Studies On The Heat Stress Induced Apoptotic-like Cell Death In Two Pleurotus Species And Mechanisms Of Transcriptional Responses To Stress In The Beauveria Bassiana

First part: Studies on the heat stress induced apoptotic-like cell death in two Pleurotus species.The mushroom industry, during the last 30 years, has developed to become the fifth largest economically important agricultural crop, after grains,crop-based oils, fruits and vegetables. Mushroom production is greater than 32 million tons with a value of over 200 billion yuan. The Pleurotus species represent the major cultivars in mushroom production(over 18%), andthe cultivation of most Pleurotus species is significantly influenced by environmental conditions, especially hot weather. High temperature(>38oC) is an important environmental factor that affects the growth and development of most mushrooms. However, the mechanism(s) for resistance to high temperature remains elusive.As part of this work, whether heat stress can activate apoptosis-like cell death in mycelia of Pleurotus was investigated. Two Pleurotus species(CCMCC00358 P. ostreatus and CCMCC00493 P. pulmonarius) were used to detect morphological features characteristic of apoptosis including nuclear condensation, reactive oxygen species accumulation, and DNA fragmentation when exposed to heat stress(42°C, 0.5h-4h). The results showed that these classical apoptosis markers were apparent in Pleurotus strains after heat treatment. Forstrain 00358, the ratios of nuclear condensation, reactive oxygen species, and DNA fragmentation were:53.8%, 32.4%, and 32.6%, respectively. Forstrain 00493, the ratioswere: 53.6%, 38.4% and 24.5%, respectively. The heat-induced apoptosis-like cell death in Pleurotus was further probed using oligomycin and N-acetylcysteine, both of which were shown to block processes leading to apoptosis. This is the first report that apoptosis-like cell death occurs in Pleurotus species as a result of abiotic stress, and that this process can be inhibited with chemicals that block mitochondrial-induced apoptotic pathways and/or with ROS-scavenging compounds. Second part: Mechanisms of transcriptional responses to stress in the Beauveria bassiana.From September 2013 to March 2015, I was supported by China Scholarship Councilto perform joint training at the University of Florida. In America, I studied a set of transcription factors involved in fungal stress, focusing on the protein-DNA and protein-protein interactions in the insect pathogenic fungus, Beauveria bassiana.Results from the study of B. bassiana would have implications for other fungi, including mushrooms.B. bassianais a mycoinsecticide used as a pest biological control agent worldwide. This filamentous fungus has emerged as a model system with which to probe diverse aspects of fungal growth, stress response, and pathogenesis.The stress response transcriptional regulator, MSN2/4 was studies, including binding of the protein to stress response elements(STRE) sequences. To investigate the binding of Bb MSN2,we expressed the Bb MSN2 in E. coli and synthesized DNA probes that included target sequences with different numbers of Msn2 binding sites(AGGGG/CCCCT)as well aspoint mutations in order to test the specificity of the Bb MSN2 recognition motif. Bb MSN2 binding was assayed by EMSA methods, leaning to the characterization of the protein-DNA binding specificity. In addition,binding to promoters of crucial genes involved in virulence and development was assayed. These results showed binding of Bb MSN2 to the promoter sequences of the protease(SSP CDEP-1) and chitinase(CHIT1) genes and Bb MSN2 interaction with both promoters with an affinity, i.e. KD values~0.39 μM. Competition experiments were performed to validate binding to the promoter sequences, confirming the specificity of Bb MSN2 binding.Multiprotein bridging factor 1(MBF1) is a transcriptional co-activator that bridgessequence-specific activators and the TATA-box binding protein(TBP). Little is known about the interaction between MBF1, TBP, and TBP binding to DNA(TATA-sequences) in filamentous fungi. The binding of Bb TBP to TATA-sequences was investigated via EMSA analyses. These data showed Bb TBP binding to TATA-sequences with a high affinity, i.e. KD value ~ 1.2 n M.Interaction between Bb MBF1 and Bb TBP+DNA was shown via EMSA super-shift assays, indicatingformation of Bb MBF1-Bb TBP-DNA ternary complexes. The interaction between Bb TBP and TATA-containing DNA sequences and Bb TBP and Bb MBF1 was further probed using electrochemical impedance spectroscopy. These data confirmed the specificity of the interactions, leading to a model where(i) Bb TBP can bind to(TATA-containing) DNA,(ii) Bb MBF1 can bind to Bb TBP,(iii) Bb MBF1 cannot bind to DNA directly, and(iv) therefore Bb TBP mediates the interactions of MBF1 to DNA.