 I am delighted with this opportunity to tell you about our work on interactions of fungi and bacteria. And in particular, our pursuits to understand evolution of fungal bacterial mutualisms and also innate immunity in fungi. So this is the work that stemmed from our pursuits of evolution of bacterial fungal symbiosis. And I should point out that fungi, as Tim mentioned, fungi are increasingly recognized as hosts to endobacteria, and in particular, early divergent fungi, mucoromicota, mucoromicota, unlike dichloria, harbor endobacteria that are ancient, highly co-evolved, and heritable. And our lab studies glomeromicotina and the symbion of glomeromicotina are vascular mycorrhizal fungi. But today I'm going to talk about our work on endosymbion of mucoromicotina. And Greg is going to tell you about endosymbion of mucorrhizalomycotina tomorrow. So our model system that we focus on is rhizopus microspores symbiosis with mycetohabitans. Mycetohabitans was formerly known as burkholdaria. It is a better proteobacterium. It is a pathogen of rice causing rice seedling blight. It harbors endosymbion, although sometimes it doesn't. Here is a hypha of rhizopus with mycetohabitans visualized by YFP expression. Here are two hyphae of, I'm sorry, two spores of rhizopus filled with mycetohabitans cells and rhizopus microspores mycelium and sporangiform sporangiform. This system lends itself to in-depth studies because unlike most of the symbiotic associations of fungi with bacteria, it is easy to manipulate. If you manipulate bacteria can be, fungi can be cured of bacteria, bacteria can be cultivated independently and put back into fungi, and both fungi and bacteria can be transformed genetically. As I mentioned, we are interested in the origins of mutualistic symbiosis and we use this system to address this question, but we also noticed that this system can be used to understand innate immunity in fungi. And so before I talk about our work, I would like to remind everybody that mucoro mycotina are ubiquitous soil saprotrophs. They are food spoilage agents, they are plant pathogens that are responsible for post-harvest crop diseases. And importantly, they are opportunistic pathogens of immunocompromised humans and they are very difficult to treat because they are resistant to most antifungal agents. There are multiple applications of mucoro mycotina, ranging from cheese making to biodiesel production, and yet with all these important applications and ecological roles, this is one of the least understood groups of filamentous fungi. So in a way we are using bacterial endosymbion of these fungi as probes to understand their basic biology. So we became interested in the Rhizopus microsporus mycetohabitans symbiosis when we read the paper published in 2005 by Christian Hirtfeck's group and this paper reported a presence of mycetohabitans, then it was called Burkholderia. This bacterium was attributed with the ability to produce a toxin, rhizoxin, which is important for pathogenesis of rice in rice seedling blood. The phylogeny of Burkholderia reveals several different species of bacteria that are associated with various hosts. Mycetohabitans is here and along with mycetohabitans Burkholderia are home for mycoavidus, again Greg will talk about this hopefully, and glomerobacter endosymbion of arbascular mycorrhizal fungi, but there are also many symbiotic associates of plants in green here and animals in blue. So, even before we started working on this system, it was known from Christian Hirtfeck's lab that these endobacteria control asexual reproduction of the fungus. So a fungus that has endosymbion will reproduce asexually in a typical way of mucoro mycotina, but when mycetohabitans is removed by antibiotic treatment, the fungus is only able to reproduce vegetatively. A graduate student, Steven Mondo in our lab, became interested in whether these endosymbion also control sexual reproduction of the fungus. And it turns out that, yes, they either control it completely. The removal of bacteria from compatible mates either completely obliterates their ability to mate, so this plate shows successful mating between compatible mates of rhizopus and the fungus, this plate illustrates mating between the partners that were cured, and you can see that there is no mating. In some cases mating is reduced, but the reduction, it is reduced by removal of endobacteria, but this reduction is significantly, lower than sporulation in between mates that have endobacteria. So seeing these patterns, we became interested in the evolution of this particular symbiosis and reconstructed the genealogy of rhizopus microsporus. And realized that this association evolved from an antagonistic interaction. The basal isolates on this genealogy are non-hosts marked in red in the phylogeny. And these non-hosts interact antagonistically when faced with bacteria here, these bacteria are isolated from the host. And here again the same image that I showed you before, previously cured host interacting with its own isolated bacteria becomes colonized, whereas the non-host isolates become stressed, inhibited and never colonized. So this led us to a conclusion that this particular symbiosis evolved from an antagonistic interaction through compensatory evolution in the host. And this was not really an original idea, the