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Transgenes to combat the infection of myrtle rust on Eucalyptus
1. Background
Myrtle rust (Austropuccinia psidii) is a is an invasive pathogenic fungus that targets the family
Mytaceae. Myrtle rust initially was found in Brazil and has become well established in South
America; it is known to infect guava, allspice and eucalyptus (Roux et al., 2013) but has been
described to affect over 125 species of Myrtaceae. The family of Myrtaceae are known for having
fruit, flowers, and particularly good wood for commercial uses; they also have a wide distribution in
tropical and temperate regions. Eucalyptus in particular is used to make cellulose and paper, grown
in large plantations of cloned trees. Due to the Myrtaceae family being of such widely used
commercially grown trees, there is a major risk to pathogenic fungi causes mass mortality
worldwide.
The infection of myrtle rust targets young, actively growing leaves and shoots, even on fruits; once
the spores encounter the host leaf, the spore will germinate, and infection will start. Infection of rust
fungus on young trees can kill shoot tips and severely impact the fruit production, often leading to
loss of fruit and tree mortality (Carnegie et al., 2016). The life cycle of myrtle rust is autoecious, it
only needs one host to complete all stages of the cycle. Once the spore has attached to the surface
of the host it will germinate, then from entry into the stoma the fungi can colonize the plant cells
and gain nutrients and lastly will reproduce and produce lesions on the leaves that can be used to
reinfect other parts of the host plant (Chock, 2020).
Tree mortality in plantations poses a major threat as most plantations, especially in Eucalyptus are
all genetically identical plants; if there is exposure to the fungus then the risk of infecting the whole
plantation is greatly increased especially considering more than 20 million hectares of Eucalyptus is
planted globally.
Most of the work that has been done on myrtle rust has involved the use of fungicides to combat the
reinfection of spores. In Australia the eucalyptus trees are a major species in their ecosystems and
loss in their forests would cause devastating impacts on their ecology. One study looked at two
species R. rubescens and R. psidioides, these species are highly susceptible to the fungus and show
fruit infection. Using a fungicide, they observed the infection rates on mature and immature leaves,
shoots and fruit. It was found that the treated trees had larger leaves than the untreated trees and
they retained their immature leaves and had decreased crown transparency (Carnegie et al., 2016).
This is encouraging work for a simple and effective way to possibly treat the fungus and stop any
reinfection from occurring.
More work done has found that some trees show a natural resistance to the fungus and have a
hyper-sensitive response (HR) that slowed and often stops the fungus from infecting the tree. The
HR is a useful mechanism used by plants to stop the spread of infections to microbial or fungal
pathogens; it involves rapid cell death around infected lesions that cut of the supply of nutrients and
restricts the growth and spread of the infection to other parts of the plant (Balint‐Kurti, 2019). Gene
expression studies have been used to find the resistance genes; the genes identified in resistance
were transcription factors, receptor-like kinases and enzymes used in secondary metabolite
pathways (Tobias et al., 2018).
Eucalyptus has a resistant gene Ppr1 that is associated with the HR to myrtle rust; it is located on
chromosome 3 and produces the protein NBS-LRR. Studies have found that the NBS-LRR protein can
hold up to 40 resistance genes (Tobias et al., 2016). Similar to the Ppr1 genes, Wrky is a
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transcription factor that positively regulates resistance genes; found in Arabidopsis, they have been
found to provide resistance to fungi (Shree P. Pandey and Imre E. Somssich, 2009). Another gene
that works for resistance is LR67 found in wheat; it codes for a sugar transporting protein (STP) that
produces resistance to rust and powdery mildew in wheat and is triggered by disease signaling or
reduced nutrition availability for rusts (Milne et al,. 2019).
For another angle on combating myrtle rust, I will look at the composition of the wax cuticle on the
leaves and determine if there is a possible way to stop the germination and infection from starting.
FATB is a gene that codes for the protein palmitoyl-acyl carrier protein thioesterase, this gene is
used in synthesizing palmitate which is expressed in the leaves cuticle and has been found as an
activator for the spores to germinate, among other chemicals (Batista dos Santos et al., 2019).
Usually this chemical is used in the leaves to form a spiky surface to deter insects from eating the
leaves. Using a knockout of the FATB will stop the germination of spores.
2. Proposal
Myrtle rust is a fungus that targets its infection site to the leaves of the host tree; naturally occurring
resistance has been shown in some trees of the Eucalyptus that have been associated with the HR.
Using the plants natural resistance mechanisms, 3 genes have been identified to trigger defense
responses against infection and one gene has been identified in the wax cuticle. These genes will be
used to look at possible way’s myrtle could be combatted.
