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How low levels of glucose and tryptophan may signal changes in development and
symbiotic potential of ectomycorrhizal fungus Laccaria bicolor
Alexandra Goetsch
2 ABSTRACT
Symbiosis between tree species and ectomycorrhizal fungi is a fundamental relationship that promotes the overall health of boreal and temperate forests. Nutrient exchange between the two organisms is mutualistic in nature however establishment of this relationship is still not fully understood. Many molecules including phytohormones, sugars, and amino acids have been shown to be involved in this symbiotic process from establishment to prolonged interactions.
Fungal auxin is known to be a key phytohormone in this interaction and it has been proposed that its production in ectomycorrhizal fungi is to aid in the fungi’s ability to establish symbiosis.
Other molecules, however, have been proposed to work in conjunction with auxin to initiate these organismal interactions, questioning the role of fungal auxin as a master regulator. In this study, the effect of sugar and tryptophan feeding on growth and ectomycorrhiza formation was assessed. Free-living cultures of model ectomycorrhizal fungus Laccaria bicolor and lines altered (either overexpressing or RNAi-silenced) for expression of the auxin biosynthesis enzymes Aldehyde Dehydrogenase, Ald1 and Ald2 were utilized. A selection of these lines were grown in contact with common symbiotic tree species partner Populus. Fungal biomass,
expression of auxin biosynthesis and transport-related genes, auxin production and the presence of ectomycorrhiza under different sugar and tryptophan concentrations was assessed. Eight out of 21 tested genetically modified L. bicolor lines secreted significantly different levels of indole- 3-acetic acid as compared to the wild type when grown in standard glucose conditions and supplemented with tryptophan. Biomass production of wild type L. bicolor and transgenic lines was positively correlated to sugar concentrations in the medium, regardless of supplementation with tryptophan or genotype. Expression analysis for four of five auxin-related genes showed reduced expression for auxin-biosynthesis genes Ald1, Ald2 , and Ald3 and the putative auxin transport gene, ABCB5, in a selected line that was RNAi-silenced for Ald2. In addition,
expression of Ald1, 2, 3 and ABCB5 in the WT fungus appeared to be up-regulated in low-sugar conditions as compared to standard sugar conditions supplemented with tryptophan. Bringing together these results allows for proposition of a first model to conclude how low glucose and tryptophan work together as signals to impact auxin pathways in the ectomycorrhizal fungus Laccaria bicolor to modulate fungal growth and affect its symbiotic potential.
3 INTRODUCTION
Plant hormones, also known as phytohormones, play a crucial role in a plant’s overall growth and development and the plant’s ability to adapt to climatic challenges. Phytohormones also have the ability to regulate a plant’s response to biotic challenges such as herbivores, viral and bacterial pathogens as well as fungi (Taiz and Zeiger, 2015). Since plants are sessile, they need to be able to tightly regulate networks of their own metabolic compounds, phytohormones, and a multitude of other molecules in order to maintain and defend themselves. Several
phytohormones are well known to be involved in a variety of plant growth processes. For instance, cytokinin’s involvement in shoot development or salicylic acid’s role in plant defense (Taiz and Zeiger, 2015). One of the most highly researched phytohormones is auxin. Auxin has been identified to be involved in a multitude of growth and development processes including control of shoot elongation, cell division and expansion, apical meristem development, lateral and adventitious root formation and development (Overvoorde et al, 2010 and Zhao 2011), and has also been shown to be involved in establishing symbiosis with various bacterial and fungal partners (Sukumar et al, 2013). The role of auxin in establishing symbiosis with ectomycorrhizal fungi will be discussed to a greater extent later in this report.
The most common form of auxin found in plants is known as indole-3-acetic acid (IAA) however there are several other forms of plant auxins including Indole-3-butyric acid (IBA) and indole-3-propionic acid (IPA) which are usually precursors to IAA (Ludwig-Muller, 2011). As molecules, auxins are characterized chemically by the presence of an aromatic ring and
carboxylic acid group, thus IAA resides naturally in an acidic state (Korasick et al, 2013). Due to their unique chemical nature, plant auxins establish bonds with other metabolites such as amino acids to form amide conjugates and sugars to form ester conjugates (Ludwig-Müller, 2011).
Forming bonds with other metabolites allows for prolonged storage of plant auxins as well as auxin transport to other tissues. Auxin conjugates are frequently taken into consideration when studying auxin regulation in plants due to only a fraction of free IAA being present depending upon the tissue type (Ludwig-Müller, 2011). Auxin transport within plant tissues has also been studied to a great extent, and has been shown that auxin transport through various efflux transporter proteins (PINs) and influx carrier proteins (AUX1/LAX) regulate root growth, specifically lateral root induction (Laskowski et al, 2008; Reed et al, 1998; and Swarup and Bhosale, 2019; Swarup et al, 2008). The role of auxin transport in root tissues and its
involvement in lateral root growth holds a particular significance in the context of mycorrhizal symbiosis of boreal forest tree species with ectomycorrhizal fungi. Pathways of plant auxin in association with its metabolism, signaling, conjugation and degradation have been shown to be impacted in roots colonized by ectomycorrhizal fungi (Vayssieres et al, 2015). Research investigating how plant as well as fungal auxin pathways including biosynthesis and transport impact root architecture is of great interest to better understand plant-fungi symbiosis and can provide insight to how these processes are controlled on the molecular level.
Mycorrhizal symbiosis mediates the exchange of nutrients between two common forest organisms: plants and fungi. Host plants supply the mycorrhizal fungus with carbohydrates in the form of sugars while the fungus provides soluble forms of nutrients (nitrogen and phosphorous) to the plant. There exist several types of mycorrhizal fungi however the two dominating forms are arbuscular mycorrhiza and ectomycorrhiza (Marcel et al, 2003, Martin et al, 2016 and Taiz and Zeiger, 2015). Ectomycorrhizal fungi are important for maintenance of boreal forest systems as they are the dominating mycorrhizal partner of tree roots and thus, were utilized for the experiments done and will be the focus of this report. Among the most common ectomycorrhizal
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fungi establishing these symbiotic relationships are those belonging to Basidiomycota or Ascomycota classifications (Smith and Read, 2008). Ectomycorrhiza are evolutionary younger than arbuscular mycorrhiza and generally form symbiosis with tree families such as Pinaceae (pines), Salicaceae (poplar, aspen), and Myrtaceae (Eucalyptus) (Taiz and Zeiger, 2015). Root systems of plants colonized by ectomycorrhizal fungi grow more slowly, are thicker due to the growth of the fungal mantle surrounding them and appear to be more branched and swollen than those of non-colonized plants. Ectomycorrhizal fungi form a mycelium mantle around plant roots and their hyphae penetrate the apoplastic space between epidermal cells and cortical cells (Horan et al, 1988). The hyphae do not penetrate the root cells specifically but instead forms a thick structure known as the Hartig Net around the root cells (Blasius et al, 1986 and Plett et al, 2013).
