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AASEN-DISSERTATION.Pdf (4.833Mb) Discovery of an extracellular stress sensory protein in Beauveria bassiana and identification of photolyase encoding phr-1 sequences in five entomopathogenic fungi A Thesis Submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Applied Microbiology in the Department of Food and Bioproduct Sciences University of Saskatchewan Saskatoon By Kelly R. Aasen © Copyright Kelly R. Aasen, April 2013. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of the Department of Food and Bioproduct Sciences University of Saskatchewan Saskatoon, Saskatchewan, S7N 5A8 Canada i GENERAL ABSTRACT Entomopathogenic fungi (EPF) are being developed as an alternative to chemical insecticides and several, including Beauveria bassiana are currently important tools in integrated pest management. However, their ability to bring about effective control of pest insects can be compromised by environmental stresses and conditions, including short-wave ultraviolet radiation (UVR) and high temperatures. The work described in this thesis: 1) identified a region of a candidate photolyase gene (phr-1) in five different genera of EPF, which encoded a portion of a putative cyclobutane pyrimidine dimer photolyase (CPD-PHR; named after the type of DNA damage they repair and the encoding gene, respectively), 2) established that in B. bassiana, tolerance to UVR or heat can be induced in conidiospores (CS) or blastospores (BS), respec- tively, by cell-free filtrate (CFF) suspected to contain an extracellular sensory component like factor (ELF), 3) identified key steps required for tolerance induction and 4) characterized a gene candidate to encode ELF (elf) and its encoded product using a combination of molecular biological and microbiological techniques, which provided evidence to suggest that the candidate gene encodes a functional ELF and propose some cellular components which may be involved in its potential interaction with the cell. Note that ELF is named after the extracellular sensory component (ESC) from Escherichia coli, but not due to known or suspected similarities in their sequence, structure or specific function, but rather because of their predicted ability, once activated by heat (in this particular case), to increase environmental tolerance in treated cells. Furthermore, this response was termed ELF/ESC induced tolerance (EIT), which is similar to the definition used by the late Dr. Rowbury and his colleagues. Currently, EIT is characterized only by an increase in ii environmental tolerance, measured as viability, following treatment with activated ESC or ELF. However, if more specific functions or roles for ESC, ELF or EIT were discovered than these definitions would require revision to accommodate such findings. In addition to the determinants of infection controlled by the fungus and host, including their molecular biology, physiology and biochemistry, the environment where they subsist is of great importance. Environmental conditions, including available moisture, wind, temperature, solar UVR and type of insect pest, all influence the ability of EPF to establish pathogenesis. High heat stress and UVR are of most importance when EPF are applied to crops during daylight hours since prolonged solar exposure can cause inactivation of fungal spores leading to decreased insect mortality. Both of these stresses and their impacts on fungi were studied during this thesis research. Sunlight is the main source of UVR in nature and its short to medium wavelengths (< 315 nm) were of most importance in this study. These wavelengths can damage DNA through the creation of cyclobutane pyrimidine dimers (CPDs) which can be lethal to cells due to their ability to halt DNA replication and gene expression. This type of damage is most efficiently repaired by CPD-PHR dependent photoreactivation, which can repair ~ 90% of CPDs but is dependent on the availability of light at 380-410 nm. The phr-1 gene has been shown to encode CPD-PHR in other fungi and was studied here in five genera of EPF. A region of a putative phr-1 that had sequence conservation with known phr-1 from other filamentous fungi was found in EPF from the genera Beauveria, Isaria, Lecanicillium, Metarhizium and Tolypocladium. This region encoded several conserved characteristics known to be functionally important for the of CPD- PHR in DNA repair. Furthermore, homology modeling was used to predict the structure/ function of the products encoded by this region of putative phr-1 and provided evidence that they iii contained structural homology with those that had CPD-PHR repair activity (E.C. 4.1.99.3). In addition, homology modeling was able to differentiate the products encoded by the putative phr- 1s in the studied EPF and a model CPD-PHR from the two other protein types belonging to the photolyase (PHR)/cryptochrome (CRY) family that are also found in fungi. The predicted molecular phylogeny of the putative CPD-PHRs was consistent with the established phylogenetic relationships of these fungi, with the exception of Lecanicillium. These results are the first for a putative phr-1 in EPF from the genera Isaria, Lecanicillium and Tolypocladium. Another phenomenon was also studied in this thesis. It had been shown in bacterial and yeast cultures that extracellular factors (for example ESCs and ELFs) are present and appear to be involved in cellular sensing of environmental stress conditions, such as high temperature and can induce tolerance to various environmental stresses, including heat and UVR. However, this phenomenon has not been studied in any filamentous fungi. This investigation was conducted to determine if CS or BS from B. bassiana were responsive to preheated CFF and possessed a phenomenon which resembles EIT to heat and/or UVR, which is characterized by an increase in the survival rates of challenged cells. The results showed that tolerances of CS and BS to UVR (under non-photoreactivating conditions) or heat (55 °C), respectively, were increased following treatment with preheated CFF. Several novel bioassays were also developed to screen for and characterize ELF candidate (EC) proteins produced by B. bassiana. These included a poly- acrylamide-based, in situ bioassay and a whole cell, pull-down assay that were employed to identify several ECs and one such candidate, EC1 (~ 28 kDa), potentially interacted with BS. Furthermore, bioassays with purified EC1 and EC2 showed that only the glycosylated EC1 possessed the ability to increase heat tolerance in BS and appeared to be an ELF based on this result, which was consistent with the current understanding of the effects of EIT. iv Analysis of EC1 and EC2 by electrospray ionization with tandem mass spectrometry (ESI-MS/MS) revealed that they were the same protein and differed by EC1 being glycosylated. An expressed sequence tag (EST) encoding peptide sequences, representing six of the eleven parent ions found in EC1/EC2 by ESI-MS/MS, was identified in the public data base of ESTs from B. bassiana. As a result, this EST was considered to be a candidate to partially encode for ELF in B. bassiana and was used to design oligonucleotide primers to amplify the remaining coding regions of the gene using polymerase chain reaction (PCR) based methods. This led to the identification of a potential 741 bp open reading frame (ORF) that encoded a total of eight out of a possible eleven parent ions derived from EC1 and EC2. In silico analysis revealed that the ORF encoded a protein with other features that were also consistent with those observed in the likely ELF, such as its nascent derived size (25.9 kDa), candidate glycosylation sites and predicted secretion signal peptide. The candidate elf was inserted into an expression cassette and introduced into the methylotrophic yeast Pichia pastoris where expression of elf could be controlled by the presence of methanol. This strategy was successful in yielding a cell line able to produce transgenic-ELF (TG-ELF) that was able to confer elevated tolerance to heat in BS to a level that was similar to ELF produced by B. bassiana. This provided additional evidence that elf encoded a functional ELF. The molecular function(s) of ELF currently remain unknown, as no sequence homo- logues of ELF with defined functions have been established. Furthermore, structural models could not confidently predict functions for the likely ELF studied here. Nonetheless, it was apparent that the ELF involved in establishing heat tolerance in BS was a small secreted cysteine rich protein (SSCP) because of its size [< 300 amino acid residues (aa)], abundance of cysteine (5.2%) and is secreted, which are characteristics consistent with other SSCPs. The ELF also v contains three, 63 aa internal repeats, which have been thought to be involved in rapid protein evolution in other organisms. Furthermore, correlative clues were found that suggested the apparent interaction between ELF and BS is of low affinity, which is consistent with other agonist-receptor interactions that are involved in signal transduction.
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