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Regulation of Amino Acid and Sugar (AA/S) Transport in Plants by Coronatine

Regulation of amino acid and sugar (AA/S) transport in plants by Coronatine.
BACKGROUND: Plants are surrounded by pathogens constantly and throughout their lifespan. To combat pathogen attack, plants have evolved a sophisticated innate defense mechanism. Innate plant immunity consists of primarily two components – pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI confers basal resistance and is mediated by pattern recognition receptors such as toll-like receptors localized in the cell surface which bind to conserved foreign molecules evading the plant, also known as microbial/pathogen-associated molecular patterns (MAMPs). ETI relies on intracellular resistance (R) proteins to detect effector molecules which are secreted by pathogens to counteract the host plant’s first line of defense 1,2.
For defense against biotrophic pathogenic bacteria such as Pseudomonas syringae via PTI, the plant immune signaling molecule salicylic acid (SA) plays a key role. However, another plant hormone Jasmonic acid (JA) which tailors a defense response against necrotrophic bacteria is known to antagonize SA signaling. Some biotrophic bacteria such as P. syringae contain a phytotoxin coronatine which can mimic jasmonic acid isoleucine (JA-Ile) and activate JA signaling pathway by activating coronatine insensitive (COI1) protein-mediated degradation of JAZ proteins. Coronatine promotes multiple aspects of P. syringaevirulence, including reopening of the stomata, proliferation in the apoplast, and rapid development of disease symptoms3.
Nutrients are important for the growth and development of both plants and pathogens. Pathogenic bacteria infect host plants to acquire nutrients. The nutrients and favorable environmental conditions help the pathogens to grow and multiply, eventually causing serious disease. Pre-existing gas or water openings such as stomata and hydathodes are the entry point for them. Pathogenic bacteria that infect leaves colonize in the intercellular space (apoplast) of the photosynthetic cells present in the leaf which is rich in nutrients that can support bacterial growth such as free carbon and nitrogen 4. This extracellular space (apoplast) acts as the first battle field between plants and the pathogens. The concentration of free amino acids and sugars (AA/S) in the apoplast results from a balance between two antagonistic cross-membrane transport processes: namely secretion and uptake. The mechanism that regulates the concentration of AA/S during pathogenic bacteria infection is still unknown. Activation of PTI is characterized by the accumulation of salicylic acid and is associated with a decrease in concentration of AA/S in the apoplast. This inhibits the bacterial growth as the concentrations of AA/S in the apoplast dictates the growth of the bacterial pathogens and the robustness of the infection. My preliminary data suggests that in presence of a bacterial phytotoxin coronatine there is an increase in free AA/S concentration in plant apoplast. Based on these observations, I hypothesize that changes in AA/S concentration in apoplast after coronatine treatment is regulated by JA signaling pathway via regulation of AA/S transporters.
I propose to test my hypothesis by (a) determining if coronatine regulates apoplastic AA/S concentration via JA signaling pathway; (b) determining the relationship between AA/S concentration in apoplast, their transport activity and susceptibility to bacterial infection; (c) Identifying the transporter genes that execute increase of AA/S concentration in apoplast during P. syringae infection; (d) Investigating the role of SA/JA crosstalk in coronatine mediated AA/S transport in plants.
AIM I. Determining if coronatine regulates apoplastic AA/S transport via JA signaling pathway
It is known that coronatine hijacks plant’s native JA signaling pathway and helps to suppress plant immune response against P. syringae but the role of JA pathway in the regulation of nutrients in the plant during infection is still unknown. I hypothesize that coronatine regulates AA/S transport to manipulate the concentration of apoplastic AA/S via JA signaling pathway. To test this, I will measure the concentration of AA/S in the apoplast of JA biosynthesis and signaling mutants (myc2, coi1), which have altered resistance to bacterial infections. In addition, the relationship between apoplastic AA/S concentration and susceptibility to bacterial infections will be determined.
AIM II. Identify the transporters that execute the changes in AA/S concentration after the activation of JA signaling pathway
The concentration of AA/S in apoplast is influenced by nutrient transporter genes present on the plasma membrane. I will identify the AA/S transporters regulated by coronatine via activation of JA signaling pathway. I will perform global gene expression analysis to identify transporter genes. To identify the AA/S transporter genes regulated by JA, I will compare transcriptomic data of JA deficient mutant and wild-type plants after treating them with coronatine. I will generate loss-of-function and gain-of-function mutant lines of these candidate genes to test their function. I will measure the apoplastic AA/S concentration after treating them with coronatine and assess their susceptibility to bacterial infection. If I observe any alteration in the concentration of the amino acids in one of the mutants or find bacterial susceptibility to be modified, I will perform functional analysis such as transport kinetics and cellular imaging to locate these genes.
AIM III. Investigate the role of SA in coronatine mediated AA/S transport in plants
The crosstalk between SA and JA pathway in plant defense response has been well documented in previous studies. However, the role of SA in JA mediated AA/S transport hasn’t been studied. I will investigate the role of SA in coronatine mediated amino acid transport in plants. I hypothesize that coronatine regulates AA/S concentration in apoplast by suppressing SA level in the plants i.e. SA/JA crosstalk is required. I will measure the AA/S concentration in the liquid exudates of SA deficient mutant (sid-2, nahG) seedlings treated with coronatine. Then I will also measure the AA/S concentration in the liquid exudates of coronatine treated nac-3 mutant which is deficient in JA mediated SA suppression. This will provide new insights on how the SA/JA crosstalk is involved in coronatine mediated AA/S transport in plants.
Background and Significance
With the rapid growth in the global population, there is an increasing demand for food. Unfortunately, about 15 % crops produced are lost due to microbial infection making it crucial to understand plant-pathogen interactions5. Our study seems to suggest a novel pathway in plant defense during infection, through AA/S transport. An understanding of how plants and pathogens recognize each other and differentiate to establish the mechanism of nutrients transport during infection and its regulation can unravel the crucial aspects of defense responses in plants in general, for which Arabidopsis has long functioned as a model organism. Unraveling this mechanism could eventually lead to insights into improving crop quality, and methods to reduce crop loss due to bacterial infection.