Hyphae are able to convert insoluble organic nitrogen and phosphorous into forms that can be utilized by plants. The Hartig Net is the site of nutrient exchange between two symbiotes and is a key feature of mycorrhiza roots (Taiz and Zeiger, 2015).
Colonization of host plant roots with ectomycorrhizal fungi results in a re-structuring of root architecture indicated by changes in lateral root growth in addition to phenotypic changes to the overall shape of the root tips when compared to uncolonized roots (Figure 1). One specific
focus of plant- fungi symbiosis is to better understand the molecular mechanisms involved in this drastic
restructuring of root architecture.
It has been proposed early on that auxin in the form of IAA plays a key role in re-organizing the root
architecture of host plants when in contact with ectomycorrhiza fungi (Slankis 1973). More recent studies have confirmed Slankis’s hormonal theory of mycorrhizal development in many other
ectomycorrhizal fungi including but not limited to: model ectomycorrhizal fungus Laccaria, bicolor (Felten et al, 2009 and Vayssieres et al, 2015); Paxillus involutus (Rudawska and Kieliszewska-Rokicka, 1997); Tricholoma vaccinum (Krause et al, 2015); H. cylindrosporum Romagnesi (Gay et al, 1994); as well as in Truffles (Splivallo et al, 2009).
Due to the significance of auxin in many plant growth and development processes, it has been hypothesized that fungal auxin might also play a significant role in establishing
ectomycorrhizal symbiosis with host plant partners (Krause et al, 2015; Gay and Debaud, 1987;
Bartel, 1997). However, further research suggests that even though auxin has been shown to play a crucial role in symbiotic establishment, there other molecules that may work in conjunction with auxin or regulate auxin biosynthesis genes to initiate these symbiotic relationships and alter
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root architecture. Some of those other types of molecules include VOCs and ethylene having been shown to be involved in restructuring of the tree roots when colonized (indirect contact studies from Felten et al, 2010) and (Vayssieres et al, 2015). It has been shown that fungal VOCs in the form of sesquiterpenes (SQTs) also contribute to reorganization of host plant root
architecture by inducing lateral root growth. These experiments also showed that direct contact with the fungus is not necessarily required to alter root structure (Ditengou et al, 2015).
Lipochitooligosaccharides (LCOs) have been shown to be involved in establishing symbiosis between ectomycorrhizal fungus L. bicolor and Poplar host plants. When expression of genes associated with a calcium-spiking pathway that is activated by LCOs was decreased, L. bicolor- Populus associations were impacted (Cope et al, 2013). In addition to these compounds it has been shown that the phytohormones, ethylene and Jasmonic acid, potentially work in conjunction during late colonization to limit formation of the Hartig Net such that the plant is not suffocated by the fungal mantle (Plett et al, 2013).
It has been suggested that ectomycorrhizal fungi, along with other microorganisms, are capable of producing their own phytohormones, which may be to promote their ability to
establish relations with plant partners. Production of auxin in ectomycorrhizal fungi as well as its processing has been a central focus to better understanding the initial stages of symbiosis.
Several ectomycorrhizal fungi have been shown to both alter host plant root architecture in addition to having auxin producing capabilities; such fungi include: Hebeloma, cylindrosporoum (Gay and Debaud, 1987 and Tranvan et al, 2000); Laccaria, bicolor (Felten et al, 2009,
Karabaghli-Degron et al, 1998 and Rincon et al, 2003); and Truffle species: Tuber borchii and Tuber melanosporum (Splivallo et al, 2009). This suggests IAA likely plays some role in altering host plant root architecture when colonized by ectomycorrhizal fungi (Sukumar et al, 2013).
Genetically altered strains of ectomycorrhizal fungi are useful tools that can provide insight as to how fungal auxin is utilized during ectomycorrhiza formation and symbiotic establishment. For the experiments performed and discussed in this report model
ectomycorrhizal fungus Laccaria, bicolor lines were generated with altered expression of the enzyme Aldehyde Dehydrogenase 1 and 2 (Ald1 and Ald2 respectively) which catalyzes the last step of IAA biosynthesis through the tryptophan dependent pathway (Figure 2). Among the lines created there are Ald overexpessors, Ald RNAi lines as well as empty vector lines which are expected to behave as the WT L. bicolor strain (S238N_UNQ). Previous experiments in the Felten group have been conducted to confirmed that the expression of these lines have been altered in the desired manner. Auxin production was first assessed in wild type L. bicolor to obtain a base level of IAA production in the mycelium. Auxin production was also quantified for the liquid media after the fungus was incubated on the liquid’s surface. Results from this
assessment however, showed that in both the mycelium and the liquid media there is large variation between the experiments and even more variation between batches of wild type L.
bicolor grown in one particular experiment.
This has led to the consideration that IAA production in L. bicolor may require exogenous addition of IAA precursor, tryptophan, in order to maintain stability. Previously discussed experiments have shown that auxin production can be triggered by exogenous tryptophan application when present in high concentrations (5 µM- 2.5mM). These results are supported by experiments performed in Rudawska and Kieliszewska‐Rokicka, 1997, however it would be of interest to test more physiologically relevant conditions (< 5 µM).
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Additionally, an important consideration for understanding auxin biosynthesis may also be that L. bicolor may react to other signals such as nutrient availability especially if auxin is important for establishing symbiosis with host plants. Sugars are important molecules that determine overall growth of plants and fungi alike. Signaling of sugars in plants has been shown to impact root growth and development by interacting with plant phytohormones (Mishra et al, 2009). Auxin alone has been well documented as a molecule that controls root growth and development (Overvoorde et al, 2010), thus it is highly likely that there would exist an interaction between these two molecules, those being auxin and glucose. Indeed, glucose molecules have been shown to act as signals that modulate the expression of auxin-related pathways in Arabidopsis (Mishra et al, 2009). Taking into consideration that fungi regulate their own available nutrients to exchange for the sugars produced by host plants, it is possible that ectomycorrhizal fungi may be capable of responding to levels of environmental glucose and thus modulate their own production of phytohormones in order to establish symbiosis. It can thus be hypothesized that phytohormone production, specifically auxin, in L. bicolor may rely on exogenous IAA precursor supplementation in the form of tryptophan in addition to induced carbon starvation (low glucose conditions) as means to initiate functional symbiosis with host plants.
AIM AND APPROACH
The overall aim of this thesis project was to better understand the role of fungal auxin with a specific focus on the potential modulating effects of tryptophan supplementation and glucose starvation conditions on symbiosis with boreal forest tree species using and ectomycorrhizal fungi. Several methods were implemented using model ectomycorrhizal fungus Laccaria, bicolor in order to gain more insight on the regulation of auxin biosynthesis and its impact on
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ectomycorrhizal symbiosis. I first set out to quantify the production of fungal auxin within the tissues as well as quantify the amount released from genetically altered lines of L. bicolor.
