1. 1
Visualising Periarbuscular Membrane Between Rice and
Arbuscular Mycorrhizal Fungi In Live Tissue
Joe Brennan
Robinson College
Supervisor: Dr. Ronelle Roth
Word count: 5992

2. 2
Visualising Periarbuscular Membrane Between Rice and
Arbuscular Mycorrhizal Fungi In Live Tissue
Abstract: Arbuscules are the site of exchange of mineral nutrients and fixed carbon between fungi
and plants during arbuscular mycorrhizal (AM) symbiosis and are surrounded by a newly generated
plant membrane, known as the periarbuscular membrane (PAM). Fluorescent tagged fusion proteins
have been used to investigate the PAM. Here we utilised multi-photon laser scanning microscopy to
visualise AM-induced proteins tagged with fluorescent reporter proteins in Oryza sativa and
demonstrated that this microscopy technique offers improved resolution and image quality over
previous fluorescent microscopy methods. The data generated provides insights into different
localisation patterns of different functional groups in the PAM.
Key terms:
Arbuscule
Periarbuscular membrane (PAM)
Trunk domain
Branch domain
Multi-photon laser scanning microscopy (MP-LSM)
Introduction
Arbuscular mycorrhizal (AM) symbiosis is an ancient mutualistic interaction between plants and fungi, shown to
have originated over 400 million years ago [1]. The ecological importance of this symbiosis is demonstrated by
its coincidence with plant colonisation of land [1] and its conservation in approximately 80% of land plant
species [2]. The symbiosis evolved as a means of optimising plant nutrient acquisition, with the fungi providing
water and nutrients to the host plant in exchange for fixed carbon [3]. The anthropological importance of the
symbiosis should not be understated. Field studies show that AM symbioses reduce losses of nitrogen and
phosphorus from the soil, facilitating a reduction of chemical inputs and environmental damage as a result of
fertiliser runoff [4]. Expanding the understanding of the molecular components and processes required to
establish and maintain this symbiosis may facilitate its manipulation and engender an enhanced presence of
this mutualism in modern agricultural settings.
AM symbiosis facilitates exchange between the plant host and the extensive hyphal networks of the fungi.
These fungi effectively enlarge the nutrient interception zone of the plant [4], providing plants with increased

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quantities of essential yet relatively immobile phosphorous and nitrogen nutrients [5]. The exchange of
resources is bidirectional, with host plants providing fixed carbon to the fungi. This exchange is possible as a
result of a large intercellular interface between fungal hyphae and plant root cells, known as arbuscules.
Arbuscules form from fungal hyphae that penetrate the inner cortical cells of the plant root and undergo
extensive branching (Figure 1A and 1B). Arbuscules remain separated from the symplast by a plant membrane
known as periarbuscular membrane (PAM), continuous with the plant cell plasma membrane. Both the
penetration of the hyphae into the root tissue and the formation of an arbuscule are associated with drastic
rearrangements of cellular contents [6],[7] and considerable membrane biogenesis [8].
Despite the ecological and anthropological importance of this symbiosis, our understanding of the
developmental and regulatory events occurring throughout the arbuscule life cycle remain limited. Previous
studies have utilised AM-induced proteins tagged with a fluorescent reporter protein to investigate these
characteristics [7],[8],[9],[10]. These studies were critical in identifying and characterising distinct domains of
the PAM [7]. These include the trunk domain (Figure 1D), contiguous with the intraradical hypha as it enters
the space formed within the inner cortical cell. Within the trunk domain of the PAM are AM-induced proteins
that localise more specifically to the trunk than the surrounding plasma membrane [7]. The branch domain of
PAM surrounds the fine hyphal branches that constitute the majority of the arbuscule (Figure 1D) and was
found to contain AM-induced phosphate transporters that localise specifically and exclusively to this
membrane in both Oryza sativa [9] and Medicago truncatula [7]. These observations suggest that
accompanying the spatial and molecular distinction between the branch and trunk domain, there is also a
functional distinction, with the branch domain potentially operating as the site of nutrient exchange between
the plant and fungi [9].
A more recent study investigated the localisation of an AM-specific SCAMP (Secretory carrier membrane
protein), AM42, in the PAM by using a pAM42:GFP-AM42 fusion protein in O.sativa [10]. SCAMPs are integral
membrane proteins thought to play roles in mediating endocytosis [11], exocytosis [12] and cell plate
biogenesis [13], although their molecular role in these processes is yet to be fully elucidated. Kobae and
Fujiwara found that the GFP-AM42 localised to the PAM trunk and branch domains of young arbuscules but
only to the PAM branch domains of fully developed mature arbuscules [10]. GFP-AM42 was also found to

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localise to the perihyphal membrane (PHM) surrounding intraradical hyphae and was a useful marker of PAM
throughout the arbuscule lifecycle.
In this study, the same O.sativa GFP-AM42 fusion line used by Kobae and Fujiwara [10] was investigated using
the novel deep-tissue live-cell imaging technique of multi-photon laser scanning microscopy (MP-LSM) to
examine the localisation pattern of GFP-AM42 during AM symbiosis. MP-LSM facilitated visualisation of GFP-
AM42 in PAM at higher resolution than achieved by conventional imaging techniques such as confocal laser-
scanning microscopy (CLSM), owing to reduced autofluorescence, deeper tissue penetration and intrinsically
high 3-dimensional resolution of MP-LSM [14].
AM42 is likely involved in membrane transport and dynamics, so the localisation pattern that GFP-AM42
displays may be a general representation of the localisation of proteins involved in these processes during
arbuscule development. AM-induced phosphate transporters PT11 in O.sativa and PT4 in M.truncatula were
found to localise to the PAM but only at the branch domains [9],[7]. This showed that different functional
groups of proteins may localise to the PAM in different ways throughout the lifecycle of an arbuscule. We
wanted to examine the PAM localisation of a protein involved in fungal recognition and signalling and so we
investigated another fluorescent reporter line, pLYK1:LYK1-RFP. LYK1/OsCERK1 encodes a lysine-motif (LysM)
receptor-like kinase essential for the recognition of fungal chitin and involved in signalling in AM symbioses
[15]. The physical localisation of LYK1 in the PAM is undefined. We used a pLYK1:GUS reporter line to confirm
the cellular specificity of LYK1 expression during AM symbiosis, and also to investigate changes in spatial
patterns of LYK1 expression at different time points during colonisation, by examining the reporter lines at
three and six weeks post-infection (wpi).
The results of imaging the pAM42-GFP-AM42 reporter line were consistent with those of Kobae and Fujiwara
[10].However, we characterised the localisation of GFP-AM42 signal at different PAM domains throughout the
fungal lifecycle at far increased resolution, owing to the advantages MP-LSM offered relative to CLSM. Spatial
analyses of Lyk1 gene expression using pLYK1-GUS reporter lines showed strong, delineated expression specific
to cortex cells containing arbuscules. Neither pLYK1-LYK1-RFP line showed RFP signal, rendering a comparison

