 Well, thank you so much Don and the organizers for the, it's a real honor to be participating in this symposium. I got a notice on my screen. Can everybody hear me? We're fine. Yeah. Okay. Yeah, so I'm going to tell you that my foray with Mortirella really began with this culture here on the right. I was in Reedus Vilgalee's lab and we had the opportunity to sequence some genomes. We had three genomes and so we wanted to maximize phylo diversity. So we picked a Bacidio mycete, an Asco mycete, and a Zygomycete. And the reason we picked this, the Zygomycete is because it was common in a lot of our next gen sequence data sets. It was common in the soil microbiome and these genomes were for metatranscriptomes of forest systems. And so we thought it'd be important to have this one. These TEMs are from Kerry O'Donnell's graduate studies at Michigan State University. And this last one is a TEM of the bacteria living in the hyphae of this genome. So when we got this genome back, there were two genomes. We got the fungal genome and the bacterial genome. So we were excited. We called it TOOFAR. So I'm going to give a little bit of background on the ecology, the biology, and then about the phylo diversity of this family or sub phylum, if you will. I'm going to, the third part's going to talk about the bacteria and then the fourth part's going to talk about morterella plants interactions. So these fungi are almost as charismatic as Kabir's, Sewillis, and Betsy's endophytes. If you've cultured fungi from soil, you've most certainly come across this rosette pattern of growth, which is distinctive of most of the genera in this family, though the form of the rosette is dependent on the diet of the fungus. There's been a lot of history on this group since the mid-1800s. Linamon was one major, made a lot of contributions looking at the sporangia forms for differentiating a lot of the species as well as some of the macro-morphological characters and described a number of species of morterella. However, there's other genera in the family, and so I had to bring it back to the phylo, and so this is from Roland Daxter. He isolated desophora from woodmouse dung in Cambridge, and he had this culture alive for years, but during his travels in the West Indies, it died, but he still had prepared slides, and he described this new genus, desophora. Lobo sporangium is another genus in the group with its distinctive sporangia shown here. Meredith Blackwell and Gerald Benny placed this in the morterellaaceae, and it's only been isolated three times. So these fungi are all synesthetic, though they can make cross walls, and they often wall off old parts of their mycelial network. They're characterized by bi-directional cytoplasmic streaming, and you can see that here. This is a genus that we described, Beniella erionia, which is shown with these kind of chained chlamyto spores. In the family, there's at least 200 species, and likely more, and the species can either be sexual, homothalic or heterothalic, so some of them can self and others need to out-cross. And following up on Matt, here's one of Matt's interesting finds in South America. This is moticellarineiformis, which is a sporocarpic morterellaaceae fungus known only from South America. That's species. We have a species in North America, and there's a third in New Zealand. So about the ecology of morterellaaceae, it's still really understood. Peter gave a good overview that they might be implicated in a kind of necromass turnover in soils. For sure, morterellaaceae are a core part of the soil microbiome. Nutritionally, they can utilize simple sugars. They're very good at utilizing chitin in their genomes and protein, their genomes are enriched in protease enzymes. A number of the groups have been shown to be endophytes and plants, and in some cases can promote plant growth. And these are industrial fungi. They're used in lipid production commercially. So my first PhD, the first PhD student in my lab, Natalie Vandepole, recently published this work, which used phylogenomics and a combination of phylogenomics and multigene phylogenetics to try to resolve what was known as morterella, the genus, which was a polyphyletic genus. And so we used monophily as a way to try to describe these different genera. So we can better ascertain maybe what their ecology is and their distribution. So we used the low-coverage genome sequencing of 70Taxa to have a highly resolved tree, which we then expanded with the use of multigene phylogenetics with a six loci. So we kind of had this super tree approach. We constrained the backbone. And so you see up top, here's Beniella, the sister group of Modicella. Here's DeSophora, which is related to Gamziella. And here comes Lobosperangium out on a branch. Morterella alpina group, polycephalus, the type species, so that we retained the genus