Second, I studied the expression levels of key genes involved in regulating the expression of fungal auxin producing enzymes and fungal auxin transporters when nutrient levels of the
growth medium were modified. Finally, I observed how these same genetically altered L. bicolor lines established symbiosis with hybrid T89 Populus tremula x tremuloides clones when grown in altered glucose conditions. For all of the described experiments the fungal cultures associated were grown according to the flow chart (Figure 3), which involved growth on solid growth media, movement of cultures to liquid growth media and finally harvesting and storage of tissues for the specified experiment.
MATERIALS AND METHODS
Biological Material
Genetically modified L. bicolor strains used in all experiments include those overexpressing either ALD1 or ALD2 and or carrying RNAi constructs for ALD1, ALD2 or both. The amount of each type of strain included 8 overexpressor lines, 10 RNAi-silenced lines, 2 empty vector (Ev) lines and the WT (S238N_UNQ) control (21 strains in total, Table 1). Tissues of these L.
bicolor strains were harvested and stored for later use in various
experiments outlined in this section.
Coculture experiments with a selection of these transgenic L. bicolor lines and WT L. bicolor were grown in contact with symbiotic tree partner, hybrid aspen, specifically Populus tremula x tremuloides UPSC clone T89.
Generation and Harvest of Fungal Material
Growth of the 21 modified fungal lines for IAA quantification in addition to the fungal lines utilized for RNA expression analysis and additional IAA quantification is outlined in
Figure 3. Tissues were initially grown on solid Pachlewski (P20) medium (pH of 5.5) (Table 2). In order to contain the mycelium growth to the surface of
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the medium, cellophane membranes were placed on top of the media prior to inoculation with a plug of mycelium from a stock culture. Cellophane membranes were prepared by boiling for 20 minutes in 1.0 L of deionized H2O with 1.0 g of ETDA to remove excess minerals present on the membranes, prior to rinsing and autoclaving. The L. bicolor strains were grown for ten days at room temperature in the dark.
After this incubation period the agar plugs used for inoculation were removed and the growing mycelium in addition to the underlaying cellophane membrane were transferred to 6 cm (diameter) round petri dishes containing 6 mL of liquid P20 medium (pH of 5.5). The liquid media conditions included 1.0 g/L glucose and 1.0 µM tryptophan (for all 21 fungal lines
quantified for IAA only) and tissues were placed on a layer of 5mm glass beads to keep the fungus on the surface of the liquid medium. Replicates of five samples were produced for each of the 21 fungal lines tested. The mycelium tissues were incubated in these conditions for three days at room temperature in the dark. After the incubation period, the mycelium cultures were harvested by removing the growing tissue from the
cellophane membrane and the liquid media was also collected. The mycelium tissues were flash- frozen via liquid nitrogen in 1.5 mL Eppendorf tubes, weighed and later freeze dried.
Approximately 2 mL of the liquid media for each mycelium tissue was collected in 2 mL Eppendorf tubes and stored at -80 ̊ C.
The fungal lines used for RNA expression analysis and the additional IAA quantification were grown in a similar manner. Tissues in replicates of eight were initially grown on solid P20 growth media per the conditions previously described and then transferred to liquid P20 media, with the underlying cellophane membrane, containing either low (0.1 g/L) or normal (1.0 g/L) glucose and supplemented with 1.0 µM tryptophan or grown in its absence (Table 3). Upon harvest after the incubation period in liquid media, four of the eight replicate mycelium tissues were collected in 1.5 mL Eppendorf tubes for IAA quantification. Tissues were freeze-dried and their weights were assessed after drying. The other four replicates of tissues were flash frozen and stored for RNA extraction and gene expression analysis. A 2 mL sample of the liquid media
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for each replicate from all the treatment groups was also collected and analyzed for IAA content.
A full workflow of these experiments is shown in Figure 3.
IAA Extraction and GS-MS Analysis
Liquid media samples incubated with and without L. bicolor were collected and analyzed for secreted IAA content. Prior to extraction 0.5 mL of cold sodium-phosphate buffer at pH 7.0 and 10 µl of 50 pg 13C6-IAA internal standard was added to 400 µl of each sample and were mixed in pre-labeled 2.0 ml Eppendorf tubes. The samples were acidified to a pH of 2.7 with 1.0 M HCl to further purify the IAA. A solid-phase extraction (SPE) method using SPE column Isolute C8-EC (500 mg/3 ml, #291-0050-B) was used to extract IAA from the samples. SPE columns were conditioned first with MeOH and then with and 1.0% Acetic acid. Samples were applied and the columns were washed with 10% MeOH in 1% Acetic acid and eluted with 70%
MeOH in 1% Acetic acid for each sample. The samples dried in a speedvac for 2.5 hours at 40˚C. Samples were methylated with 0.2 mL of 2-propanol, 1 mL of dichloromethane and 5 µL of 2 M trimethylsilyl-diazomethane in hexane to further stabilize the IAA in each sample. The samples were dried overnight at room temperature in a fume hood. Samples were transferred to GC-vials with MeOH and evaporated in a speecvac at 40˚C for 20 minutes. Finally, the samples were silylated by adding equal parts ACN and BSTFA + 1% TMCS, heated for 30 minutes at 70 ̊ C and dried in a speedvac at 40˚C for 15 minutes. The final solutions were dissolved in heptane and analyzed using Agilent GC/MS Triple Quad 7000 and quantified using Agilent Mass Hunter software.
Mycelium tissues were also harvested and analyzed for IAA content. For this analysis freeze-dried mycelium tissues were pre-ground in 1.5 ml Eppendorf tubes containing two 3mm glass beads on a bead mill (Retsch MixerMill 301) for 3 minutes at 30 Hz. Approximately 2.0 mg of ground mycelium powder was added to clean pre-labeled 1.5 ml Eppendorf tubes and were extracted and analyzed in a similar manner as described above for liquid media samples.
Prior to application to the SPE columns each sample was centrifuged for at least 1 minute to pellet unwanted particles and the supernatant was applied to the columns.
Data Analysis and Normalization
Quantified IAA values were normalized to the concentration of internal standard added to each sample to obtain values defined in pg of IAA/sample. Thereafter the data for the liquid media samples were normalized to the weight of the full-grown mycelium in which the media was collected to obtain values defined in pg of IAA/ mg of mycelium. Data obtained for IAA content in the mycelium tissues was also normalized to the concentration of internal standard added and then normalized to the quantity of ground mycelium powder that was used in the extraction. A student’s T-test was performed on experimental data associated with the 21 fungal strains to determine if the amount of IAA present in the tissues as well as the amount of IAA secreted was different respective to the genetic alteration.