  1. Overview of plant immunity

In nature, plants live in diverse and complex environments in which they constantly encounter a variety of insect herbivores and pathogens which have a wide array of lifestyles and infection strategies. Biotrophic pathogens absorb nutrients from the living host plants while necrotophic pathogens kill host cells to feed on the content. The first line of protection against pathogens is performed by physical barriers, the cuticle, and the cell wall6. Some of the pathogens enter intracellular space in leaves via preexisting openings such as stomata, hydathodes and they may ultimately reduce biomass, decrease fertility, or even kill the plant7. Plants have evolved two strategies to detect pathogens during infection. The first tier of the plant immune system is through pathogen perception via the recognition of conserved microbial elicitors such as bacterial flagellin or fungal chitin known as pathogen-associated molecular patterns (PAMPs) by receptor proteins called pattern recognition receptors (PRRs) localized in the plasma membrane8. Stimulation of the PRRs leads to downstream signaling and activation of the defense response called PAMP-triggered immunity (PTI). Some of the pathogens have developed the ability to suppress PTI and promote disease by secreting virulence effectors in the extracellular matrix or into the plant cells.  Plants that are not able to detect these effectors are susceptible to a pathogen, resulting in effector-triggered susceptibility (ETS)9. Pathogenic bacteria deliver effectors into host cells using type III secretion system (TTSS). These effector molecules can dampen plant immune response to allow successful pathogen colonization via suppressing PTI signaling and/or via manipulating the transcription of plant genes by binding in the promoters. The second tier of perception involves recognition of these effector molecules by intracellular receptors encoded by ‘disease-resistance’ (R) genes and this recognition induces effector-triggered immunity (ETI)1. Activation of the ETI response typically leads to a hypersensitive response (HR) which is characterized by rapid induction of host cell death in the area of initial infection, which also kills the pathogen and limits the spread of infection. PTI is a generally slower response and effective against non-adapted pathogens whereas ETI is a faster response which is active against adapted pathogens. Both PTI and ETI leads to several defense mechanisms, including stomatal closure, generation of reactive oxygen species (ROS), cell wall reinforcement, production and secretion of antimicrobials, changes in hormonal balances, transcription reprogramming10.