5. 5
of localisation of functional groups of proteins in the PAM, beyond that of AM42 and PT11/PT4, impossible at
present.
Materials and Methods
Transgenic rice reporter lines
The preparation of the pAM42:GFP-AM42 line is described in the ‘Materials and Methods’ section of [10]. In
the pAM42:GFP-AM42 reporter construct, the GFP gene is fused to the 3’ end of the AM42 gene and the fusion
is under the control of the native AM42 promoter. Two hemizygous, first generation lines of pLYK1:LYK1-RFP
were provided by the Paszkowski laboratory. In the pLYK1-LYK1-RFP fusion construct, the RFP coding sequence
is fused to the 5’ end of the LYK1 gene. A hemizygous pLYK1:GUS first generation reporter line was also
Figure 1.
(A) From [17] An overview of the general stages involved in the development of an arbuscule. A hypha extends from a
spore and penetrates the epidermis of the root cell (rhizodermis). The hypha grows in the apoplastic space between cells
and penetrates the outer and inner cortex of the root tissue. At the inner cortex, a hypha enters the apoplastic cavity
within an inner cortical cell, formed as a result of drastic cellular reorganisation. The hypha differentiates into a highly
dichotomously branched, disperse network of hyphae; the arbuscule.
(B) Image of a WGA-stained arbuscule. White arrow indicates a intraradical hypha. Blue arrow indicates the highly
branched hypha of the arbuscule. Scale 10μm.
(C) 1μm image slice of B. Individual hyphal branches of the arbuscule visible. Scale 10μm.
(D) From [7]. A representation of the sub-domains of the PAM. This shows a cortical cell with an arbuscule within. The
dotted lines surrounding the grey hypha of the AM fungus represent the trunk domain of the PAM. The unbroken black
line contiguous to the dichotomous branches of the hypha represents the branch domain of the PAM.
A B
C
D

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provided by the Paszkowski laboratory. All transformed rice lines were Oryza sativa cv Nipponbare. (See
Appendix 1).
Seed sterilisation and germination
Seeds were sterilised in 3% bleach for ten minutes then washed in distilled water once followed by three
washes in autoclaved water. Using aseptic technique, seeds were transferred to 0.3% bacterial agar plates.
Agar plates were sealed with Parafilm and incubated at 30°C for 2-3 days. By 2-3 days all seeds would
germinate.
Plant growth and fungal inoculation
Seedlings were transferred to 5cm petri dishes sprayed with black paint with bored holes in the lids through
which the aerial tissue of the rice plant would grow. The closed lids reduced water loss and growth of
contaminating algae. The pots contained Chelford 16/30 sand (acid washed). Plants destined for fungal
colonisation (tester plants) were added to 5cm black petri dishes containing ‘Nurse plants’, which expedite the
establishment of AM symbiosis in the newly added plants. Nurse plants were 4-5 week old O.sativa cv
Nipponbare plants that had been colonised by the AM fungal symbiont and had grown extensive roots. Upon
planting, tester plants were inoculated with a fungal spore solution containing 500 spores of Rhizophagus
irregularis (Premier Tech Canada). Mock inoculated tester plants were not inoculated with R.irregularis and did
not form AM symbioses. Seedlings destined to be mock tester plants were added to petri dishes containing
only sand and lacking nurse plants. 1ml of distilled water was added around the roots. The plants were grown
in a growth chamber with a 400W/m
2
12h day (28°C ) / 12h (20°C) night cycle at constant 60% humidity.
Plants were watered twice a week with 2.5μM phosphate Hoaglands solution and once a week with water. To
water the plants, the lids were removed and approximately 10-15ml of Hoaglands solution added to the
surface of the sand.
At 3-4 wpi, plants were selected for analyses using fluorescence microscopy and trypan blue staining to confirm
colonisation.

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DNA extraction and genotyping
1-2cm of leaf material from one-week old tester and mock plants were added to 2ml Eppendorf tubes with a
glass bead and lysed using a TissueLyser II (Quiagen). Samples were kept frozen with liquid nitrogen upon
removal from the plant. 200μm of sucrose solution (50mM Tris-HCl with pH 7.5, 300mM NaCl and 300mM
sucrose) was added to each leaf extract and the solution was heated at 98°C for 10 minutes. Samples were
then centrifuged for 2 minutes at 14,000rpm and stored at -20°C.
For detailed information on lines and primers used see Appendix 1. 2μl of DNA extract supernatant was used
for each PCR reaction. For a negative controls, 2μl of milliQ water was used instead of DNA extract. For
wildtype controls, Nipponbare DNA was used.
Seeds of the pAM42:GFP-AM42 line provided by Kobae were reported as homozygous for the fusion gene.
Genotyping of this line was carried out to ensure correct genetic background. For each sample, the presence of
GFP and Hygromycin resistance (HygR) sequence in the DNA extract was examined using PCR. Both
pLYK1:LYK1-RFP lines were hemizygous. The lines were tested for the presence of RFP and HygR sequence in
the DNA extracts using PCR. The pLYK1-GUS line was hemizygous. The line was tested for the presence of
HygR sequence in the DNA extracts using PCR. 0.8% agar gels were run with 1kb ladder and imaged with
GeneSnap image acquisition software.
Trypan blue staining
Trypan blue staining was carried out to detect the presence of AM fungi and assess colonisation levels. Rice
plants were gently lifted from the sand using forceps. Roots were gently washed in distilled water to remove
sand without damaging fragile large lateral roots (LLRs). Root samples were excised and incubated for 30
minutes in 10% KOH at 95°C. The KOH was removed and the roots were rinsed with distilled water 3 times.
Roots were then incubated in 0.3M HCl for 30 minutes then removed from HCl and incubated in 0.1% trypan
blue for 8 minutes at 95°C. Roots were removed from trypan blue and washed in 50% acidic glycerol. Roots