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RNA Extraction, DNA Digestion and RNA Quality Assessment
RNA Extractions were carried out on four replicates of frozen mycelium tissues from WT L. bicolor and an RNAi-silenced strain of L. bicolor (RNAi_Ald2_5) grown on either low (0.1 g/L) or normal (1.0 g/L) glucose conditions. Samples were ground in a bead mill (Retsch MixerMill 30) for 3 minutes at 30 Hz with one 5 mm stainless steel bead per tube. Ground tissue samples were extracted following the manufacturer’s instructions for the NucleoSpin RNA Plant and Fungi Kit with the only modifications being made for the sample grinding process and the volume of PFB binding buffer to account for the sample type.
Extracted RNA samples were diluted to 200 ng/μL prior to removal of genomic DNA using the DNA-free Kit procedure according to the manufacturer’s
instructions. The integrity of the digested RNA was assessed by mixing 10 μL RNA with 2 μL of Midori Green loading dye and running samples in a 1% agarose gel for approximately 1 hour at 120 V. RNA quality was assessed by measuring sample concentration via
NanoDrop (Table 4).
cDNA Synthesis and qPCR Reactions
Upon assessment of digested RNA concentration and quality a BIO-RAD iScript cDNA Synthesis Kit was used to reverse-transcribe 500 ng of RNA into cDNA per manufacturer’s instructions. The reaction mixtures were incubated in a thermal cycler using the program
described in Table 5.
Primer efficiency was assessed for five auxin related genes in addition to four reference genes adhered to their gene target (Table 6). A small aliquot (1 µL) of every cDNA sample was mixed and diluted to produce a 5x serial dilution series including five dilutions. A qPCR reaction mixture was prepared using 7.5 µL of SyberGreen- (Roche), 5.5 µL primer mix (equal parts of each respective forward and reverse primer templates diluted to 1.6 µM from stock concentration) and 2 µL of cDNA dilutions (five dilutions decreasing with a factor of 5) or water for non-template controls were added to a white BIO-RAD qPCR plate in two technical replicates. The plate was centrifuged and amplified in a
BIO-RAD CFX Maestro 96 thermocycler according to the program described in Table 7.
For gene expression analysis, each cDNA sample was diluted by 1/10th their original concentration and used in
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individual qPCR expression for the genes previously described in Table 6. The diluted cDNA for each sample was tested using two technical replicates for each primer pair and were prepared into the qPCR reaction mixture previously described for primer efficiency tests. For individual gene expression analysis one primer pair was assessed per plate for all samples. The qPCR mixtures were then aliquoted into a white BIO-RAD qPCR plate, vortexed and centrifuged prior to placement in the thermal cycler and samples were amplified under the conditions specified in Table 7.
Primer Efficiency and Gene Expression Analysis and Normalization
The stability of primers was assessed by analyzing the melting curves, i.e. checking for the absence of shoulders which would indicate primer dimers, and the primer efficiency values from the cDNA dilution curve amplification. Primer efficiency was considered acceptable if the efficiency value fell between 80 % to 120 % and primers dimers were absent from the melting curve (absence of peaks < 75˚C). Peaks detected at lower dilutions (1/5th, 1/25th and 1/125th) were still acceptable if they fell within the acceptable efficiency range and lacked primer dimers.
The quality of individual gene expression was assessed in a similar manner but by only looking at the melting temperature peaks. Again, if consistent shoulder-less peaks were detected from the melting curve for each primer pair that qPCR reaction was considered acceptable.
Blank water controls were also assessed and were acceptable if no expression peaks were detected. Melt curve data for all genes was then processed using the gene study program within the same BIO-RAD CXF software. The Gene Study program calculated gene expression data and normalized the data to the reference genes Histone 4 (H4) and Elongation Factor 3 (EF3).
Although four reference genes were amplified only the two previously mentioned genes were used for normalization as their gene stability was considered acceptable with M-values of 0.424 (calculated from the Gene Study application in the BIO-RAD CXF Maestro software) for both
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genes. A One-way ANOVA, Tukey Post Hoc statistical test was performed to determine if any statistical differences could be observed between varying glucose and tryptophan treatments for each gene.
Poplar and L. bicolor Functional Analysis experiments
To investigate the impact of mycorrhiza formation of
ectomycorrhizal fungi in the presence of poplar plant hosts, two functional analysis experiments also known as contact experiments were conducted.
Contact experiments take anywhere from 44-51 days to complete depending upon growth of the poplar explants and fungal mycelium.
Approximately 5-6 jars of growing poplar plants (Populus tremula x tremuloides UPSC clone T89) were cut between the internodes and placed vertically into growth boxes containing ½ MS media (2% sucrose and 8.0 g/L plant agar) and were grown at 23°C/
18°C, 16h light/ 6h dark in a growth chamber for at least 21 days. After this incubation period or once the explants start to develop roots, they are transferred to 12 x 12 cm square plates between two cellophane membranes containing ½ MS media (containing 2% sucrose and 10 g/L plant agar) and allowed to grow for ten more days in order to develop lateral roots. The cellophane membranes are placed underneath and on top of the plant roots to ensure the growth of roots is on top of and not into the media in addition to maintaining root hydration. While the explants develop lateral roots, L. bicolor cultures are prepared by growing approximately seven stock fungus plugs on top of long rectangular cellophane membranes on solid P20 media (pH 5.5) (Table 2) for ten days. These cellophane membranes are also prepared by boiling them in 1.0 g ETDA/ liter of water to remove excess minerals.
Once the fungal cultures and poplar explants have grown for ten days, they are both ready for initiation of contact. Two to three poplar explants were carefully removed from the growth plates and placed on top of a clean cellophane membrane sitting on P20 media containing 1.0 g/l MES sodium salt (pH 5.8). The MES salt acts as a buffer since the fungal cultures quickly acidify the media which can induce stress reactions in the plants. The cellophane membranes holding the fungal cultures were placed upside down onto the poplar roots to allow for a direct contact of poplar roots to the fungus. A control plate of poplar explants with clean cellophane membranes not containing any fungus was also prepared. Initial contact photos were taken of the plates which were then incubated upright in a growth chamber at 23°C/ 18°C, 16h light/ 8h dark.
A black plastic seal was placed around the bottom of the plate to induce mycorrhiza formation with the roots.
Fixation of Tissues from Functional Analysis Experiments
Para-formaldehyde (PFA) fixation buffer was prepared prior to tissue fixation. A PFA buffer composed of 40 g/L para-formaldehyde powder dissolved in 1x PBS buffer and was prepared per in-lab protocol. The PFA solution was adjusted to a pH range of 6.9- 7.0 with concentrated HCl. The finished PFA buffer was aliquoted into 10 mL falcon tubes and stored at -
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20 ̊ C. For tissue fixation the stored PFA buffer was thawed to room temperature. Aliquots of 1 mL of PFA were placed into 1.5 mL Eppendorf tubes and experimental material was harvested and placed into this PFA buffer. The tubes were then placed in a vacuum for three cycles of applying and removing vacuum for five-minute intervals to ensure the fixative penetrated the tissues. Tissues were left for 2 hours up to overnight in PFA fixative at 4 ̊ C. After fixation, the PFA waste was discarded and the tissues were rinsed three times with 1x PBS buffer. Samples were then stored in the PBS buffer at 4 ̊ C until further processing.