compensatory, mutualism evolution through compensatory evolution has been proposed by Duran and Rolf Hextra. But we provided additional evidence that indeed the fungus is addicted for reproduction to its bacterial symbiot. So this is illustrated or apparent looking at the extent mutualism and the bacteria are responsible for facilitation of host reproduction, symbion removal causes loss of asexual reproduction. And reduction of sexual reproduction. Now, we speculate that this association involved from an antagonistic interaction in which ancestral workhold area or ancestral mycetohambitons used and manipulated fungal hosts to extract energy from their hyphae. And this realization or this speculation made us think very carefully about interactions of non-host isolates with bacteria, which in turn led us to a realization that fungi possess innate immunity mechanisms. So what happened? We are to really understand a little more about how fungi, both hosts and non-hosts interact with the symbion of the host conducted transcriptional profiling experiments, interacting previously cured host with mycetohambitons isolated from a host. And we also interacted a non-host that with the bacteria isolated from the host in at two time points before the partners came into contact and after the partners became close in physical proximity. And I'm not going to bore you with the details of transcriptional profiling. I am going to tell you about the observations that we made and because these observations are based on transcriptional profiling, we were able to really use them only to formulate hypothesis to be tested and those hypothesis are about molecular dialogues between host and non-host fungi and bacteria that are capable of colonizing the host. So pre-contact, bacteria react to both hosts and non-hosts in a very similar way as if they did not care whether they are interacting with a mutualist or antagonist. They engage expression of genes and coding type two and type three secretion system effectors and in turn hosts respond with cell wall remodeling and non-hosts respond with cell wall remodeling or I should say they change expression of genes that are involved or in cell wall biosynthesis but these changes are quite different. Mutualists behave as if they were preparing for accepting the symbiont whereas antagonists seem to strengthen their cell walls in protection against bacterial entry. And these patterns continue to the physical contact phase so again the non-hosts strengthen their cell walls and in addition they produce reactive oxygen species and when we compare the patterns of expression of genes that are involved in reactive oxygen species biosynthesis the non-hosts seem to engage in a very potent reactive oxygen burst whereas the hosts produce a reactive oxygen species but then they seem to quench them. And this is the only hypothesis, the reactive oxygen, the difference in reactive oxygen species production between host and non-host fungi is the only hypothesis that we actually tested empirically tested functionally but before I tell you about that I just wanted to mention that this difference between reactive oxygen species output is also apparent in the symbiont or bacterial gene expression patterns. And the bacteria that interact with the non-host seem to respond to reactive oxygen species stress and these reactive oxygen species come from this potent reactive oxygen species burst that the non-host produces. So as I said this is the only hypothesis that we tested in this particular system. We quantified reactive oxygen output by using a nitro blue tetrasolium staining, nitro blue tetrasolium when it's reduced by superoxide radicals it turns blue. So the areas of fungal mycelium that are colored blue are accumulating reactive oxygen species. So this panel, this panel, I cannot point, this panel shows the non-host interacting with bacteria and you can see that there is quite a bit of color development at the edge of the colony, as well as the inhibition of the colony growth and bacteria are also producing reactive oxygen species in interaction with the fungus, and this is much different from the non-host mog inoculated with bacteria. This does not really develop intense coloring, and this is reflected in measurements of color development. So non-host interacting with mycetohabitans produces significantly larger reactive oxygen species burst and host quenches reactive oxygen species. So with this, we started thinking that these patterns really resemble innate immunity responses in both plants and animals. So both raw reactive oxygen production and cell wall remodeling. And these, these responses, we believe, although we did not really have, we have not done really rigorous experimentation, but just by growing other non-host, new chromicotina fungi, we see the same pattern of inhibition of fungal colony growth when interacting with mycetohabitans bacteria, and we see it in mucosircinaloides, and importantly, mucosircinaloides is the model species for mucoro mycotina, and can be manipulated genetically, and also in the sister species of rhizopus, or rhizin. So innate immunity provides the first line of defense against pathogens, both in animals and plants, and it really relies on pattern recognition receptors, both extracellular and intracellular in animals, to detect