Leaf specific promoter used for gene constructs
For the chosen leaf specific promoter C1, it has been found in cotton leaf multan virus (CLCuMV). It
was found to be 3-5-fold higher in expressing in the leaves than CaMV 35S promoter (Xie et al.,
2003). This should target the expression of the hypersensitive response to the leaves and stop the
primary infection of the fungus by cell necrosis before it is able to reinfect other areas of the host. If
the C1 promoter doesn’t efficiently express the gene in the leaf, another leaf specific promoter
would be substituted or 35S can also be used for general expression.
Resistance through gene expression of a TIR1-NBS-LRR resistance gene Ppr1 (Puccinia psidii
resistance gene 1)
Ppr1 is associated with the HR in plants; it is found on chromosome 3 and produces NBS-LRR protein
which are recognition proteins that can hold up to 40 resistance genes that coordinate the HR. The
way in which Ppr1 and HR work together, is through the natural resistance method of cell necrosis.
When plants sense a possible threat, the best way to stop the spread of infection is to cut off the
supply to the threat and they do this by selectively choosing cell death of the infected cells before
there is time for the fungus to start germinating and spread to other parts of the plant (Junghans et
al., 2003).
The aim of using Ppr1 is to introduce a mechanism that is already known to cause natural resistance
in the Eucalypts against rust fungus but is not found in all the species and plants. An advantage of
the Ppr1 gene, is that it is not specific to rust fungus but is most likely evolved from a similar
pathogenic fungus that is native to Australia (Tobias et al., 2016); this could mean possible resistance
to other infectious fungus.
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Increasing rust resistance through overexpression of WRKY transcription factors from Arabidopsis
The WRKY transcription factor positively regulates the expression of genes that express resistance
genes. This has been shown in Arabidopsis natriuretic peptide receptor 1 (NPR1), AtWRKY52
contains a TIR-NBS-LRR domain which acts with RPS4 to provide immunity to fungi diseases. The
WRKY gene will act in defense responses to myrtle rust, using positive and negative regulators. In
Arabidopsis genes AtWRKY33 and AtWRKY70, both positively modulate systemic acquired resistance
(Shree P. Pandey and Imre E. Somssich, 2009).
In normal stress responses plants activate three signaling pathways; salicylic acid, jasmonic acid and
ethylene (Jiang et al., 2016). WRKY transcription factors have a known role in regulating these
pathways in defense responses. The purpose of using WRKY is to add a regulator into Eucalyptus to
activate the signaling pathways when there is an infection. When WRKY genes are overexpressed
you often get plants that are resistant to certain pathogens and fungi. In rice blast, the
overexpression of WRKY22 showed increased resistance to the fungi and showed it was a positive
regulator (Shree P. Pandey and Imre E. Somssich, 2009). The overexpression of WRKY throughout
the plant should regulate resistance to pathogens and fungus.
Using broad spectrum rust resistance from the Lr67 gene coupled with NLR for enhanced resistance
The Lr67 gene codes for a sugar transporting protein (STP); it is present mostly in adult plant
development and gives partial resistance or slows pathogen growth but is not associated to the HR
response (Milne et al,. 2019). The resistance of Lr67 has been shown to be broad resistance to rust
species (Spielmeyer et al., 2013). This resistance gene by itself will only confer partial pathogen
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resistance but could be more potent if coupled with NLR, giving it a more durable resistance. Lr67
belongs to the STP13 clade; this clade functions as a high affinity hexose-proton symporter but has
been found in many species to provide defense responses especially to infections. It is the change
from Lr67 susceptible to Lr67 resistant that renders it uncappable of being a hexose transporter and
enables it to work for resistance defense. It has been shown in barely that Lr67 progeny with high
expression showed no pustule formation, showing good resistance to infection (Milne et al,. 2019).
How this change may provide the resistance is not exactly known but it is possible that altered
carbon partitioning by Lr67res that causes nutrition to be limited for the fungal haustoria and that
cuts off the fungus from being able to grow and reproduce. Alternatively, it could be the reduction in
hexose that leads to a changed hexose/suc ratio that triggers sugar signaling that is similar enough
to pathogen invasion activity and then that causes a defense response.
Knockout of FATB gene to alter the composition of the leaf cuticle as seen in Arabidopsis
FATB is the only gene that is naturally occurring in the plant leaves. It codes for the protein that
synthesizes Palmitate which has been shown in other studies in myrtle rust invasion. FATB knockout
plants show reduction in the wax of the leaves by up to 20% and 50% in the stems, showing it’s an
important synthesizer of saturated fatty acids in wax biogenesis (Bonaventure et al., 2003). The
uredospore’s from the rust interact with the leaf cuticle and the chemical signal from the leaves
trigger the germination of the spore and infection of the host (Batista dos Santos et al., 2019).