RESULTS
Transgenic Ald1 and Ald2 Lines of L. bicolor Altered Overall Tissue Growth
Mycelium tissues grown in liquid p20 medium were weighed to assess overall growth of the transgenic L. bicolor lines compared to the WT control line (n = 5). Out of twenty-one modified lines (Table 1), four of the ten RNAi-silenced lines and, all the lines overexpressed for either Ald1 or Ald2 as well as both empty vector lines were identified to be significantly different in weight when compared to the WT control (Figure 4A and B). From this data it can be
concluded that all the overexpressed lines as well as both the empty vector lines overall grew more tissue mass when compared to the WT control. One double mutant line
(RNAi(I)_Ald1Ald2_7) grew more tissues mass compared to the WT control. In addition, one line silenced for Ald1 (RNAi_Ald1_10) and two lines silenced for Ald2 (RNAi_Ald2_5 and RNAi_Ald2_6) also grew more tissue mass compared to the WT control. No modified lines that grew less tissue mass than the WT control were observed to be statistically significant.
Transgenic Ald1 and Ald2 Lines of L. bicolor May Alter IAA Secretion and IAA Production
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To determine whether IAA (indole-3-acetic acid) secretion was altered in the transgenic L. bicolor lines, IAA concentration was measured for each of the liquid media samples (n = 5) for each transgenic L. bicolor lines by GC-MS and compared to the wild type (Figure 5A and B). In total eight of the twenty-one modified fungal lines showed significantly altered IAA concnetrations in the medium when compared to the WT control (Student’s T-test p-value <
0.05). For two Ald1 overexpressor lines (Ald1OE_1 and Ald1OE_7) and one double RNAi- silenced line (RNAi(I)_Ald1Ald2_7) higher IAA levels were detected in the medium, while three RNAi-silenced lines, one silenced for Ald1 (RNAi_Ald1_25) and two silenced for Ald2 (RNAi_Ald2_5 and RNAi_Ald2_20) released less IAA into the media. In addition, the medium underneath the empty vector 7 line for the first batch (Figure 5A) contained more IAA while the medium underneath the empty vector 9 line in the second batch (Figure 5B) secreted less IAA.
Although the T-tests indicate statistical significance for secreted IAA between the lines, it is important to note the variability of this data between batch one and batch two which is easily observed for the empty vector lines, which were grown in both batches.
IAA content was also quantified inside the mycelium in the same two batches of samples as mentioned above (n = 5) of each transgenic L. bicolor lines (Figure 6). However due to experimental issues regarding the extraction and low IS response in the GC-MS detection processes no data was obtained for the second batch of samples and the data represented for batch one is not conclusive for specified fungal lines (described later) and thus no statistical analysis was performed on this set of data. Although the data for the IAA content within the tissues is not conclusive, it appears that for one line in particular, namely Ald1OE_1, higher levels of IAA were detected in the analysis when compared to the WT control line, however it cannot be stated whether or not this difference in IAA content is statistically significant. As previously mentioned, this data is not reliable due to low IS response in several replicates for five of the lines assessed. These include: all replicates of the WT control line, one to two of the five replicates for lines Ald1OE_4 and Ald1OE_5, (indicated by a diagonal fill pattern in Figure
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6) and four to five of the five replicates for lines Ald1OE_7 and Ev9 (indicated by a blank outlined bar in Figure 6). Due to this low IS response, comparisons for statistical analysis with other transgenic L. bicolor lines could not be made.
Increased Glucose Concentration regardless of the Presence of
Tryptophan Increased Overall Growth in Both L. bicolor Lines To assess the impact of sugar nutrition and availability of the auxin precursor tryptophan, overall growth was assessed for mycelium tissues of an RNAi-silenced transgenic Ald2 line (RNAi_Ald2_5) and the WT L.
bicolor grown in varying media conditions (Figure 7). There were no significant differences in growth between the RNAi line compared with the WT control line for each individual media condition tested. However, when glucose concentrations in the media were increased L. bicolor lines showed increased biomass regardless of the presence of tryptophan determined by a One-
way ANOVA, Tukey Post Hoc Test (p-value <
0.05). There were no significant differences in growth for both lines when comparing the presence of tryptophan for either low (0.1 g/L) or normal (1.0 g/L) glucose concentrations.
However, when the RNAi-silenced line was supplemented with tryptophan and a higher glucose concentration in the media there was a significant increase in growth which was not determined to be
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significant for the WT exposed to the same conditions (One-way ANOVA, Tukey Post Hoc Test, p-value < 0.05).
Expression of Auxin-related Genes in WT L. bicolor may be Altered by Increased Glucose Concentrations when Supplemented with Tryptophan
A RT-qPCR-based expression analysis of the auxin-related genes Ald1, 2, and 3 and ABCB3 and 5 was performed to determine if auxin biosynthesis (Ald1-3 expression) and auxin secretion (through assessment of expression of the putative auxin carriers: ABCB3 and ABCB5) were altered when WT and RNAi-silenced L. bicolor lines were grown in varying media conditions (Figure 8). For most genes, expression levels appeared to be reduced in all media conditions tested for the RNAi-silenced line compared to the WT control line. Expression of Ald1 and 2 was significantly lower in the RNAi-silence line compared to the WT control line when the glucose concentration in the media was normal (1.0 g/L) and tryptophan was absent (One-way ANOVA, Tukey Post Hoc test (p-value < 0.05)). There was also lower expression of Ald3 and ABCB5 in the RNAi-silenced line compared to the WT control when glucose
concentrations were low (0.1 g/L) and tryptophan was also absent (p-value < 0.05). Ald2
expression was significantly lower in the RNAi-silenced line when glucose conditions were low regardless of the presence of tryptophan. Overall, there appears to be a trend that when glucose concentrations were increased, and tryptophan was supplemented, the expression of most tested genes in WT was reduced. This trend of expression was significant for Ald2, 3 and ABCB5 when compared to the expression of the WT control line in low glucose conditions supplemented with tryptophan (p-value < 0.05). While there was not a significant difference in Ald1 expression there is still reduced expression in the WT control when the glucose concentration is increased and supplemented with tryptophan, this expression trend does not appear to be the case in the
17
RNAi-silenced line which had consistent levels of expression regardless of supplementation with tryptophan. It did not appear that expression levels of most of these genes (apart from Ald3) in either L. bicolor line were altered by increased glucose concentrations alone. It was observed that there were no significant differences in ABCB3 expression for any media condition tested or genotype of L. bicolor grown.