morterella for the alpina clade. Linnomania is what we would call morterella elongata. So a lot of my talk is now talk, I'll use the new name Linnomania. And Podella in orange is another plant-associating group. So Podella, morterella, and Linnomania are often isolated from plant rhizosphere. Now morterella ac are known to associate with bacteria. In fact, nearly 20% of isolates will harbor bacteria, either external or internal. And so here is kind of a little bit of a meta-analysis that combines data from our lab with those from Aliska Okrasiska and Takashima, who recently published on these endobacteria. So in blue, peribercal dairea, these are kind of facultative endobacteria associated with the hypo-plane there in mucora, umbilopsis, and morterella ac. Teresa Palauska talked about rhizopus and their bacteria. These are pink here, myceto-habitins, and those are endobacteria. And the AMF in green here is shown candidate as glomerobacter. These are in the gigasperaceae, gigaspera and scutelospora. And then in red are those bacteria that I'll be talking about, peribercal dairea related. These are mycoavidus, and there's three main clades of that. We might call them species, but they're actually quite diverse. So how do you know they're endobacteria? To really demonstrate this, transmission electron microscopy, or fish, are used to confirm this. And so here's two different groups that we show from Alessandra D'Zero's work. And then fish is shown here from the Takashima and all paper in 2018 showing these bacteria inside the hyphae and inside sporangia spores. So we've gotten a number of genomes back. And if we take their genes out and align the orthologous genes, then we can look at a kind of presence absence. And this is kind of a little bit of a crude way to look at it. But what's fascinating to me, so the first one here, mycoavidus species 3, this one actually appears to promote the growth of the host. So when the host has this bacterium, it grows a bit faster. The two below it, one of those is from Linomania elongata in North Carolina. The other one is from Linomania elongata, same host, but isolated from Japan. And so here we have the endosymbion of the fungus. And if you look at it, they're not completely the same. But wow, there really is a lot of conservation here, which to me is kind of amazing. We never detected any plasmids. Their genomes are kind of small, 2.5 to 2.8 megabases with a little over 2,000 genes. We've been able to use some associative mapping techniques similar to what's used in GWAS to look at genes under positive selection in these bacteria. And there's not a lot. We had a short list. But of those that we found, they tend to be involved in energy and amino acid metabolism. I should say a lot of them are amino acid oxytrophs. Like for instance, to grow them, you have to supplement with cysteine and other metabolites. So there's a second group of endobacteria that I want to talk about. These are the molecule-related endobacteria. And this was a passion of Alexander Desiro who came to work in my lab as a postdoc. He had worked with Palabonfonte and characterized MRE in our muscular mycorrhizal fungi. And then he discovered them in endogony, which is an ectomycorrhizal fungi in the endogonyllis. And so he was really wanted to know, are they in mortiurelacy as well? And in fact, he was able to demonstrate that they are only in 3% of our cultures. We went through hundreds of cultures, but he found them in nearly every clade at the time, we would call a genus now. And there were three main groups of these endobacteria, molecule-related. It has these beautiful SEM done in Robbie Robertson's lab, where you can actually see the cells dividing. So they are actively growing in these fungi. We've been generating some genomes from these. These are very hard to work with because they are so divergent from each other. But they do have a, there is a clade that is fungal-specific. So this is work of Julian Lieber, where we're looking at, we're doing kind of a comparative approach based on these orthologs. But the challenge is a lot of these are just hypothetical proteins. We just got the Unitig last week of the smallest genome yet of one of these MRE was 326 kilobases. There's between 1,100 to 1,400 genes of these bacteria. They can have plasmids and one isolate has a mitovirus in it as well. If you look at what they are really there, what we know about the genes that are there is a large part of the genomes involved in translation, replication, repair, and protein metabolism. So the question of course arises, what is their impact on the host? And so we've looked at this in a number of different ways. Jesse, Julian published early on or a few years back anyway, showing that when you remove these bacteria, the fungus is a little bit relieved and it grows more fluffy. You get