12/24/22:
Messages from the mycorrhizas

Now that you're all familiar with the leading cast of this mycological movie, let's dig into the relationship dynamics between fungi and plants. As I mentioned earlier, the symbiosis between fungi within the clade Glomeromycotina and their plant hosts has been widely recognized as one of the most ubiquitous mutualisms found in nature. Over 80% of land plants are thought to form AM associations, and many of these plant hosts include important food crops. In fact, farmers and researchers have used AM inoculated soils to increase crop yields of grains and vegetables. The outcomes of these relationships are well known: plants are supplied with phosphorus, nitrogen, water, and in some cases a higher resistance to other pathogens and environmental extremes, as courtesies of their fungal partners. In return, fungi receive a payment of around 10-20% of the plant's total photosynthate production. The amount of nutrient and sugar exchange vary widely and depend on many variables such as the species involved, environmental conditions, seasonal fluxes, and so on. These results are relatively easy to observe and measure when compared to the tiny signaling molecules that initiate this affair. So, who made the first move?

Disclaimer: I want to make it clear that my anthropomorphizing of the characters in this film should in no way be taken seriously. My aim is to make these posts enjoyable, but also to tell a story that is easy to understand and recall at later times. If a stranger next to you cries out: "UGH, if only I understood mycorrhizal associations, then all of my problems would be solved and I could save the world," it would fill me with joy to know that you could solve that stranger's highly specific problem. This is all to say, mycorrhizal associations aren't driven by any conscious decision of the species involved. The nutrients exchanged between organisms aren't traded on a one-to-one ratio. Fungi aren't FedEx-ing phosphorus to the plant and waiting for a payment in the form of sugar or Bitcoin. This is a highly dynamic system where many things are regulated simultaneously. Both fungi and plants have evolved to work together, but they have also evolved for survival and replication of their individual genes and species.

Back to our regularly schedueled programming on the molecular dialogue that's orchestrating the symbiosis between fungi and plants. Before fungi colonize their host plant's roots, they need to ensure that they have adequate space and a specific type of tissue in which they can house their hyphal webs. To investigate the steps of mycorrhizal colonization, Gutjahr et al. performed an experiment on rice plants and AM fungi and found that fungal signaling initiated the growth of lateral roots, which have a larger diameter and more cortical tissue. This is important because the cortex is the area of the root colonized by AM fungi. By showing that the architecture of the rice roots was modified before penetration of the fungal hyphae, scientists concluded that their must be signaling during the pre-symbiotic stage to prime the host root and initiate root growth.

Below you can see images of AM root colonization from Yuan and colleagues. Roots and fungi are stained using a blue dye, the chitin in the fungal cell walls retains the blue color, allowing us to examine the filamentous hyphae (string-like cords) and vesicles (orbicular structures) formed from fungal tissue.

While specifics regarding the fungal signaling molecules that cause such phenomena like the lateral root branching described above remain mostly unknown, there has been some headway into isolating and identifying these pre-symbiotic compounds. Chitooligosaccharides have been of particular interest to researchers and have a variety of applications. They are found to promote mycorrhizal symbioses by suppressing plant host immunity and initiating changes in root metabolic pathways and tissue development. These chitin-based compounds have even been isolated from commercial shrimp fisheries and fungal sources to use as agricultural biofertilizers. Current research is being done to find sustainable ways to isolate chitooligosaccharides from large-scale mycelial production. This is one example of how fungi are proactively sending signals into the environment while house-hunting for their future living quarters. What are plants doing to ease the move?

In the last blog update, I mentioned branching factors and strigolactones, which are a class of compounds found in root exudates that facilitate mycorrhizal colonization. For quite some time, the impacts of these mysterious root compounds on hyphal branching were observed and known. Due to the extremely low concentrations produced by plants and their chemical instability, they have been tricky to isolate and identify. Exciting work in the last two decades has identified the chemical structures of strigolactones and elucidated their roles in both mycorrhizal development and other ecosystem relations. One important driver of strigolactone excretion seems to be phosphorus depletion. From what we know about mycorrhizal fungi and their nutrient scavenging abilities, this makes perfect sense. If a plant is unable to meet their phosphorus needs, why not exude an itty-bitty compound into the soil and propose to a fungal stranger: "I will let you move in if you pay me rent in phosphorus." However, as yet another reminder of how ecology and interactions are never that simple and romantic, this strategy doesn't always benefit plants. Seeds of parasitic weeds also germinate in response to strigolactones and can have devastating impacts on crops such as legumes, sunflowers, tomatoes, tobacco, and many cultivated cereals. Below is a diagram depicting how this process works.