Reduction of Ald2 Expression in L. bicolor May Impact Lateral Root Growth in Co-cultures With Hybrid Aspen Under Varying Glucose Conditions
To assess whether exogenous glucose impacts ectomycorrhiza formation depending on auxin biosynthesis, hybrid aspen (T89) clones were grown in contact with RNAi-silenced L.
bicolor (RNAi_Ald2_5) and WT L. bicolor in the presence of low (0.1 g/L) or normal (1.0 g/L) glucose conditions in the medium (Figure 9A and B). It is important to note that observations of this experiment can only be made in a qualitative manner and mycorrhiza formation cannot be
further assessed due to insufficient experimental replication because of poor plant rooting. These tissues have
however been fixed and prepared for potential quantitative mycorrhiza formation in the future. It appears that hybrid aspen grown in contact with WT L. bicolor has increased lateral root growth when grown in low glucose conditions compared to plants grown in contact with the WT L. bicolor on normal glucose conditions. This is also observed in the control (plants grown in the absence of a L. bicolor).
However, when comparing the growth of hybrid aspen grown in contact with the RNAi-silenced L. bicolor, there does not seem to be any qualitative difference in lateral root growth between the low and normal glucose conditions, with the exception of thicker root
structures in the normal (1.0 g/L) glucose condition in contact with the RNAi- silenced L. bicolor compared to the clone grown in low glucose conditions. When observing the growth between the plants grown in contact of both L. bicolor types on the normal glucose concentration specifically, there seems to be more lateral roots in the plants grown in contact with the RNAi-silenced L.
bicolor compared to the plants grown in contact with the WT L. bicolor as well as
18
the control plants without fungus even if the length of these roots appears to be similar between the plants grown in contact with L. bicolor. Overall, it may be possible that lateral root growth and formation is impacted by contact with L. bicolor lines altered in auxin biosynthesis in addition to varying glucose media conditions. Further quantitative analysis would have to be performed to determine these differences more specifically.
L. bicolor Altered in Ald Expression Initiates Contact with Hybrid Aspen Clones and Forms Mycorrhizal Roots
To investigate the impact of mycorrhiza formation of modified and WT L. bicolor in the presence of Poplar tremula x tremuloides (UPSC clone T89) another functional analysis
experiment was conducted. This contact experiment involved making contact between and selected altered L. bicolor lines of which 1 overexpressing line (Ald1_OE_1), 1 RNAi line (RNAi_Ald2_5), 1 empty vector line (Ev9) and the WT control were selected (Figure 10A and B). Due to poor rooting only one of each type of L. bicolor line could be selected from for the contact experiments. Although quantitative data could not be assessed at this time due to lack of time and lack of experimental replicates, root tip tissues have been fixed and stored for future measurement and analysis of quantitative data for mycorrhiza formation in these roots.
Qualitatively it can be observed that all the modified fungal lines appeared to make contact with their respective plant partner and formed mycorrhiza roots indicated by short, rounded root tips as compared to the longer, pointy root tips of control plants without fungus. It can also be observed that out of the plants in contact with the fungal lines, lateral roots in contact with Ev9 L. bicolor appeared to be longer compared to roots in contact with the other modified fungal lines.
19 DISCUSSION
Growth, IAA Production, and IAA Secretion of Transgenic Ald1 and Ald2 L. bicolor Lines Supplemented with Tryptophan
Growth assessment of transgenic L. bicolor lines in typical P20 media supplemented with tryptophan indicates that most transgenic Ald1 and Ald2 lines (All overexpressors and empty vector lines in addition to four out of the ten RNAi-silenced lines) grew more in comparison to WT regardless of the genetic modification made (Figure 4A and B). Studies that compared fungal growth of transgenic ectomycorrhiza could not be easily accessed, thus growth
phenotypes for these L. bicolor lines would be best characterized by further replication of the experiment with emphasis on growth measurements such as mycelium diameter and dry weight biomass. In addition, studies that do use transgenic ectomycorrhizal lines such as overexpressors (Gay et al, 1994) measure growth, more particularly mycorrhiza formation when in contact with plant hosts and thus does not provide a good comparison for growth of free-living transgenic fungal cultures. Other studies that do measure growth in pure cultures of ectomycorrhizal fungi look more into supplementation with the precursors to IAA as well as IAA inhibitor impact on fungal growth and provides insight to how IAA may be involved in fungal growth but provides less insight for transgenic modifications to ectomycorrhiza fungi specifically (Krause et al, 2015).
Since the transgenic L. bicolor lines in this experiment were supplemented with the IAA precursor: tryptophan it was expected to observe increased secretion of IAA in the overexpressor lines and decreased IAA secretion in the RNAi-silenced lines compared to the WT and empty vector lines. This hypothesis was confirmed for some lines but not in all transgenic lines grown under these conditions (Figure 5A and B). Only two lines of Ald1 overexpressors secreted significantly higher amounts of IAA compared to the WT and only three RNAi-silenced lines secreted significantly lower amounts of IAA in comparison. Previous studies in free-living ectomycorrhizal fungi H. hiemale showed increased IAA production when tryptophan (1 mM) was applied exogenously to the growth media (Gay et al, 1989). Another study in
ectomycorrhizal fungi T. vaccinum showed increased [13C6] IAA production when treated with
13C6-labeled L-tryptophan (0.50 mM) (Krause et al, 2015). In addition, tryptophan-
overproducing mutants of the ECM fungus H. cylindorsporoum were also able to overproduce IAA. Thus IAA-overproducing mutants of this ECM fungus have the ability to utilize excess endogenous or exogenous tryptophan for IAA production (Durand et al, 1992 and Gay et al, 1994). The result of increased IAA secretion in the some overexpressor lines of L. bicolor may be reasonable considering that overexpression of ald genes may lead to increased IAA
production by the mycelium since these genes regulate the IAA biosynthesis pathway. A previous study had shown that ald1 expression in T. vaccinum was upregulated in an
overexpressing mutant when treated with IAA precursors such as tryptophan compared to the wildtype T. vaccinum (Krause et al, 2015). There are however many levels of regulation controlling each process from Ald gene expression to molecular secretion of IAA. IAA
molecules are generally present in low concentrations in living organisms due to their ability to conjugate with amino acids and sugars thus a direct link cannot be made to infer that
overexpression of Ald genes correlates directly to IAA secretion since not all free IAA is directly secreted (Ludwig-Müller, 2011). Unexpectedly, four RNAi-silenced lines secreted significantly more IAA compared to the WT and even more striking was the significant increase in IAA secretion from the Ev7 line specifically. Increased IAA secretion in the specified RNAi-silenced
20
and especially the empty vector lines were unexpected results considering that RNAi-silenced lines were hypothesized have reduced IAA secretion if one of the main enzymes in the IAA synthesis pathway were knocked down (Figure 2) (Daguerre, Felten et al. unpublished). The empty vector lines were expected to behave as the WT regarding IAA secretion. Thus, the large variability among these lines is particularly noteworthy. It is this variation among empty vector lines in addition to the WT between the two batches that calls into question the reliability of the these data for IAA secretion for the transgenic L. bicolor lines, considering that previous experiments in the Felten group reported similarly variable results.
