James Kronstad

The corn smut fungus, Ustilago maydis, is the premier basidiomycete model for the study of biotrophic plant-pathogen interactions. In fungi, monothiol glutaredoxins are central regulators of key cellular functions such as iron homeostasis, cell wall integrity and redox status via their interactions with other proteins as well as iron-sulfur clusters and glutathione. In this study I characterized the novel roles of the monothiol glutaredoxin Grx4 in the biology of U. maydis. In addition to its roles identified in other fungi, Grx4 is necessary for normal pathogenesis by U. maydis on its plant host, Zea mays. Mutants expressing a conditional allele of grx4 under the control of the arabinose-induced/glucose-repressed promoter Pcrg, exhibited decreased virulence on maize which correlated with grx4 transcript levels at the time of infection. Additionally, perturbations were detected in homeostasis and perception of the essential nutrients iron and nitrogen following grx4 repression in glucose-containing media. Furthermore, grx4 repression strongly altered secondary metabolism, leading to induction of melanin and itaconic acid biosynthetic genes and accumulation of these metabolites in vitro. A subset of Grx4-regulated multicopper oxidase genes with possible roles in secondary metabolism, (particularly genes potentially involved in melanin biosynthesis) were targeted for further reverse genetic study. This aspect of the work identified the key laccase enzyme required for melanization and laccase activity by U. maydis in a variety of conditions, Lac2. It also identified hitherto unknown roles in virulence for this gene family. Together, these data suggest that glutaredoxins could play important roles in the virulence of plant pathogenic fungi in addition to their established roles as key regulators of fundamental cellular processes. The involvement of Grx4 in the production by U. maydis of industrially relevant secondary metabolites (e.g., itaconic acid) also renders these findings of biotechnological interest.

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The opportunistic fungal pathogen, Cryptococcus neoformans, must survive within a human host to cause disease. Stress responsive proteins such as heat shock proteins facilitate adaptation to the host environment mitigating proteotoxic stress induced by elevated temperature and the immune system. We undertook the characterization of J domain protein (JDP) co-chaperones with the prediction that JDPs would contribute to processes required for virulence. To this end, deletion mutants were generated for three type III JDPs, which lack glycine phenylalanine rich and zinc finger domains. The in vitro and in vivo phenotypes of these mutants were characterized to gain insights regarding their functions. These JDPs were tagged to interrogate their localization using fluorescent microscopy and identify binding partners using affinity purification-mass spectrometry. This approach allowed the characterization of Mrj1 as a mitochondrial JDP which supports mitochondrial respiration by facilitating electron flow through complexes III and IV of the electron transport chain. Mitochondrial function supported by Mrj1 also contributed to maintenance of cell wall architecture and elaboration of capsule. In a mouse model of cryptococcosis, mutants lacking MRJ1 were avirulent. The ER co-chaperone, Dnj1, was identified as a JDP which facilitates the elaboration and secretion of virulence factors at host temperature. Accordingly, a mutant lacking DNJ1 proliferated more slowly compared with the wild type in the lungs and had decreased dissemination to the brain in a mouse model of cryptococcosis. Dnj4 was characterized as a nuclear co-chaperone which interacts with histones 3 and 4. Deletion mutants lacking DNJ4 were hypersensitive to the DNA damaging agent hydroxyurea, prompting characterization of the transcriptional response to hydroxyurea in the wild type and mutant. This analysis revealed a role for Dnj4 in up-regulation of DNA repair genes in response to DNA damage. Mutants lacking DNJ4 were also impaired in the in vitro elaboration of key virulence factors but had no defect in virulence in a mouse model of cryptococcosis. Taken together, this study of JDPs demonstrates that co-chaperones contribute to distinct biological processes in C. neoformans. Importantly, Mrj1 and Dnj1, which have divergent amino acid sequences from human JDPs, represent promising targets for antifungal drug development.