micro-associated molecular patterns, mumps, and once the presence of mumps is detected, that signal is transduced to the nucleus and the cellular responses are elicited. And importantly, both animals and plants have evolved innate immunity systems, but those innate immunity systems are products of convergent evolution. So both animals and plants have extracellular and intracellular receptors, they have signal transduction modules, and they have response modules which consist of reactive oxygen species production, production of antimicrobial peptides, and program cell death. But again, those specific components of these modules are completely different because they evolved convergent. So we started thinking about, well, how about fungi? Do fungi have innate immunity responses? And our experiments with rhizopus microspores, the non-host isolates, suggest that they do. So we started looking at the literature and trying to figure out what is known about innate immunity responses in fungi. And a study from Nick Talbot's group suggested that fungi don't really have those pattern recognition receptors that are so common, both in animals and plants. Instead, fungi seem to rely on adenylate cyclase genes and proteins for perception of microbial molecular patterns and maybe other receptors that are still unknown. So here is the cartoonish representation of adenylate cyclase in candida albicans. And the key module of this modular protein is a loose and rich repeat structure that is responsible for perception of peptidoglycan. Which is a component of bacterial cell walls. The signal from the loose and rich repeat module is transduced to the cyclase domain of adenylate cyclase and the signal cyclic AMP is produced. Cyclic AMP is a signaling, common signaling molecule in fungi. And the cellular response is produced. We thought about this signaling process and we found an inhibitor of downstream signaling from adenylate cyclase inhibitor that affects functioning of protein kinase A. And we started playing with this by co-cultivating host and non-host. Rise up with microspores and here I'm showing you images from the non-host. So when we co-cultivated non-host with myceto habitants, YFP expressing myceto habitants in the presence of inhibitor, myceto habitants were able to enter the host. I'm sorry, non-host high fee. Without the inhibitor, as previous experiments demonstrated myceto habitants was not able to enter the fungus. So this suggests that there are innate immunity mechanisms that are involved in mutualism functioning in raise up with microspores and perhaps other fungi and we are pursuing it. But more importantly, we are really interested in whether program cell death is a component of innate immunity response in muco-romicotina fungi. So program cell death is both in fungi, I'm sorry, in animals and plants, a kind of an ultimate way of eradicating invading microbes. In animals, it is manifested by pyroptosis and necroptosis and in plants by our familiar hypersensitive response. So program cell death can be very easily diagnosed. It is diagnosed by detection of reactive oxygen species, production or burst by, you can detect cell death with common cytological kits like live dead kit, they are shrinkage by just staining DNA or staining nuclei to visualize their shrinkage fragmentation and DNA diffusion and then somewhat more sophisticated methods once those first screens allow for detection of program cell death. Now, importantly again, muco-romicotina are capable of program cell death. In fact, muco-sercinoloides was one of the first fungi where program cell death was reported and it was reported to be caused by lovastatin anti-cholesteroid drug and here are germinating spores of muco-sercinoloides germinating without lovastatin and with lovastatin and hopefully you can see in the live dead viability assay there are clear differences in cellular appearance and also nuclear fragmentation in propidium iodide. So we went on and reconstructed hypothetical innate immunity and program cell death signaling networks in muco-sercinoloides and so obviously as expected muco-sercinoloides has adenylate cyclases and multiple copies of it. It has another potential receptor pattern recognition receptors of microbial molecular patterns and as evidenced by the appearance of program cell death, it has the machinery that is responsible. The question is, is there a connection between innate immunity machinery and the machinery that is responsible for program cell death and I am hoping this is a subject of active research in our lab and I am hoping that I will be able to report in very near future whether there is this connection. So in conclusion, we believe that early divergent fungi respond to bacteria in a way that resembles innate immunity responses defenses in plants and animals, and in particular early divergent fungi are capable of producing reactive intrusion species and cell wall remodeling when challenged by bacteria and we are interested in discovering whether program cell death is part of fungal innate immunity and going back to the mutualistic interactions, whether bacteria that are able to overcome defenses and enter the cells, how they manipulate their host and control their reproductive biology and with this I would like to thank our collaborators and our sources of funding.