Assessment of IAA extracted from the mycelium turned out to be much more variable compared to the IAA extracted from the underlying medium (Figure 6). Due to experimental issues with the extraction process for these tissues, as well as issues quantifying IAA, reliable values associated with IAA production in transgenic L. bicolor lines could not be determined.
There are a few sources from the overall experimental procedure that could be identified to explain this variation. Working from the source least likely to most likely to cause variation one such source could be from the mass-spectrometer from one batch to the next. The instrument undergoes routine maintenance and is frequently used for non-IAA related quantification. The changing of columns and methods for different compounds could impact the variation of IAA analysis especially if analysis batches are interspersed between instrument set ups for different compounds. Since this issue is not easily solved by instrument scheduling, due to difficulty in planning for all the analysis of a particular experiment to be performed all at once, a solution could be to have a biological internal standard. This standard would be an internal control of pooled wildtype mycelium tissue that could be extracted and analyzed with every batch of samples. It would provide a baseline for how much IAA is theoretically present in the WT fungal tissues and would hopefully inform the user if a batch of samples quantified for IAA is not reliable and that it should be reanalyzed. The next likely source of variation could be the IAA extraction method itself. Although this method has been validated and reproducible results have been obtained for plant material it is possible this method is not as effective at extracting IAA for fungal tissues. Finally, and quite possibly the source causing the most variation is the culture growth system used for these experiments. As previously stated, tissues are incubated on solid P20 media and then transferred to liquid media of the same type. Tissues are kept on cellophane membranes which are kept flat in the liquid media by addition of glass beads to the plate prior to transfer. The physical transfer of tissues to this liquid media environment could induce stress and induce secretion of stress compounds which could possibly interfere with IAA production. In addition, the tissues are not fixed to these glass beads and are free to “float about” the media, thus tissues could end up submerged underneath the media which could impact mycelium structure and metabolism. A possible solution could be to eliminate this transfer step and grow tissues solely on solid media however, this would mean secreted IAA could no longer be quantified only IAA production in the tissues could be.
Growth and Expression of Auxin-related Genes in Transgenic Ald2 RNAi-silenced L. bicolor line Compared to WT Control in Altered Media Conditions
Growth was assessed for L. bicolor lines grown in either low or normal glucose conditions in the presence or absence of tryptophan. It can be observed that both the RNAi-silenced line and the WT fungus grew more in normal glucose concentrations than in low glucose concentrations regardless of supplementation with tryptophan (Figure 7). This increased growth in higher glucose conditions is expected, since there is more sugar available for growth but also suggests
21
that supplementation with 1 μM of tryptophan appears to have no impact on growth in these two lines. However, in previous experiments in the Felten group, where higher amounts of exogenous tryptophan were applied, a reduction of L. bicolor growth was observed. This finding is
inconsistent with previous studies indicating that tryptophan supplementation induces growth of other ectomycorrhizal fungi (Gay et al, 1989). However, the concentrations of tryptophan used in these experiments were much higher (1 mM) than the physiologically relevant conditions that were simulated in the experiments for this report (1 μM). An interesting but unconclusive remark regarding growth of the RNAi-silenced line is that it appeared to increase when in media
supplemented with tryptophan regardless of the concentration of glucose, these differences in growth however are not significantly different when compared to growth in media without tryptophan for both glucose concentrations. This pattern of growth was not consistent in the WT fungus. This observation may suggest that glucose is playing a more interactive role in fungal growth than the addition of IAA precursors alone. It has been shown in Arabidopsis, thaliana seedling roots that auxin and glucose do work in conjunction to regulate transcription of auxin- related genes. However, the extent of the role each molecule plays in this regulation is still poorly understood and may not be the same type of regulation in fungal tissues (Mishra et al, 2009).
Assessment of expression data for five auxin-related genes indicates that transcription of three of them (Ald2, Ald3 and ABCB5) were significantly down-regulated in WT L. bicolor grown in standard glucose conditions when supplemented with tryptophan as compared to standard glucose concentration in the absence of tryptophan or low glucose with or without tryptophan (Figure 8). This expression pattern was not observed in the RNAi-silenced L. bicolor line for these conditions. Due this significant down-regulation of Ald2 and 3 (auxin biosynthesis genes) as well as ABCB5 (auxin transporter gene) in the WT fungus, it could be theorized that glucose and tryptophan may be working together to regulate the specific processes related to their corresponding auxin pathways. In Arabidopsis thaliana, glucose was shown to affect expression of several auxin genes relating to biosynthesis, perception, signaling and transport (Mishra et al, 2009). In my experiment, for most media conditions, down-regulation of Ald1, Ald2, Ald3 and ABCB5 was observed for the RNAi-silenced line compared to the WT fungus. In general, lower expression of Ald2 and Ald3 can be expected since this line was knocked down for Ald2. There does not appear to be a clear pattern of expression for all the genes tested between the two lines for any one specific media condition. For instance Ald1 expression was only significantly different between the two lines for one of the four media conditions tested (normal glucose conditions in the absence of tryptophan) as was a similar case for Ald3 and ABCB5 expression with significantly different levels of expression between the two lines for one media condition (in this case low glucose in the absence of tryptophan). Whereas Ald2
expression was significantly different between the two lines for three of the four media
conditions tested (both low glucose conditions and normal glucose conditions in the absence of tryptophan), however as previously stated this may be due to reduced Ald2 levels in this line.
Growth of Hybrid T89 Aspen Clones in contact with transgenic L. bicolor lines on altered glucose media as well as normal P20 media
Based on qualitative observations of growth of hybrid aspen plants in contact with wildtype or Ald2 RNAi-silenced L. bicolor lines it can be concluded that despite Ald
modifications the fungus was able to colonize the roots (Figure 9A and B). Mycorrhizal roots are typically shorter, thicker, and rounder than non-mycorrhiza roots (Felten et al, 2009). These
22
modifications were observed in the host plant roots grown in contact with transgenic L. bicolor regardless of glucose concentration in the media and the genotype of L. bicolor the host was establishing symbiosis with (either the Ald2 RNAi-silenced line or the WT). One observable difference in growth of the plants in contact with WT L. bicolor was that, under low glucose conditions, lateral roots appeared to grow longer compared to the plants grown in contact on normal glucose conditions. Previously it has been shown that lateral root growth of host plants is reduced when in contact with L. bicolor (Felten et al, 2009 and Vayssieres et al 2015). However, these studies do not investigate the effect of varying sugar conditions. It had been hypothesized that plants grown in contact on low glucose conditions would have more mycorrhiza roots since the low sugar availability to the fungus would induce a more immediate need for symbiosis with plant hosts. Time constraints for these experiments inhibited the ability to analyze the extent of mycorrhization in these plants, thus future experiments in these conditions should be replicated.