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were suspended in 50% acetic glycerol (1:1 mixture of glycerol / 0.3M HCl) and mounted on slides. Slides were
photographed using an Olympus Bx43 microscope and QCapture Pro image-analysis software.
EVOS and 2-photon microscopy
To check for fluorescence arising from the fluorescent protein tag, plant roots from fluorescent reporter lines
were checked using an EVOS-FL digital microscope. Plants were gently removed from sand with forceps and
the roots were washed in distilled water. The plants were placed in empty petri dishes and a small volume of
distilled water was added to cover the roots. The EVOS microscope allows the user to examine the samples
using transmitted light, a GFP channel and an RFP channel. Comparison of the signal between GFP and RFP
channels allows one to determine whether signal is unique to a particular channel and can be attributed to the
presence of a fluorescence reporter protein, rather than autofluorescence arising from endogenous cellular
structures such as the cell wall or cell damage. Autofluorescence appears in both channels. Fluorescence
unique to the green channel likely arose from GFP and fluorescence unique to the red channel likely arose from
RFP. The root system of the sample was analysed for the presence of signal unique to the appropriate reporter
protein. If signal was identified, the root area was mounted in distilled water under a 26x40mm coverslip using
vacuum grease to seal the sides of the coverslip and the region of interest marked on the petri dish for
immediate imaging.
Positive pAM42:AM42-GFP root samples and pAM42:AM42-GFP mock samples were analysed using MP-LSM.
Whole plants in petri dishes were placed in the detection chamber and imaged using an Olympus 25X
objective. GFP and RFP were excited at 927nm and 1040nm wavelengths, respectively, using a femtosecond Ti-
Sapphire laser. To detect GFP fluorescence, emissions below 560nm were collected, whilst RFP fluorescence
was collected from emissions above 648nm. Root target areas were located using epifluorescence to generate
signal from the fluorescent reporter protein. 560- nm for GFP detection and 927nm laser for excitation.
648+nm for red fluorescence detection. Laser power of between 3-10% was used for most images. Z-stacks of
various depths were taken using 1μm slices for overviews and 0.5μm slices for regions of interest. Images were
denoised using ndsafir denoising software [16] to sharpen the image and reduce background fluorescence.
Images were processed using IMARISx64 8.1.2 image-analysis software (Bitplane). Further processing and

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labelling of images was carried out on Fiji/ImageJ. For time-lapse images, plant samples were kept at room
temperature and submerged in 2.5μm phosphate Hoagland’s solution in the petri dish and illuminated using an
LED lamp.
GUS staining
For GUS staining of pLYK1-GUS samples roots were immersed in 1ml of staining solution containing 100mM
NaPO4, 10mM EDTA, 0.5mM K4Fe(CN)6, 0.5mM K3Fe(CN)6, 0.1% Triton X-100, 1mM XGlcA cyclohexylammonium
salt (X-Gluc), vacuum filtrated 3 times for 5 minutes and incubated in the dark at 37°C for 48hours. Roots were
then washed in distilled water and destained in 50% EtOH for two days before the samples were imaged with a
KEYENCE VHX5000 digital light microscope.
WGA staining and imaging
GUS-stained root samples or samples imaged with the MP-LS microscope were stained with wheat germ
agglutinin (WGA), a stain for the chitin of fungal cell walls. Roots were suspended in PBS solution containing
0.2μg/ml WGA-AlexaFluor488 (Invitrogen), covered in foil and left at 4°C until imaged. Before imaging, samples
were removed from the WGA stain and suspended in 1μg/ml propidium iodide (PI) solution for 2 minutes and
30 seconds, to stain plant cell walls. Roots were removed from the PI solution and fixed on slides. Root
samples were imaged using a LEICA TCS-SP5 confocal laser-scanning (CLS) microscope. A 63x water immersion
objective was used. Excitation wavelength for WGA was 488nm and was collected at 500-550nm. Excitation
wavelength for PI was 594 and was collected at 610-750nm. Images were processed using LEICA Application
Suite and IMARISx64 8.1.2.
RNA extraction and concentration measurements
Root samples were taken from washed plant roots at 4-5wpi and frozen at -80°C. Root samples were kept
frozen and ground into a fine white powder. The samples were suspended in 1ml TRIzol (ThermoFisher) at
room temperature for 5 minutes after which 0.2ml 100% Chloroform was added and mixed well. The samples
were centrifuged at 14,000rpm at 4°C for 15 minutes and the aqueous phase was transferred to a tube
containing 0.5ml isopropanol and mixed by inverting. The samples were centrifuged at 14,000rpm at 4°C for 10

10. 10
minutes and the resultant pellets were washed repeatedly in 75% EtOH and DEPC water. 4M LiCl was added
and the samples were precipitated at -20°C for 1 hour. The samples were centrifuged at 14,000rpm at 4°C for
20 minutes and washed again according to the same method described above. The pellets were air-dried and
dissolved in 20μl of autoclaved milliQ water.
To check the concentrations of RNA from each sample, a Nanodrop2000 spectrophotometer was used.
Results
GFP-AM42 signal is specific to colonised plants
To confirm that the GFP signal observed with MP-LSM is due to the fluorescent protein reporter construct and
to confirm that AM42 is expressed only in colonised cells, a number of pAM42:GFP-AM42 ‘mock’ plants were
not inoculated with R.irregularis and were imaged with the MP-LS microscope at 3-4 wpi. These plants had
been genotyped and the presence of the transgene insertion was confirmed (Figure 2).
Figure 2. Gel of PCR products of pAM42: GFP-AM42 plant sample DNA amplified with GFP or Hygromycin
resistance (HygR) primers.
1, 2 and 3 represent the positive control, wildtype and negative control products of PCR with GFP primers.
4, 5 and 6 represent the positive control, wildtype and negative control products of PCR with HygR primers.
In this gel, 8 pAM42:GFP-AM42 plant samples were genotyped. The arrows indicate which primers the DNA
were amplified with. The positive control for GFP primers failed to show a band. GFP sequence was
unavailable to act as a positive control, so a DNA extract of a transgenic rice line with a GFP reporter
construct was used. This extract had been thawed and refrozen numerous times so the DNA may have
degraded resulting in a failure to amplify by PCR and thus failed to produce a band.
There are four more wells for HygR because genotyping was carried out twice for some samples using PCR
buffer from a previous round of genotyping to assist in the event of troubleshooting a failed gel.