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The mechanism of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsid egress from the nucleus to the plasma membrane leading to the formation of budded virus (BV) is not known. AC141 is a nucleocapsid protein and has been shown to be associated with β-tubulin. We hypothesized that nucleocapsid proteins associate with the lepidopteran microtubule and kinesin-1 during egress. Kinesin-1 is a motor protein that moves along the microtubules and carries cargo. Experiments showed that nucleocapsid proteins associate with kinesin-1 during infection. Downregulation of kinesin-1 by siRNA results in reduced BV production. These studies support that AcMNPV utilizes kinesin-1 and microtubules for nucleocapsid transport and BV production.GP64 (integral membrane protein) and ME53 associate at the plasma membrane and are believed to be at budding foci of nucleocapsids. AC141 was shown to associate with the ME53-GP64 complex at the plasma membrane and potentially facilitates the budding of nucleocapsids. The interaction between AC141, GP64, ME53 may enhance the cellular relocation of ME53. AcMNPV-encoded viral ubiquitin (vUbi) and AC141 (a predicted E3 ubiquitin ligase) have been shown to be required for efficient BV production. We hypothesized that vUbi interacts with AC141 and this interaction is required for BV production. Deletion of both ac141 and vubi restricted the infection to a single cell. AC141 was ubiquitinated by either vUbi or cellular Ubi and this interaction was required for optimal BV production. Virion fractionation showed that a nucleocapsid protein of 100 kDa, potentially AC66, is specifically ubiquitinated with vUbi in BV and but not in occlusion derived virus (ODV). These data suggest ubiquitination of nucleocapsid protein acts as a signal that determines how a nucleocapsid is directed to become a BV or ODV.Polyubiquitin chains are formed by the internal lysines present within the ubiquitin and serve as a signal for various cellular pathways. Mutations of lysines to arginine showed that vUbi is involved in cellular processes mediated by K6 and K27 polyubiquitin chains. The collective results of this study provide significant new data on the role of viral and host proteins and the mechanism by which baculovirus nucleocapsids egress from the nucleus to form BV.

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Virion assembly and disassembly are crucial aspects of the virus multiplication cycle, however, relatively little is known about these processes in plant viruses. While the former helps to produce multiple copies of stable infectious progeny virions, the latter is required for release of the encapsidated viral genome into a host cell for initiating new rounds of virus multiplication. In this thesis, I aimed to study Cucumber necrosis virus (CNV) particle assembly and disassembly and the role of CNV coat protein (CP) and host HSP70 homologs in these processes. It was found that CNV infection of Nicotiana benthamiana causes a significant upregulation of HSP70 homologs, and that, in turn, HSP70 is co-opted by the virus at several stages of the multiplication cycle to promote various aspects of the infection cycle including viral RNA, CP and particle accumulation. HSP70 homologs were also found to assist CNV CP in chloroplast targeting possibly to attenuate chloroplast-mediated plant defence and thereby allow further spread of the virus. It was also determined that the HSP70 homologue, Hsc70-2 is bound to CNV virions and that this association appears to facilitate the uncoating efficiency of CNV particles likely via triggering a conformational change in particles. This is the first report that a plant virus utilizes HSP70 homologs for disassembly.A highly basic “KGRKPR” sequence in the ε-region of the CNV CP arm was also examined for its role in virion assembly and encapsidation of viral RNA. Through mutational analysis, it was found that the basic residues promote T=3 versus T=1 virion formation and encapsidation of full-length viral RNA in vivo. Moreover, mutants lacking 2-4 of the basic residues encapsidated proportionately greater amounts of host RNA suggesting the role of these basic residues in selection of viral RNA during assembly. It was also shown that heat shock enhances transcription of heat-inducible ONSEN-like retrotransposons known to be induced during CNV infection. Since retrotransposons are known to play an important role in genome variation, the described studies may be helpful in understanding the importance of plant viruses in inducing genome variation and perhaps adaptation of plants to changes in the environment.

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Cryptococcus neoformans is a fungal pathogen and the causative agent of meningoencephalitis in immunocompromised and AIDS patients. Iron acquisition and the maintenance of iron homeostasis in C. neoformans are important aspects of virulence. Here, the monothiol glutaredoxin Grx4 was identified as a binding partner of Cir1, the master regulator of iron-responsive genes and virulence factor expression in C. neoformans. We confirmed that Grx4 binds Cir1 in vitro and in a yeast two-hybrid assay. RNA-Seq performed on the grx4 and cir1 mutants, and the WT strain, under low or high iron conditions identified genes involved in iron metabolism and expanded the concept that Grx4 plays a central role in iron homeostasis. A grx4 mutant, with deletion of the glutaredoxin domain, displayed iron-related phenotypes similar to those of a cir1 mutant, including elevated activity for cell surface reductases, sensitivity to high iron levels and increased susceptibility to phleomycin. Importantly it was shown that a grx4 mutant was avirulent in a mouse model of infection. We also observed that after 48 hour of starvation grx4 mutant had a growth defect in an iron-supplemented media. Interestingly, the growth defect of the grx4 mutant was rescued by exogenous GSH. We therefore further investigated the role of GSH in C. neoformans. Deletion of the GSH2 gene encoding the second enzyme in GSH biosynthesis resulted in a significant decrease in intracellular GSH levels in gsh2 mutants compared to the WT. This also resulted in loss of elaboration of major virulence factors including production of capsule and melanin, growth at host body temperature, and enhanced susceptibility to antifungal drugs. In conclusion GSH is required for expression of major virulence factors and defense against iron starvation-induced stress. GSH also influenced cell wall integrity. These results indicate that GSH plays a major role in the virulence of C. neoformans, and suggest a crosstalk between Grx4 and GSH as components of iron homeostasis and virulence expression. Overall, our findings provide further understanding of the regulation of virulence in C. neoformans and suggest novel drug targets for anticryptococcal therapy.