Another qualitative difference in growth of the host plants can be observed when it’s in contact to the RNAi-silenced line as compared to the WT fungus, on normal glucose conditions.
Lateral root length appears to be similar for these two individual plants however there appears to be a higher number of lateral roots for the plants grown in contact with the RNAi-silenced L.
bicolor line compared to the plants grown with the WT fungus. This however needs more replication and quantification to be confirmed. It has also been shown in previous studies that contact with L. bicolor increases lateral root induction in host plants (Poplar: Felten et al, 2009, P. abies: Karabaghli-Degron et al, 1998 and P. abies: Rincon et al, 2003). However, the higher number of lateral roots for the plant grown in contact with the RNAi-silenced line is interesting considering this line of L. bicolor theoretically produces less auxin, a compound suggested to be involved in establishing symbiosis with host plants. Although this line of L. bicolor appeared to establish symbiosis with host plants it would be of interest to investigate the extent to which mycorrhizal symbiosis occurred. Previous studies have shown that IAA-overproducing mutants of ectomycorrhizal fungi form drastically thicker mycorrhizal mantles, and Hartig Nets
penetrating more cell layers compared to WT fungus (Gay et al, 1994 and Gea et al, 1994).
Considering that IAA-overproducing fungi appear to have more pronounced symbiotic
characteristics it could be expected that RNAi-silenced lines of ectomycorrhizal fungi may have less pronounced features of symbiosis and my not establish symbiosis as well as WT fungi.
Further contact experiments providing quantitative and anatomical data would have to be done with this RNAi-silenced L. bicolor line to better understand the extent to which symbiosis is established with host plants.
Observations of hybrid aspen root growth when in contact with three selected lines of transgenic L. bicolor lines and WT L. bicolor on standard P20 media indicated that each fungal line appears to have established symbiosis with the host plants (Figure 10A and B). This is indicated by the shorter, rounder, thicker roots of the host plants compared to the pointed, thin roots observed in the control host plant root (Figure 10B). Consistent with the interpretations of the previous contact experiment it is not possible to deduce from these results to what extent each of these fungal lines established symbiosis with their host plants. It can be hypothesized that the overexpressor line shown here could have more features indicating symbiosis and could perhaps be more established with host plants compared to the WT based on the observations in Gay et al, 1994 as well as Gea et al,1994. However, these studies were done with a different type of ectomycorrhizal fungus (Hebeloma, cylindrosporum) and only looked at IAA-overproducing mutants. Again, further experiments collecting quantitative and structural data from these L.
23
bicolor lines would provide more insight to the extent of symbiosis for transgenic L. bicolor lines with hybrid aspen.
CONCLUSION
Taking into consideration the experimental findings of this report, a rough model has been proposed to explain how various physiological signals such as tryptophan and sugar impact primarily fungal IAA biosynthesis in addition to their effects on fungal development and
symbiotic potential of ectomycorrhizal fungus Laccaria, bicolor (Figure 11). This model relies on two basic assumptions: 1) That IAA biosynthesis in L. bicolor is not capable of being
positively regulated by outside signals. IAA production in the fungus is by default switched “on”
and addition of outside signals work to switch IAA biosynthesis “off” or have no impact, 2) There may not be transcriptional regulation of Ald expression in Laccaria as is it known in plants with regulation through auxin transcription factor binding. It is possible that, in Laccaria, IAA works to reduce Ald enzymatic activity through feedback inhibition. This conclusion is
suggested since there was not any significant down-regulation of Ald expression (Figure 8) in conditions that were observed to be correlated with increased IAA production (low glucose with tryptophan supplementation). The individual effects as well as the compounded effects of signals such as sugar and the IAA precursor: tryptophan were taken into consideration for interpretations of this model. First the consideration of sugar signals alone. From presented results it can be observed that standard glucose conditions resulted in regular growth of the fungal tissues whereas low glucose conditions resulted in reduced growth of fungal tissues in wildtype L.
bicolor (Figure 7). Previous experiments by members of the Felten group have also shown this reduction in fungal growth on media with lower than normal sugar concentrations compared to standard glucose concentrations. It has been shown in prior experiments that IAA secretion is promoted and even significantly increased when glucose concentrations are low compared to normal concentrations at least for WT L. bicolor. Thus, supporting the proposed model that when fungal tissues are grown in standard sugar concentrations, signaling by way of glucose alone, is not inducing IAA biosynthesis. It is the simulation of carbon starvation that is inducing the fungus to potentially synthesize more IAA and release it into the media.
Second the consideration of tryptophan signaling alone. Prior experiments in the Felten group indicated treatment with tryptophan significantly increased IAA secretion in free-living L.
bicolor. These experiments supplemented tryptophan at higher concentrations than those of my own experiments. When testing more physiologically relevant conditions (> 0.5 mM of
tryptophan) there was no significant induction of IAA secretion from free-living mycelium regardless of whether glucose conditions were of standard concentrations or of lower concentrations. It has been shown that exogenous tryptophan treatment can induce IAA production in other ectomycorrhizal fungi (Rudawska and Kieliszewska‐Rokicka, 1997), however, this does not appear to be the case for L. bicolor at least at the tested conditions (1.0 μM tryptophan) and could suggest there might be a threshold for IAA production in the fungus as well as a threshold for detection with the methods used for quantification. Finally,
consideration when these two molecules are present together in the media. Previous experiments in the Felten group showed that when glucose conditions are low and tryptophan is
supplemented, there is a significant increase in IAA secretion by the fungus compared to normal glucose conditions as well as a reduction in growth of the fungus when glucose conditions are low. Although fungal growth does not directly correlate to IAA production, these two processes can be considered in the context of one another. Perhaps it can be proposed that less resources
24
are allocated to fungal growth when resources are scare (aka low sugar availability) and more is allocated to IAA production in an effort to attract a plant host and establish symbiosis.
ACKNOWLEDGMENTS
I would like to take this opportunity to thank my thesis project supervisor, Judith
Lundberg-Felten for supervision and project guidance in addition to members of the Felten group that provided laboratory support and feedback: Raghuram Badmi, Jamil Md Chowdhury, Lill Eilertsen, Carnia Lubrecht, Jingjing Zhou. A special thanks to Yohann Daguerre for guidance and laboratory supervision of this project. An additional thanks to Karin Ljung as well as Roger Granbom for their discussion and laboratory support for the GC-MS. A friendly thanks to Tinkara Bizjak for company in the lab and moral support. A thank you to Umeå University for awarding the Umeå University Scholarship during the academic term in which I completed my thesis work. And finally, big thank you to my parents Thomas Goetsch and Cynthia Fawcett for supporting me every step of the way during my Master’s Program.
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