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Trypan blue imaging of root samples of mock plants showed no evidence of fungal structures or colonisation
(Figure 3A,B). Roots of mock plants were selected for MP-LS microscope imaging (Figure 3C,D,E). In both the
560-(GFP) and 648+ (autofluorescence) channels, the only structures visible were cell walls, a structure known
for high autofluorescence. No structures resembling the signal of GFP-AM42 seen in [10] were visible, despite
the presence of the fluorescent construct in the mock plant genome (Figure 2). The lack of GFP-AM42 signal in
mock plants shows that the protein does not accumulate in uncolonised cells, making it an excellent marker for
the PAM. The roots imaged with MP-LSM were then stained with WGA-AlexaFluor488, to detect the presence
of any fungal structures and PI to stain the cell walls of the rice root cells, and then imaged using TCS-SP5 CLS
microscope (Figure 3F,G). The only signal obtained was of cell walls stained with PI, indicating an absence of
any fungal structures (Figure 3F,G).
Figure 3.
(A,B) Trypan blue stained large lateral root (A) and crown root (B) of pAM42:GFP-AM42 mock root tissue. Scale
50μm.
(C,D,E) False colour MP-LS microscope image of a large lateral root of a pAM42:GFP-AM42 mock root. C shows
signal collected from 560nm- (GFP) channel, false coloured green. D shows signal collected from 648nm+
(autofluorescence) channel, false coloured magenta. E is an overlay of D and E. Scale 50μm.
(F,G) TCS-SP5 CLS microscope image of pAM42:GFP-AM42 large lateral root (F) and crown root (G) stained with
WGA and PI. No fungal structures are evident. The only signal is that of the 610-710nm channel (PI) showing cell
walls in red. Scale 50μm.

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For pAM42:GFP-AM42 plants inoculated with R.irregularis, trypan blue staining and MP-LSM was carried out on
root samples at 3wpi. Imaging at 3wpi allowed us to image early stages of fungal colonisation and kept root
autofluorescence low as the cells were relatively young. Like ‘mock’ pAM42:GFP-AM42 plants, inoculated
plants had been genotyped to confirm the presence of the transgene (Figure 2). The staining showed the
presence of arbuscules and confirmed colonisation (Figure 4A). Pre-screening of roots was carried out using
an EVOS-FL digital microscope and revealed signal unique to GFP and these root areas were imaged with MP-
LSM. Fluorescence signal corresponding to GFP-AM42 fusion protein was seen similar to that described in [10]
(Figure 4B). Following MP-LSM imaging, roots containing the regions of interest were excised and stained in
WGA-AlexaFluor488 and PI to confirm that the fluorescence signal detected corresponded to AMF colonised
cells. TCS-SP5 CLSM imaging of these roots showed clear intraradical hyphae and arbuscules (Figure 4C). It was
difficult to locate the exact cells imaged with MP-LSM, however the WGA/PI staining confirmed colonisation in
the same root sector.
Figure 4.
(A) Trypan blue stained section of large lateral root from a colonised pAM42:GFP-AM42 plant. Black arrows indicate
arbuscules. Yellow arrow indicates an intraradical hypha. Scale 20μm.
(B) MP-LS microscope image of heavily colonised large lateral root of a pAM42:GFP-AM42 plant. The image is an overlay of the
560- and 648+ channels, false-coloured green and magenta respectively. White arrows indicate arbuscules. Scale 30μm.
(C) WGA and PI stained section of the same large lateral root from (B), imaged with a TCS-SP5 CLS microscope. PI stains plant
cell walls red. WGA-AlexaFluor488 stains fungal cell walls green. White arrows indicate arbuscules. Blue arrows indicate
intraradical hyphae. Scale 50μm.

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The GFP signal seen in the colonised pAM42:GFP-AM42 plants was not observed in wildtype (Nipponbare)
colonised plants (Figure 5A-C), allowing us to conclude that the signal observed was due to GFP-AM42 labelling.
The GFP-AM42 signal observed in arbusculated cells was largely unique to the -560 (green) channel, suggesting
that this signal was not the result of autofluorescence. Autofluorescent signal is seen in both the -560 and
648+ channels, thus the green signal was likely arising from the distinct localisation of GFP-AM42 (Figure 5D-F).
Figure 5.
(A, B and C) MP-LS microscope image of wildtype (Nipponbare) roots colonised with R.irregularis. No signal
unique to the 560- (GFP) channel can be seen. The fluorescence signal is from cell walls. Scale 50μm.
(D, E and F) MP-LS microscope image of an arbuscule in the root of a pAM42:GFP-AM42 inner cortical root cell.
The signal resembling the branch domain of PAM is mostly unique to the 560- (GFP) channel. In the 648+
(autofluorescence) channel, a small amount of autofluorescence from the PAM can be seen. In this channel a
number of autofluorescent bodies are highly visible, indicating that this is an older stage IV arbuscule. Scale
10μm.

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GFP-AM42 localises to functionally distinct domains of perifungal membrane
Higher-magnification images of arbuscules using the MP-LS microscope allowed detailed examination of the
localisation patterns of GFP-AM42. To estimate arbuscule ages, we looked for a number of fluorescent
features and compared the morphology of the labelled PAM with the morphology of arbuscule developmental
stages (Figure 6).
It was observed that GFP-AM42 localises on the PHM surrounding intraradical hyphae (Figure 7A,B,C), although
this pattern was rarely seen, observed in only 5 arbuscules out of 68 imaged, and in one instance the labelling
was unassociated with an arbuscule. The localisation of GFP-AM42 to PHM was observed only from Stage II
(Figure7A-C) to early Stage IV (Figure 7D-F) arbuscules.
Figure 6. Diagram from [17].
A representation of the developmental stages of an arbuscule in a root cortical cell. The different stages are
associated with different PAM morphology and different localisation patterns of GFP-AM42 in the PAM.
Stage I involves the drastic cytoskeletal reorganisation as the nucleus moves within the cell. No GFP-AM42 signal is
visible at this stage. At Stage II, a hypha branches off from the intraradical hypha and enters the space formed in the
apoplastic cell, forming the initial arbuscule trunk. The PAM surrounding a Stage II arbuscule can be seen in Figure
6A-C. At Stage III, low order branching of the trunk domain occurs. The transition from Stage III to Stage IV can be
seen in Figure 6D-E. At Stage IV the arbuscule is mature and highly branched. Figure 5D-F shows the PAM
surrounding a late-stage Stage IV arbuscule. As the arbuscule collapses, between Stage IV and V, GFP-AM42 signal is
lost from the PAM (Figure 10) and once the arbuscule has collapsed (Stage IV), the GFP-AM42 is aggregated and
appears as lobes of intense signal (Figure 9).
Other indicators of arbuscule age can be visualised with MP-LSM. As the arbuscule progresses from early to late
Stage IV, small autofluorescent bodies, thought to be acidocalcisomes [20] appear and increase in number and
intensity, visible in Figure 5E-F and Figure 10. Another change observed as the arbuscule progresses from early to
late Stage IV is that the arbuscule becomes more autofluorescent, with increased signal in the 648+ channel. These
features are useful diagnostic tools for estimating the age of an arbuscule.

