1 gene of in genome of T. harzianum T35, but alternative splicing of ThPIL1 generates two transcript isoforms, ThPIL1-α and β. Spliced transcript ThPIL1-β retains an unspliced first intron that carries a premature termination codon. The expression characteristics of the both isoforms of ThPIL1 gene in the mycelium grown at different temperatures were investigated using quantitative real-time PCR (qRT-PCR). The results showed that they are differentially expressed in mycelia exposed to different temperatures, the major product being ThPIL1-α. The transcripts of ThPIL1-β accumulated markedly when the Trichoderma mycelium was subjected to heat (40 °C and 45 °C). The expression level of the ThPIL1-α was highest in treatment when mycelium was grown at 45 °C for 2 h, and was approximately 4-fold higher than at 45 °C for 1 h. The highest expression level of ThPIL1-β was detected at the same temperature, and very negligible expression was found at lower temperature. These results provide new insight into roles in the expression regulation of fungal eisosome genes and suggest that ThPIL1 gene may play a role in growth of T. harzianum T35 under the heat stress response to environmental stresses.