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Assembly is one of the major steps in the virus multiplication cycle. Recognition of viral RNA by coat protein (CP) is one means to ensure specific packaging of viral RNA over host RNA for the production of infectious virus particles. Viral RNAs possess specific sequences and/or structures [origin of assembly sequences (OASs)] which serve as high-affinity binding sites for the CP. In this thesis, I aimed to identify the OAS of Cucumber necrosis virus (CNV). Serendipitously, it was found that besides viral RNA, CNV also encapsidates host RNAs albeit to a lower level (~0.1%). Therefore, I extended my research to characterize the host RNAs present in CNV virions and in virus-like particles (VLPs) formed during agro-infiltration with CP.Characterization of encapsidated RNAs showed that both CNV virions and VLPs contained a variety of host RNA species, the most predominant being chloroplast encoded RNAs. Remarkably, certain retrotransposon or retrotransposon-like sequences were among the most efficiently encapsidated nuclear encoded RNAs, indicating that CNV virions may possibly serve as a vehicle for horizontal transmission of retrotransposons to new hosts and thereby significantly influence genome evolution. To my knowledge, this is the first report of a plant virus encapsidating retrotransposon-like sequences and complements the recent findings that Flock house virus, a small icosahedral insect virus, also encapsidates retrotransposons. Interestingly, analysis of the relative encapsidation efficiency of CP mRNA in VLPs was found to be high, indicating that the CNV CP ORF may contain an OAS(s). Towards the identification of an OAS in CNV RNA, a 1.2 kb segment encompassing the 3’ terminus of the replicase ORF and 800 nt of the CP ORF was found to stimulate encapsidation of heterologous chimeric viral RNA during co-infection with CNV. However, smaller portions of this region failed to facilitate encapsidation. Interestingly, two chimeric viral RNAs expressing CNV CP were efficiently encapsidated suggesting that the CP ORF may contain an OAS(s). This result raises the possibility that the microenvironment where virus replication and encapsidation occurs may play a role in the specificity of encapsidation, supporting the involvement of multiple factors in the specificity of CNV RNA assembly.

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The pathogenic yeast Cryptococcus neoformans causes life-threatening meningoencephalitis in immunocompromised individuals. The ability of C. neoformans to cause disease depends on the elaboration of virulence factors including a polysaccharide capsule, melanin deposition in the cell wall, the ability to grow at 37°C, and the secretion of extracellular enzymes. The cyclic-AMP/Protein Kinase A (PKA) signal transduction pathway is a key regulator of virulence in C. neoformans and may also regulate the trafficking of virulence factors. The influence of PKA1 expression on the intracellular and extracellular proteomes and identification of Pka1 phosphorylation targets using phosphoproteomics have not been investigated for C. neoformans. In our current study, I performed quantitative proteomics using a galactose-inducible/glucose-repressible expression strain of the PKA1 gene to identify regulated proteins in the secretome and proteome. During investigation of the secretome, five proteins showed changes in extracellular abundance upon Pka1 induction. These included the Cig1 and Aph1 proteins with known roles in virulence, as well as an α-amylase, a glyoxal oxidase, and a novel protein. Targeted proteomics of these Pka1-regulated proteins allowed us to identify the secreted proteins in biological samples suggesting their potential as biomarkers of infection. During investigation of the intracellular proteome, I identified a broad and conserved influence by PKA1. Furthermore, an analysis of protein-ptotein interactions emphasized the impact of PKA activity on several clusters of proteins involving translation and the ribosome, the proteasome, and diverse metabolic processes. Lastly, a phosphoproteomic study identified six potential targets of Pka1 phosphorylation including the master iron regulator, Cir1. Construction of site-directed mutants showed that Pka1 phosphorylation of Cir1 impacted the production ofcapsule and melanin, cell size, and the ability to grow under low iron conditions. Overall, thedata presented in this thesis have contributed a better understanding of the broad and conserved influence of Pka1 on cellular regulation and secretion in C. neoformans, and the discovery of potential biomarkers may facilitate the monitoring of disease progression. Additionally, the identification of new Pka1 phosphorylation targets present opportunities for the development of a molecular understanding of the regulation of virulence as well as novel therapeutic strategies for treatment of cryptococcosis.