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GFP-AM42 could also been seen to localise to the trunk domains of PAM (Figure 7). Interestingly, this
localisation was only observed in stage II to stage IV arbuscule PAM, but not late stage IV arbuscule PAM or
PAM of arbuscules that were senescing (transitioning from stage IV to V). Examining z-stack slices of images of
stage IV arbuscules was useful in identifying trunk domain containing GFP-AM42 (Figure 8A-C). By examining
Figure 7.
(A,B,C) MP-LSM image of GFP-AM42 signal on the PHM surrounding an intraradical hypha and trunk domain of a
stage II arbuscule, showing no branch domain. White arrows indicate PHM. Yellow arrow indicates trunk domain
of PAM. The arbuscule is young, as no branch domain is visible, there are no autofluorescent bodies and
autofluorescence of the arbuscule is low. Scale 5μm.
(D,E,F) MP-LSM image of GFP-AM42 signal from an early stage IV arbuscule. PHM, trunk domain of PAM and
diffuse signal on young branch domain are visible. White arrows indicate PHM. Yellow arrows indicate PAM trunk
domain. Blue arrows indicate young PAM branch domain. The arbuscule is early stage IV as branch domain
containing GFP-AM42 is visible but there are no autofluorescent bodies and autofluorescence of the arbuscule is
low. Scale 10μm.

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individual z-stack slices, GFP-AM42 signal could be observed on trunk domain that was obscured in the 3-d
stack view by the dispersed signal of GFP-AM42 localised to PAM branch domains (Figure 8D).
The most commonly observed and distinct localisation area was that of the branch domains of PAM, with
localisation to this domain seen in 67 out of 68 imaged arbuscules. In general, the localisation of GFP-AM42 to
branch domains appears diffused along the PAM with small foci of varying intensity. In particular areas, PAM
containing GFP-AM42 surrounding an individual hyphal branch can be distinguished (Figure 9). As for when
examining labelled PAM trunk domain, analysing a slice image of a z-stack can provide finer detail of the PAM
domain structures and localisation patterns (Figure 9A-C). GFP-AM42 does not appear to localise specifically to
hyphal tips but is instead distributed with a degree of uniformity along the PAM branch domains (Figure 5F,9C).
Figure 8. High magnification of an arbuscule of a pAM42:GFP-AM42 plant.
(A, B, C) MP-LSM image of PAM trunk and branch domains labelled with GFP-AM42 of a stage IV arbuscule. The image is a
0.5μm slice in the z dimension of a stack containing 103 0.5μm slices. White arrow indicates trunk domain of PAM. Blue
arrows indicate branch domains of PAM. The arbuscule is younger than late stage IV as it shows low autofluorescence and
few autofluorescent bodies. Scale 10μm.
(D) Whole stack (51μm in z-axis) of MP-LSM image slices of the same arbuscule from A, B and C. 560- and 648+ channel
overlay. Branch domain of PAM containing GFP-AM42 is visible but trunk domain cannot be easily distinguished. Scale
10μm.

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GFP-AM42 signal provides insight into spatial-temporal degradation dynamics of AM42
Given the transient nature of arbuscules, and the drastic rearrangements of PAM that occur in accordance with
arbuscule development and degeneration, fluorescent PAM protein reporter lines present interesting targets
for live-cell time-lapse imaging. As a protein hypothesised to be involved in arbuscule accommodation,
visualising the temporal dynamics of AM42 will provide insight into the spatial-temporal degradation dynamics
of PAM formation and degradation. The MP-LS microscope facilitated time-lapse imaging of GFP-AM42 over
long periods as the lower energy photons limit photodamage to the focal plane [14].
Time-lapse imaging shows that GFP-AM42 reporter protein labels the PAM throughout the life cycle of
arbuscules. The 68 individual arbuscules imaged, alongside time-lapse imaging, facilitated a detailed view of
GFP-AM42 localisation patterns in PAM of stage II (Figure 7C), stage IV (Figure 5F) and stage V (Figure 10)
arbuscules. Highly senescent arbuscules (stage V) displayed weak, diffuse GFP-AM42 signal in the arbusculated
cell, with lobes of concentrated signal (Figure 10). These lobes are likely areas where the PAM has folded upon
itself, aggregating AM42-GFP and generating a strong region of signal.
Figure 9. Arbuscule of a pAM42:GFP-AM42 plant.
(A,B,C) MP-LSM 0.5μm slice image of a stage IV arbuscule. GFP-AM42 can be seen in PAM branch domains surrounding
individual fine hyphal branches. The signal is diffuse on the PAM and can be seen not to concentrate at PAM adjacent to
hyphal tips. B shows that the within the cell signal in the 648+ (autofluorescence) channel is very low. GFP-AM42 can be
seen in PAM branch domain surrounding individual hyphal branches, indicated by white arrows. Scale 10μm.
(D) Whole stack (65μm in z-axis) of MP-LSM image slices of the same arbuscule from A, B and C. -560(GFP) and +648
(autofluorescence) channel overlay. GFP-AM42 labelled branch domain of PAM is visible as a larger, more diffuse area.
Finer structures cannot be distinguished relative to the 1μm slice images of A.B and C. Cell walls can be seen to be highly
autofluorescent. Scale 10μm.