  1.  Nutrients transport in plants

1.2.1 AA/S transport across the plasma membrane
AA/S are synthesized in photosynthetic mesophyll cells of leaves and secreted in the apoplast. This AA/ S are then either transported to the sink tissues by vascular tissues or can be transported back to mesophyll cells. This movement of the amino acids and sugars within and between the cells requires membrane proteins functioning as exporters and importers. Two superfamilies of amino acid importers have been defined in plants: a. the amino acid, polyamine, and choline transporter superfamily (APC) and amino acid transporter family (ATF) superfamily. The APC family consists of CAT (cationic amino acid transporter) family and LAT (L-type amino acid transporter) family. The ATF superfamily is further divided into 6 subfamilies: AAP (Amino acid permease), ProT (proline transporter), LHT (Lysine histidine transporter), ANT1-like (aromatic and neutral amino acid transporter), AUX (auxin resistant) and GAT (c-aminobutyric acid transporter) 11. Only few membrane proteins that function as facilitators for amino acid export have been identified. UmamiTs (Usually Multiple Amino Acids Move In And Out Transporter) are newly identified amino acid exporters in Arabidopsis. The exporters UmamiT11, UmamiT14, UmamiT28, and UmamiT29 are identified to have a secretory function which is necessary for the embryo development in the seeds12.
Two major families of sugar transporters have been described: MFS (Major Facilitator Superfamily) and SWEETs (Sugar Will Eventually be Exported Transporters). MFS superfamily includes STPs (Sugar transporter Protein) and SUTs (Sucrose H+ Symporter). Both of them transport sugar into the cell via H+ symport mechanism13. The SWEETs are uniporter for glucose and sucrose. Pathogenic bacteria have evolved a mechanism to hijack SWEET transporters in Arabidopsis for nutritional gain14. Also functional mutations n nSTP1 and STP13 increase glucose concentration in the apoplast and susceptibility of Arabidopsis mutants to bacterial infections15. This indicates that these transporters play a vital role in plant resistance against the pathogen.
1.2.2 Long distance transport of AA/S
Plants can uptake N from soil via transporters present in roots (AAP1, AAP5, LHT1, LHT6, ProT2)16 but that amount is not enough. Thus, transport of AA/S from source to sink is one of the major determinants of plant growth. Long distance transport of AA/S occurs in the phloem sieve elements of the vascular system. The process of nutrient uptake by phloem in mature tissue is called phloem loading. This phloem loading can occur through plasmodesmata and cell cytosols (symplastic) and/or across plasma membranes via intervening walls (apoplastic). SWEETs function as sucrose exporter in mesophyll cells while SUC2 imports them to companion cells from apoplast17. After phloem loading, the transport direction goes along with concentration gradient in the sink tissues. At the sink tissues, phloem unloading takes place via transporters (e.g. SWEET1, SWEET12, UmamiT11, UmamiT14). Other transporters (e.g. AAP1, AAP8, CAT6, CAT9, SUC2) will uptake AA/S from apoplast to support growth and development of sink tissue12,18.

  1. Overview of the Jasmonic acid signaling pathway

The plant hormone jasmonate (JA) is a fatty acid derived hormone which regulates different aspects of plant growth, development, and immunity. Along with its derivatives, JA plays a critical role as signaling molecules to activate defense responses against herbivores and necrotrophic pathogen 19. JA molecules also act as a regulatory molecule in the developmental process of plants including sex determination, fertility, root elongation and fruit ripening 20.

  1.      JA signaling in plant development

JA has a central role in plant development and reproduction. Application of 0.1µM Methyl jasmonate (JA derivative) reduced 50% root length in wildtype (WT) Arabidopsis thaliana while mutants deficient in JA signaling pathway (jar1) didn’t show such response 21. Repeated wounding led to increased production of JA that suppresses the growth of Arabidopsis by inhibiting mitosis in young leaves and meristems 21Arabidopsis mutants deficient in JA synthesis (fad1,dad1) and perception (coi1) are male sterile due to arrested stamen development during anthesis 22. External JA treatment can restore stamen development in JA biosynthesis-deficient mutants but not in JA signaling deficient mutants 23.

  1.      JA signaling in plant immunity

Wounding of plant tissues by insect feeding causes the rapid synthesis of active JA molecule via an octadecanoid pathway which induces defense response pathway, defense gene regulation and defense-related volatile and secondary metabolites which can repel herbivores24,25. JA signaling is required for induced immunity in rice to root-knot nematode26 and biotrophic parasite Xanthomonas oryzae27. Tomato and Arabidopsis mutant (jai-1) lacking JA-Ile receptor is susceptible to root rot disease caused by soil fungus Pythium irregulare28,29.  Arabidopsis plants lacking COI1 gene are more susceptible to necrotroph pathogen such as Botrytis cinerea30 whereas same plants are more resistant to biotroph Pseudomonas syringae DC 3000 tomato p.v.31.