Discovery of chitooligiosaccharides, strigolactones, and other compounds involved in mycorrhizal signaling have helped inform models for fungi-host interactions and the physiological changes that occur in both symbionts. Colonization occurs in stages: pre-symbiotic compounds are released by roots and fungi, first contact between root and fungi induces hyphae to differentiate into hyphopodium, a penetration hyphae emerges from the hyphodium and invades the rhizodermal cell layer, and the last stages concerning how the fungus spreads within the root depends on the type of mycorrhizal fungi involved. If the new tenant is from the endomycorrhizzal group covered earlier, the fungus will spread longitudinally within the cortex and form arbuscules inside the plant's cortical cells. If ectomycorrhizal, the fungus won't penetrate deeply into the root cell, and instead puts its energy into forming a mantle of hyphal cells covering the outside of the root, and Hartig net hyphae inside the root. From what I could find when doing research for this post, the depth of penetration of the Hartig net depends on the species involved. For angiosperm associated mycorrhizas, it seems the Hartig net doesn't stray far from the root's surface and forms a network between the uppermost epidermal cells. As for gymnosperm associates, the Hartig net can extend deeper into the root, branching further down between cortical cells like the following image shows. While we're on the topic of EM interactions, let's look at research that has focused on their pre-symbiotic signals and the physiological impacts on their root symbionts.

Reminiscent of the experiment described earlier investigating the impacts of AM fungal signaling on rice plants, Felton et al. tested an EM species with poplar trees and found similar results. Prior to physical contact with poplar roots, fungal signals initiated lateral root formation in the trees. The study was taken a step further by analyzing gene expression and hormone levels within the root tissue. Fungal compounds increased auxin levels in the apex of the root while also increasing apical and lateral polar auxin transport. Not only did these exogenous fungal compounds increase plant growth hormone activity, they also impacted their transport through the plant, leading to the initiation and growth of lateral roots. What's even more interesting are the broad effects of these signaling molecules. Felton and colleagues also monitored the changes induced in non-mycorrhizal plants and found that they experienced the same lateral root formation. Recent developments have dug deep to identify where the genes for these compounds exist within the fungal genome in an effort to describe their function and evolution more clearly.

These images show an increase in lateral root formation in poplar seedlings as mycorrhizal colonization increases. Scientists used a dual staining method which dyes the fungal cells green and the plant cells magenta.

In 2008, more than a decade after its fungal kingdom cousin Baker's yeast (Saccharomyces cerevisiae) was sequenced, Laccaria bicolor became the first multicellular fungus to have its genome sequenced. The bicolored deceiver is a cosmopolitan species that forms associations with many EM plant communities, making it an ideal model organism for fungal studies. In fact, the paper referenced above chose L. bicolor as its EM representative, and because of my strong intuition/psychic powers, I can confidently proclaim that the following publication you'll see summarized will also involve the fibbing fungi. When analyzing the genetic makeup of this common mycorrhizal mushroom, Martin and colleagues finally put a face to the name in regards to those mysterious signaling molecules I've been baiting you with for the duration of this post. The genome also told an interesting story involving high amounts of repeated sequences and the loss of some carbohydrate-active enzymes, but we'll cover that in the following section. Back to the signaling molecules, mycorrhizal fungi are known to release tiny compounds into the soil and their host plant's tissue called mycorrhiza induced small secreted proteins (MiSSPs). Martin found evidence of the regions that code for MiSSPs, but the exact function of these proteins were unknown at the time. It was also shown that a proportion of these regions were only expressed in symbiotic tissues, a finding that Pereira et al. would extrapolate on in a 2018 study analyzing the secretome of another common EM species, Cenococcum geophilum.

Cenococcum geophilum is one of the most well-studied fungal species because of its abundance and wide distribution in forest ecosystems, and therefore has a lot to tell us about mycorrhizal symbioses. Pereira and crew found over 200 MiSSPs involved in the interactions between C. geophilum and two different plant hosts, pine and poplar trees. Most of these gene regions and the MiSSPs they code for showed the same levels of regulation regardless of the host. This provides insight into C. geophilum's symbiotic strategy - the signaling molecules secreted aren't host specific. However, the opposite has been shown in other mycorrhizal fungi. Some may only associate with one plant species. Others will regulate the production and secretion of different MiSSPs depending on what plant host they are associating with. Returning to Martin's earlier findings of repeated sequences and the loss of carbohydrate-active enzymes, C. geophilum's genetic code narrated a similar story.

What are repeated sequences and what do they tell us about the evolution of mycorrhizal fungi? Envision flipping through the pages in a book and finding a paragraph full of repeating A's, C's, G's, and T's. After a couple indecipherable lines of text, the writing returns to normal and your pleased to find that your book wasn't written in some secret code. You flip the page again only to come across an entire chapter of these random four letter repeats. Although you can't make any sense out of what seems to be a mishap of the publishing company, what you're looking at isn't an error nor evidence of the author's cat walking across their keyboard. It serves a purpose! Okay, my metaphor falls short here, because I can't come up for any logical reason why an actual book would contain repeated strings of letters, BUT, in the case of DNA and genomes, this a common occurrence. When we sequence a species genome, we are essentially reading the genetic biography of that species, and the language in which the book is written is in base pairs of DNA or RNA. Scientists have questioned the function of repeated sequences, which typically don't code for proteins (or, using my earlier example: words), and now believe they play important roles in expression and regulation of other genes. In the case of fungi, repeat rich regions have been found to evolve at faster speeds compared to other regions of their DNA. This allows for higher mutation rates and subsequent gain and/or loss of genes that assist fungi in interacting with their plant hosts.

Above you can see the smallest units of DNA, nucleotides (bases), and the hydrogen bonds they form with their complimentary base. For further explanation look to the glossary.