18. 18
Time-lapse imaging provided an opportunity to investigate the changes in GFP-AM42 localisation in a single
arbuscule, rather than comparing snapshots of arbuscules of different ages in a single root. From the time-
lapse images of a stage IV arbuscule, it was observed that the trunk domain lost GFP-AM42 signal earlier and at
potentially a faster rate than the branch domain (Figure 11). The loss of GFP-AM42 signal spread along the
trunk domain towards the branch domains of the PAM (Figure 11). A general loss of GFP-AM42 signal from the
branch domains can be seen 44h after the time-lapse began (Figure 11C).
Figure 10. Collapsing arbuscule of a pAM42:GFP-AM42 plant.
(A, B, C) MP-LSM image of a colonised pAM42:GFP-AM42 plant root showing a highly degraded, senescent arbuscule,
(transitioning from stage IV to V) indicated by white arrows. Diffuse GFP-AM42 signal can be seen throughout the inner
cortical cell, with lobes of highly concentrated signal, likely where PAM has collapsed. Blue arrows indicate GFP-AM42
labelling of senescent branch domain of PAM that has not yet begun collapsing in an adjacent senescent arbuscule.
Scale 10μm.

19. 19
A marker of the endocytic pathway failed to co-localise with GFP-AM42
The specificity of GFP-AM42 signal to colonised cells and the detailed labelling of the PAM at different
arbuscule developmental stages imaged using MP-LSM suggests involvement of AM42 in AM symbiosis,
however, the function of AM42 remains unknown, as Tos17-insertion knock-out mutants of AM42 failed to
show a phenotype [10]. Given the putative roles of plant SCAMPs in endocytosis and exocytosis, we attempted
to probe the molecular function of AM42 by staining colonised root tissue of colonised pAM42:GFP-AM42
Figure 11.
MP-LSM time-lapse images of a colonised pAM42:GFP-AM42 root showing a single stage IV arbuscule over 63 hours. Scale
10μm.
(A) Arbuscule at the beginning of the time-lapse (0 hours). GFP-AM42 signal on PAM branch domains can be seen. There is an
area in the upper half of the arbuscule which has less signal, potentially trunk domain.
(B) Arbuscule 24 hours after the initial image was taken. Little difference in signal between the two can be seen. It is likely the
arbuscule is still active and has yet to begin senescing.
(C) Arbuscule 44 hours after the initial image was taken. A dispersed loss of GFP-AM42 signal can be seen. The loss is most
significant in the upper half of the arbuscule, where the signal was weakest initially, as seen in A.
(D) Arbuscule 63 hours after the initial image was taken. The GFP-AM42 is weaker and more diffuse than in C. New puncta of
intense fluorescence are visible however, potentially where PAM surrounding fine hyphal branches have collapsed.
More autofluorescent bodies are visible as the time-lapse progresses and the arbuscule progresses from stage IV to V.

20. 20
plants with Fm4-64, a live-tissue stain of the endocytic pathway. Live, colonised root tissue was incubated with
Fm4-64 and a 50minute time-lapse was carried out. Time-lapse images of this root tissue failed to show co-
localisation of the stain with GFP-AM42 signal (Figure 12). The Fm4-64 stain stained the outer cells of the
colonised LLR (Figure 12A), but failed to penetrate the root tissue and reach the inner cortical cells housing the
labelled arbuscules (Figure 12B-I). A solvent-only negative control for this protocol was not carried out as the
dye failed to penetrate the tissue.
Figure 12.
MP-LSM time-lapse images of an arbuscule in pAM42:GFP-AM42 live root tissue stained with Fm4-64 dye. Overlays are of 560-(GFP)
and 648+(Autofluorescence) channels.
(A) An overview of the colonised root area immediately after staining with Fm4-64. Increased 648+ channel signal at the outer cortical
cells on the bottom edge of the image. Scale 50μm.
(B,C) Zoom on mature arbuscule in A. 20 minutes post staining. Scale 30μm.
(D,E) 30 minutes post-staining. Scale 30μm.
(F,G) 35 minutes post-staining. Scale 30μm.
(H,I) 40 minutes post-staining. No change in signal is observed in the 648+ channel, where the Fm4-64 dye is detected, suggesting the
dye has not reached the imaged cells. Scale 30μm.

21. 21
No LYK1-RFP signal was observed in colonised roots of two independent transgenic LYK1-RFP lines
at 3 weeks and 12 weeks post-infection
The high resolution images of GFP-AM42 signal in arbusculated cells and the published data of phosphate
transporter localisation at the PAM [9],[7] would have facilitated interesting comparisons of localisation of
LYK1-RFP at this membrane. This would have provided insight into the patterns of localisation of different
functional groups of proteins at the PAM.
AM root colonisation was observed in the roots of inoculated pLYK1:LYK1-RFP plants using trypan blue staining.
EVOS-FL fluorescence imaging was carried out on the roots of pLYK1:LYK1-RFP plants at 3wpi of both lines that
had been positively genotyped for RFP sequences. In both lines, no signal was observed that was unique to the
RFP channel. Root samples from the same plants were analysed again at 12wpi and stained with trypan blue,
to see if LYK1-RFP signal could be observed in older colonised tissue. Colonisation was still apparent in these
roots at 12wpi. The intact roots of these specific plants were imaged with the EVOS-FL microscope and no
fluorescence unique to RFP was observed. RNA extraction was carried out using root tissue samples from
plants at 6wpi, in order to investigate whether the LYK1-RFP transgene was being transcribed and to what
degree. The concentration of RNA was too low, at <90ng/μl, for cDNA synthesis to be carried out effectively.
Colonised roots of a pLyk1-GUS line show delineated GUS expression
cDNA synthesis of RNA extracted from colonised pLYK1:LYK1-RFP plants was not possible owing to low
concentrations of RNA, which made it difficult to identify whether the lack of signal observed in colonised
pLYK1:LYK1-RFP plants was due to low expression of the construct. However, the pLYK1:GUS line used the
same promoter as the pLYK1:LYK1-RFP lines and confirmed that the LYK1 promoter is active in colonised tissue.
Colonised crown roots (CRs) and LLRs of a pLYK1:GUS line displayed GUS expression that was specific to
individual cells at 2.5wpi (Figure 13A,B). This staining was observed in very small proportions of total root
lengths. WGA/PI staining of these root sectors demonstrated considerable fungal colonisation (Figure 13C,D).