more bio-bass. And so that was in the BREs. We see the same patterns. And it's not for all BREs. As I mentioned, there's one group that seems to promote the fungal host growth. If we look at MREs, we see a similar pattern where, well, actually, we see that morterella, this species here, which is alpina, it actually grows faster, more bio-mass at four degrees than at 22 degrees. And what we see here is it's only at room temperature that there's a change, a statistically difference in their growth rate. And we use QPCR to demonstrate that that's because at this room temperature range, the bacterial population is much higher. So at these low temperature or high temperature, the bacteria don't really, they're not as active within the mycelium. So following up on this in collaboration with Powell Mitchell, we were able to put these isolates cured or not. So we use antibiotics to remove the bacteria cured or not in these chambers for real-time, time-resolved emission analysis. And here's just kind of a cool example, because here we have them and we're ramping up the temperature in the incubator every few hours. So at 20 degrees, you can see, I'm just showing one example here. This is butyric acid, which is a known metabolite from mycoplasmas. And so as you ramp up the temperatures, the activity of these bacteria goes kind of off the charts here, especially at 30 degrees. It doesn't even go up to 37, but they would probably lose that signal. How about the fungal metabolome? So this is using metabolomics. This is an S-plot that is looking at how you can discriminate cured from wild-type isolates. So this is cured here as in red. And these are the metabolites that can discriminate the two isolates. And they're largely, when there's no bacterium, you largely get a lot more acidic polyunsaturated phospholipids. When the bacterium's there, there's a drastic change in the metabolism. It's not just a few metabolites being broken down. There's a huge shift in the fungal metabolism, with stearic acid being particularly abundant in MRE-infected isolates, as well as polyunsaturated phospholipids. Most of these metabolites are not well characterized. So how about mating? So it's been shown, Teresa, yesterday showed that with mycetohabitins, that they are required for mating in rhizopus. We see the opposite here for our burkholderia-related endobacteria. So if they're present, it suppresses, it prevents mating in morterella. And so this is, we showed this in a heterothalic species, litimania elongata. Takashima showed this last year. They published it in a homothalic fungus where it was sterile until they cleared the bacteria out with antibiotics. And then they got zygospore production. So that was kind of cool. And it showed this same thing that our work was showing. On the other hand, MRE do not have any impact on preventing sexual reproduction. And they are most definitely thought to be vertically transmitted. So this third section, I'm going to talk about some of these interactions of morterella with plants. This is work here. I'm going to highlight from Davis Matthew, who's interested in fisco-mitrium patents and its impact on how morterella interacts with the moss and how the bacteria is, there's a role of the bacteria in this interaction. And I'm just going to tell you that there's not, we didn't see any impact of the MRE or BRE on the host response. However, we did see differential effects of the different fungi on the host. And so Beniella has had more of a parasitic response on the plant. So this is based on plant genes that were overly expressed or significant differences in stress response, immunorespons, apoptosis, where Linemania elongata seem to be more of a mutualist or a friendly interaction with DEGs involved in carbon metabolism, nitrogen growth, development, and decrease in stress. This is, we wanted to see about with flowering plants. So this is work that Natalie van de Pol did with Arabidopsis and morterella. So from the same soil sample, we isolated two fungi, one of them the same species, one carried MRE, the other carried BRE. And what she showed here was there is a phenotype in the seed mass and the total seed number of those plants grown with morterella. We had been using millet and uninoculated, you know, millet not inoculated in no millet and as these two different types of controls. We were concerned that millet might, it can be allelopathic, it can also be a nitrogen sink. So she moved to these plate assays and so there's no millet confounding factor. And what we see is there's a, there's a significant impact. So these are colonized, these are not colonized negative control. The aerial biomass is significantly improved. But again, similar to what Davis found is we didn't see any impact on the endobacteria. And so how does it affect the aerial plant growth? So she did RNA-seq