This is the only gene that will need to be knocked out in the plant to get the desired phenotype. This
will be done with gene editing to silence the FATB gene and reduce the production of palmitate in
the leaves. In doing this, the transgenic plants should have an altered leaf wax cuticle that will keep
any spores present in an inactive state, therefore stopping the infection.
One issue that has been seen in Arabidopsis is severely reduced growth as a result of an increase in
the FATB pathway and the double mutation showed a complete elimination of fatty acids that led to
the loss of photosynthetic ability (Lightner et al., 1994). In the study of FATB reduction, the plants
have shown slow growth and deformed seeds with low viability, with only 40-50% less saturated
fatty acid content (Bonaventure et al., 2003).
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Transforming the genes into the host plant
To make the transgenic plants, Agrobacterium will be used to insert the gene constructs into the
DNA of the Eucalyptus. Agrobacterium in nature can integrate its own DNA into a host plant; it uses
its TDNA which contains a small circular DNA sequence that will move through the plant cytoplasm
and into the nucleus. In the nucleus the Ti plasmid integrates into the host DNA and the genes that
have been introduced can be expressed by the host. The method for transfer of Agrobacterium is
quite simple; leaves from the Eucalyptus will be damaged by small cuts and then introduced to the
Agrobacterium for 10-15 minutes. The leaves will then be removed and hormones can be introduced
to speed up the growth process so the leaves will differentiate to eventually form whole plants and
then the genes of interest can be tested (Deblaere et al., 1985).
For Ppr1 and WRKY the mechanisms involved are quite similar so they can be introduced into the
plant via the same method and this is by Agrobacterium transformation. I have chosen to use a leaf
specific promoter as I want the expression of the resistance to be specific in the leaves as that is the
first point of fungal contact. When they are transposed into the plant genome, they should be
constantly expressed and in high numbers so when there is an infection it is ready to work.
Lr67 can use the same method of Agrobacterium but the promoter region may need to be more
specific to the Eucalyptus as it involves sugar transport proteins and the overexpression of this might
decrease the plant health. Using CA35S would not be wise as it needs to be very specific in the
region it is being expressed, C1 leaf specific promoter could be more suitable.
FATB needs to be edited via CRISPR as the gene is knocked out and not overexpressed. The gene will
need to be analyzed to find the active sites of the gene to target. Once the active sites have been
identified and are suitable for the PAM site for Cas9, it will be transformed into the plant via
Agrobacterium using a leaf specific promoter and 35S for the kanamycin. U6 will be used for its
ability as an RNA polymerase III promoter that it uses to drive small hairpin RNA in vector-based
RNAi (Nie et al., 2010). This will disrupt the normal expression of the gene and knockout the
production of FATB in the plant.
To assess the localization of the genes in the plants, fluorescence in situ hybridization will be used.
The progeny will be grown on the kanamycin resistant agar so the leaves that have the target genes
can be identified. Using wild type plants for comparison, the plants will be inoculated with the
pathogenic rust and observations of lesions and necrosis will be evaluated using a scale of severity.
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3. Risk assessment
The risk for using the knockout FATB gene is the potential for dwarfism phenotype, this could
majorly impact the paper industry. Eucalypts are the most widely planted hardwood plants in the
world, they are used for cellulose and paper due to its rapid growth and wood quality with high
yields from each tree. Plantations worldwide depend on the Eucalypts to be high yield and if the
reduction of FATB were to cause dwarf trees, this would lower the production and yield.
FATB KO plants showed defective seeds; the size compared to WT seeds were on average half the
diameter, while the bolting time was also delayed. The mutant plants had delayed elongation of the
stems and overall weight of the plants was reduced in comparison to WT. However, there wasn’t any
evidence to show a deficit in photosynthesis capability in the KO plants that contributed to the slow
growth rate (Bonaventure et al., 2003). This rules out a major impact of the structure of the wax
cuticle but more work would have to be done to see if the plants are more susceptible to insects or if
there is any loss of moisture and nutrients from the leaves that are going to impact the plants long
term.
The tradeoff between the low yield and possible leaf effects would probably not make this a very
desirable solution for the myrtle rust and depending on what exactly occurs to the phenotypes. Any
of the other genes would be able to provide possible protection without changing the phenotype
like FATB.
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