22. 22
2 out of 5 mock pLYK1:GUS plants showed some areas of strong GUS activity at 2.5wpi. The staining was not
specific to individual cells and covered large areas of root tissue (Figure 14A). Subsequent staining of these
specific mock roots with WGA/PI, and visualisation with TCS-SP5 CLSM revealed that there were no fungal
structures present on these roots (Figure 14C). Some colonised pLYK1:GUS roots also displayed the heavy GUS
staining that covered a wide area of root tissue non-specifically (Figure 14B). WGA/PI staining of these roots
showed no recognisable fungal structures in these specific areas, but considerable root damage was evident
(Figure 14D). Root damage was also evident in the stained mock root areas (Figure 14C). The death of a batch
of pLYK1:GUS plants before they were due to be GUS stained and subsequent failure to germinate more seeds
Figure 13.
GUS and WGA-AlexaFluor488/PI stained sections of colonised pLYK1:GUS roots.
(A) GUS stained colonised crown root. GUS expression can be seen as delineated dark grey and blue patches, indicated by
black arrows. Scale 200μm.
(B) GUS stained colonised large lateral root that was attached to the crown root in A. GUS expression seen as dark grey
delineated patches, indicated by black arrows. Scale 200μm.
(C and D) TCS-SP5 confocal microscope image of root sections from A and B, stained with WGA-AlexaFluor488 and PI. White
arrows indicate arbuscules. Intraradical hyphae can be seen. PI stains cell walls reds. WGA-AlexaFluor488 stains fungal cell
walls green. C is from the same section of crown root as A. D is from the same section of large lateral root as B. These may
not be the same cells, however. Confirms the presence of arbuscules in the GUS-stained root regions. Scale 50μm.

23. 23
of that line rendered a comparison of pLYK1:GUS expression at different time points post inoculation
impossible.
Discussion
Consistent with the results obtained in [10] our results confirmed that GFP-AM42 is an effective marker of the
PAM throughout the lifecycle of the arbuscule. No GFP-specific labelling patterns were observed in GFP-AM42
plants that were not inoculated with R.irregularis or in wildtype Nipponbare plants that were inoculated and
colonised by R.irregularis. Previous research found no difference in GFP signal between GFP-AM42 coleoptiles
and wildtype Nipponbare coleoptiles [20] supporting the expression data showing that AM42 is induced
specifically during mycorrhiza colonisation. The MP-LS microscope enabled visualisation of labelled arbuscules
Figure 14.
(A) GUS stained mock pLYK1:GUS crown root showing damage and non-specific GUS staining. Scale 100μm.
(B) GUS stained colonised pLYK1:GUS crown root showing very dark and non-specific GUS staining. Scale 100μm.
(C) TCS-SP5 CLSM image of the same root visualised in A, stained with WGA and PI. Cell damage visible as intense red signal.
1μm slice. Scale 50μm.
(D) TCS-SP5 CLSM image of the same root visualised in B, stained with WGA and PI. Some fungal or contaminant debris and
cell damage visible. Scale 50μm.

24. 24
at a higher resolution and magnification than achieved by in [10] allowing additional insights into the labelling
patterns of GFP-AM42 at different stages of arbuscule development.
The appearance and loss of GFP-AM42 signal may be associated with the age of the membrane it is localised to.
GFP-AM42 was observed to localise to the PHM surrounding intraradical hyphae adjacent to arbuscules (Figure
7C,F). In cases where this labelling pattern was observed, the arbuscule adjacent to the labelled PHM was in
stage II, III or early stage IV, indicating that this labelling pattern was only observed in young arbuscules.
Consistent with this spatial-temporal dynamic, trunk domain of PAM labelled with GFP-AM42 was only
observed in stage II, III and early to mid-stage IV arbuscules (Figure 7,8). The trunk domain of PAM forms
before the branch domains (Figure 6), as the arbuscule branches grow from the trunk, which grows first into
the apoplastic cavity formed by the rearrangement of the root cell cytoplasm [17]. Time-lapse imaging
indicated that the trunk domain of an arbuscule loses GFP-AM42 signal before the branch domain (Figure 11).
This supports the observation that PAM trunk labelled with GFP-AM42 is observed in stage II to mid-stage IV
arbuscules, but is not observed in late stage IV arbuscules or stage IV arbuscules that are senescing. Time-lapse
imaging shows that branch domains of PAM are the last to lose GFP-AM42 signal (Figure 11). Thus it appears
that the differences in GFP-AM42 labelling of different PAM domains and PHM may be due to the relative ages
of the fungal structures contiguous to these membranes, with GFP-AM42 signal diminishing as the newly
formed membrane becomes older, implying a role in the initial development of the membrane. To further
investigate this dynamic, one could carry out line intensity plots along regions of continuous labelled PAM from
time-lapse images as the PAM ages. This finding supports and develops the theory of Kobae and Fujiwara who
hypothesised that AM42 is a general element involved in the organisation of the fungal symbiont rather than
specifically functioning in the development of PAM [10].
67 out of 68 visualised arbuscules displayed PAM branch domain labelled with GFP-AM42. In contrast, in only
12 of these arbuscules was GFP-AM42 labelled trunk domain observed and in only five arbuscules was labelling
of PAM observed adjacent to the PHM. In one instance, labelling of PHM and trunk domain was observed
without any branch domain labelled, suggesting that the arbuscule was stage II (early) and branch was yet to
develop. The relatively infrequent observation of PHM and trunk domain of PAM containing GFP-AM42
compared to branch domain containing GFP-AM42 highlights that only a minority of arbuscules observed were