experiments and looked at some of these genes and it appears to be involved something with plant hormones, modifying oxen, ethylene, and ROS response pathways. And my final little vignette, I'm going to talk about how morterella interacts with micro algae. And this is work that was done by a postdoc in my lab, Xi'an Du, who's now a professor in Hawaii. They worked on my lab, the lab next to mine worked with micro algae. And we'd known that flocculation is a big thing. So we wanted to see if morterella could help bio flocculate these micro algae. And sure enough, we found that some groups did, especially the linimonia clay, it was very effective. And you can see these hyphae kind of turn green. When you put them in a jar with these algae, they flocculate to the fungal mycelium. Not only that is, this is kind of cool. Clamidomonas has flagella. So it actually swims very quickly to the fungus mycelium when they're put together. And they form this kind of biofilm together. And it's a protective matrix. So we did this experiment with the oxidation where we put bleach on the fungi. And immediately if there's no, if it's just the algae, they bleach in a second. But if they're grown in this biofilm, they don't bleach and they persist for days, still active in photosynthesizing. What's the role of bacteria on this flocculation? We didn't publish this because this was really, our results were different. We come in, they look green, they're flocculating, we come in the next day and they'd be white, they bleach out, and then sometimes they turn green again. We did look into it and it appears that with the endobacteria, there's a higher ROS content. There's a lower viability of the algal cells, less chloroplast. So we think there's a negative response of the endobacteria. But the challenge is, is we have to monitor, we need to use QPCR so we know what the endobacteria population is throughout these studies. If you just grow nanochloropsis alone, it has a smooth cellular surface. But if you grow it in the presence of a fungus, that outer surface is removed and it has these irregular tube-like extensions that actually make contact with the fungal cell wall. And so here you can see the cells all around, flocculating all around. They stick to the mycelium. We did some carbon tracing studies with C14 where we labeled the algae and then put them with the fungus and we did this in a couple different ways. We had a no-contact control where there was a membrane between them that solutions could move between. We also used heat-killed cells, so we weren't lysing the cells, but we would kill them so they're not living. And it turns out that the signals pretty strong in those cells that directly interact and they have to be alive. And the heat-killed cells, those were put in the vial too. So what this means is this is a biotrophic interaction. It's not a saprotrophic decay kind of interaction going on. We did a similar thing with nitrogen where we were able to show that nitrogens move in both ways between the partner. It looks like a little bit more is moving to the algae than to the fungus. In here, we didn't see that a contact was necessary for this exchange. So this is kind of the coolest thing to me was this long-term effect of co-cultivation. When carbon is limited, these algae can become internalized within mortarella hyphae and they can persist for months in this condition. Whether these fungal cells are still, I don't know how much cytoplasmic streaming is going in when they become chock full of algae like this, but the colonies are alive. And we have some nice TEM that kind of show this condition. So here's the fungal cell wall showing continuous, and these are the algae actually living inside the fungi. You can see their chloroplasts are large, they're healthy, they're happy. And here you can see the fungal mycelium has cytoplasm. So it is, this one's alive at the time of sectioning. And as far as I know, I'm not aware of any fungi ever really taking up algae or being shown to do so. So in conclusion, there's now 13 reference genera in mortarella ACE. And these fungi, all of the genera can host bacteria, and they're fairly common. And so this kind of evidence supports this idea of an early invasion. So the fact that these clades, both BRE and MRE are in our vascular mycorrhizal, the glomer amicotina, bucar amicotina and mortarella amicotina, we suspect so it was an early invasion. So let's see, BRE but MRE inhibit mortarella mating. Linomania appears to interact with vascular and nonvascular plants, kind of as a mutualist, and it might be a bio-troth association with algae. But these interactions aren't affected by the bacteria. So with that, I want to thank folks in my lab who did this work, other collaborators, the Zygolife Consortium, and JGI for helping with a lot of this sequencing. Thank you.