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Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, a life-threatening disease which affects an estimated 1 million people worldwide annually. Iron acquisition is an important but poorly understood aspect of the pathogenesis of C. neoformans. In particular, no heme uptake system has thus far been characterized in this fungus, although it has been shown to utilize heme as an iron source. A previous study identified the transcript for the extracellular mannoprotein CIG1 as the most abundant message in iron-starved cells with marked down-regulation by iron repletion, thus suggesting a possible iron-related role for Cig1. In the current study, it was found that deletion of CIG1 resulted in an extended lag phase in low iron medium with heme added as the sole iron source. Additionally, the cig1Δ mutant was more resistant to toxic heme analogs than the wild-type or complemented strains implying a role for Cig1 in heme uptake. Western blot analysis and immunofluorescence microscopy identified Cig1 at the cell surface and in association with extracellular vesicles. A heme pull-down experiment, absorbance spectroscopy and isothermal calorimetry also demonstrated that Cig1 is a potential heme-binding protein. Importantly, deletion of CIG1 led to attenuated virulence in a mouse infection model in absence of the high-affinity iron uptake system. More detailed studies on Cig1 revealed that the length of the lag phase of a cig1Δ mutant in low iron medium supplemented with heme was dependent on the inoculum size in support of a cell density-dependent heme acquisition system. Similarly, growth at acidic pH rescued the heme defect of a cig1Δ mutant indicating the presence of a Cig1-independent pathway at low pH. The transcription factor Rim101 may function in this pathway. Finally, expression of a Cig1 truncated polypeptide established a role for Cig1 in secretion and cell wall integrity. In this context, a strain overexpressing CIG1 produced an enlarged capsule and secreted more extracellular vesicles than the wild-type strain. Overall, the data presented in this thesis have contributed to a better understanding of heme uptake and secretion in C. neoformans and the results may facilitate the development of new strategies to treat cryptococcosis.

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Autographa californica multiple nucleopolyhedrovirus expresses two majortransregulatory proteins IE0 and IE1 immediately upon infection. IE0 differs from IE1only by 54 additional N-terminal amino acids (aa). Either IE0 or IE1 can support viralreplication; however both are required for a wild-type infection. It is unknown what thedifferent functions of IE0 and IE1. Both IE0 and IE1 can transactivate viral early genesand support viral DNA replication. It is therefore hypothesized that by the addition of Nterminal 54 aa, IE0 acquires different transactivation activity on viral genes and interacts with different viral or host partners. To test this hypothesis, functional comparisons between IE0 and IE1 and the identification of their interaction partners in infected cells were performed.Comparisons of subcellular localization and transactivation activities between IE0 and IE1 showed no difference. However analyses of the nucleocapsid content of occlusion derived virions (ODV) revealed that IE0 and IE1 appear to regulate the number of nucleocapsids per ODV. Deletion within the IE0 specific N-terminal 54 aa did not affect IE0 transactivation dramatically but reduced its ability to support viral DNA replication.Analyses of interacting proteins did not identify any proteins that were specific to eitherwith IE0 or IE1. However, the viral protein AC16 (BV/ODV-E26) was shown to bind toboth IE0 and IE1 via a binding domain at IE1 72-99 aa. Mutation of the binding domainenhanced budded virus (BV) production by viruses expressing only IE0 but not IE1.Deletion of ac16 however resulted in increased levels of IE0 relative to IE1 as the onlyobservable impact. These results would therefore indicate that AC16 regulates ie0expression. Deletion of ac16 and the overlapping gene ac17, interestingly resulted in asignificant delay of viral gene expression for up to 12 hours. However, the delay of viralgene expression was only observed with BV-infected cells and not in cells infected bytransfecting viral DNA. AC16 and AC17 are therefore critical for rapid gene expressionduring the very early events of infection, and highlight the fact that proteins interactingwith IE0 and IE1 play key roles in baculovirus biology.

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