25. 25
younger than mid-stage IV. Perhaps for future experiments, plants grown in Chelford 16/30 sand with
R.irregularis inoculum should be observed at 2wpi rather than 3-4wpi was carried out in our investigation, in
order to characterise more stage II and III arbuscules and further classify the spatial-temporal dynamic
observed here.
Given the proposed role of plant SCAMPS in endocytosis, exocytosis and cell plate formation [12],[13], it is
likely that the potential role of AM42 in the initial development of PHM and PAM is related to the trafficking of
endosomal or exocytic vesicles. Pumplin et al. hypothesised that the development of the distinct sub-domains
of PAM is due to polarisation of the bulk secretory pathway favouring vesicle fusion with the developing PAM
rather than the PM, coincident with expression from AM-specific promoters [8]. Given the upregulation of
AM42 in colonised root tissue, it may be the case that AM42 is involved in the reorganisation of secretion,
either by directing exocytic vesicles towards the PAM or endocytosis of PAM and recycling of membrane and
proteins. Our attempts to probe the role of AM42 failed to provide definitive answers, owing to technical
failure of the protocol we employed (Figure 12). Co-localisation of Fm4-64 dye with GFP-AM42 signal would
have indicated a role of AM42 in endocytosis, however, the stain failed to reach the inner cortical cells of the
root. A higher concentration of the stain or a longer incubation period may facilitate improved penetration of
Fm4-64 into the root tissue so that it can stain the endocytic pathway of inner cortical cells. Immunogold
labelling may reveal the sub-cellular localisation of AM42, allowing it to be determined whether the SCAMP
protein integrates into the PAM and/or if it labels vesicles tethered to the PAM, as is seen in EXO70I [18].
EXO70I was found to play an important role in correct delivery of PAM proteins, and is also involved in cellular
processes that SCAMPs are proposed to play a role in such as cell plate formation [19]. EXO70I was found to
localise to PAM branch domain adjacent to hyphal tips [18], as was PT11 [20], a different localisation pattern to
that of AM42. It may be that AM42 is involved in secretion of alternative PAM proteins to EXO70I, possibly
during early stages of arbuscule development.
Tos17-insertion knockout mutants of AM42 displayed wildtype phenotypes with regards to arbuscule number
and morphology [10]. Other phenotypes of the AM42 knockout mutants, such as plant nutrition status, were
not investigated [21] . Analysis of these phenotypes in the knockout mutant may assist identification of the
role of AM42. Given the effectiveness of MP-LSM at investigating fluorescent proteins in the PAM, crossing the

26. 26
AM42 knockout mutant line with an AM-specific fluorescent reporter line such as pPt11:PT11-GFP represents a
promising strategy to investigate AM42 function. Previous studies have shown distinct localisation of PT11 and
other AM-specific phosphate transporters to the branch domains of PAM [9],[7]. Disruption of this localisation
pattern in an AM42 mutant would indicate a role of AM42 in the reorganisation of secretion to the PAM.
The spatial-temporal localisation pattern of AM42-GFP is clearly different to that of fluorescent protein-tagged
AM-specific phosphate transporters, suggesting a difference in localisation in the PAM and PHM between
functional groups of AM-specific proteins. Our attempts to investigate the localisation pattern of LYK1, an AM-
specific putative LysM-domain containing receptor kinase, failed. This prevented a comparison of the
localisation patterns of a rice AM signalling protein (LYK1), an AM-specific nutrient transporter (PT11) and an
AM-specific protein potentially involved in PAM secretory dynamics (AM42). This comparison may provide
insight into the regulatory dynamics and redirection of protein transport to the PAM, during arbuscule
development as proposed by Pumplin et al. [8]. The failure of RNA extraction experiments prevented our
examination into the lack of RFP signal in colonised pLYK1:LYK1-RFP plants that genotyped positively for LYK1-
RFP. Given that no signal was observed in both lines, it may be the case that there was an error with the
construct. RT-PCR would identify whether the construct is being expressed. Given that LYK1 has been
identified in maize and rice proteomics as a protein specific to arbusculated cells [20] and that we failed to
identify why no RFP signal was observed, a LYK1 fluorescent-reporter fusion still represents an interesting tract
of investigation.
The pLYK1:GUS reporter line showed that LYK1 is being expressed in colonised roots (Figure 13) under the same
conditions and using the same promoter as the pLYK1:LYK1-RFP plants we investigated. Specific GUS activity
did not appear to be a common occurrence in the colonised plants, however, as it is induced only in colonised
cells. The presence of arbuscules in the root sectors displaying specific GUS activity was confirmed by staining
these sectors with WGA/PI. Damage to root cells or overstaining in both colonised and mock plants resulted in
GUS staining, distinguished from signal arising from GUS expression by non-specific staining of a large root area
or a very dark stain (Figure 14). Investigating the expression of LYK1 at different time points post-infection may
provide insight into the function of LYK1 in the AM symbiosis.

27. 27
We have demonstrated that in live tissue, fluorescent-reporter proteins such as GFP-AM42, imaged with MP-
LSM are an effective approach for investigating localisation of AM-specific proteins in the PAM and beyond.
These localisation patterns appear to reflect the function of the protein. Comparing the localisation of
different functional groups of AM-specific proteins may engender a wider understanding of the molecular
mechanisms underlying arbuscule development and function and how functionally distinct proteins are
delivered to the PAM during arbuscule development. This may facilitate an increased understanding of the
symbiosis in ecological contexts and enable its manipulation in agricultural frameworks.
Acknowledgements
I would like to thank Dr. Ronelle Roth and Denise Hartken for their help and supervision throughout the
project, and Anne Bates for her assistance with practical techniques. I would also like to thank Dr. Uta
Paszkowski and the M1 lab for being very welcoming and helpful throughout the project and Yoshihiro Kobae
for providing the pAM42:GFP-AM42 seeds.
Appendices
Appendix 1 - Primer Information
Transgenic Line Primer information Primer Manufacturer
pAM42:GFP-AM42
Homozygous
Originally O.Sativa cv Nipponbare
From Kobae lab (Japan)
GFP F1 : 5'-GTA AAC GGC CAC AAG TTC AG-3'
GFP R1 : 5'- GAA GAA GAT GGT GCG CTC CTG G-3'
Microsynth
HYG F1: 5'-GGT TAT CGG CAC TTT GCA TCG GCC-3'
HYG R1 : 5'-GAT TTG TGT ACG CCC GAC ACT CC-3'
IDT
pLYK1:LYK1-RFP
Line names: UP10.3 and UP10.6
Hemizygous
Originally O.Sativa cv Nipponbare
From Paszkowski lab
LYK1 F353: 5'-ATG GCG ATA TGG GTG ACA TT-3'
mRFP-151-R: 5'-CCC TTG GTC ACC TTC AGC TT-3'
IDT
Microsynth
HYG same as those for pAM42:GFP-AM42
pLYK1:GUS
Line name: UP11.4
Hemizygous
Originally O.Sativa cv Nipponbare
From Paszkowski lab
HYG same as those for pAM42:GFP-AM42

28. 28
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