  1.      Effects of JA signaling in nutrients transport

JA influences carbon and nitrogen transport in plants via an unknown mechanism that is not fully understood. Jasmonate application resulted in more photosynthetic product export from local and systemic leaves to root and shoot in Populus tremuloides32. Application of Methyl Jasmonate resulted in a decrease in soluble sugars and increased amino acid pool in tobacco leaves33. This decrease could either be the result of increased use of sugars for induced plant defenses. This increased transport of nutrients to roots and stems may reduce nutrients resources form the predators and lead to greater tolerance against herbivores.
1.4 Coronatine
Coronatine (COR) is a non-host specific phytotoxin molecule produced by several strains of P. syringae34. The COR is a polyketide molecule consisting of two components, coronafacic acid (CFA) and coronamic acid (CMA) joined by an amide bond between the acid group of CFA and the amino group of CMA35. CMA is derived from L-alloisoleucine whereas CFA is synthesized from a cyclopentenone compound.
1.4.1 COR activates JA-signaling pathway by mimicking JA-Ile
JA (+)-7-iso-JA-Ile (JA-Ile) is a very important component of JA signaling pathway. When the levels of JA-Ile are low, Jasmonate ZIM-domain (JAZ) repressor proteins are stable. JAZ repressor proteins recruit general co-repressor TOPLESS (TPL), usually via adaptor protein Novel Interactor of JAZ (NINJA) and interact with transcription factor MYC236,37. This interaction prevents the activation of JA signaling pathway. When the levels of JA-Ile is high, it binds with CORONATINE INSENSITIVE 1 (COI1) protein, an F-box protein which recruits skp1-Cul1-F-box protein (SCR) ubiquitin E3 ligase complex38,39. This process leads to poly-ubiquitylation and subsequent degradation of JAZ by the 26s proteasome. JAZ degradation relieves repression of MYC2 and permits the expression of JA responsive genes40. COR mimics the most active isoleucine conjugate of JA-Ile both structurally and functionally. The cyclopentanone ring of COR is a stereoisomer of the JA-Ile. It has been shown that coronatine can activate JA signaling pathway in the plants41.
1.4.2 COR suppresses SA-signaling pathway
COR suppresses SA through MYC2 dependent activation of three NAC (petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) transcription factors (ANAC019, ANAC055, ANAC072)  via JA signaling pathway. These transcription factors decrease the level SA by repressing the expression of isochorismate synthase, a gene required for SA biosynthesis and by inducing the expression of benzoic/salicylic acid carboxyl methyltransferase which converts SA to inactive, volatile methyl SA42. Thus, COR can increase the virulence of biotrophic pathogens by hijacking JA-signaling pathway and suppresses SA mediated defense by exploiting JA/SA crosstalk.
Preliminary studies
Coronatine treatment leads to an increased amino acid concentration in the seedlings exudates and transcriptional changes of AA/S transporter genes
I measured the amino acid concentration in exudates of WT Arabidopsis grown in liquid MS culture after mock/coronatine treatment. Exudates collected after 24 hours of coronatine treatment showed increased amino acid concentration as compared to mock treatment (Fig 1).  This data demonstrates that coronatine increases the amino acid concentration of apoplastic AA/S. I also measured the effect of coronatine in the uptake of amino acids. I prepared protoplasts (cells without cell-wall) from leaves of WT plants and incubate them in media containing cold Proline spaced with 14C labeled Proline. As plant cells tend to accumulate AA/S, there will be an increasing level of 14C inside cells that is detected in a Liquid Scintillation Counter as counts per minute (CPM). After 1, 3, 5 hours of incubation, protoplasts are sampled, washed and lysed for counting. I found that coronatine treated protoplasts were compromised in amino acid uptake as compared to WT. To understand how this happens, I examined global gene expression data (; Danna Lab, unpublished) of coronatine treated leaves, and found that there are 22 AA transporter genes and 3 sugar transporter genes that respond to the coronatine (Table). They are the candidate genes that play role in the changes in AA/S concentration in the apoplast.
COI1 is required for the coronatine mediated amino acid transport in the apoplast
For the activation of JA signaling pathway, binding of coronatine to COI1 is essential. The coi1 mutant Arabidopsis is fully insensitive to jasmonates and coronatine. I measured amino acid concentration in exudates of a coi1 mutant of Arabidopsis grown in liquid MS media treated with mock/coronatine treatment for 24 hours. I observed that there is no change in amino acid concentration in coi1 mutant treated with mock and coronatine (Fig2A). This suggests that in absence of COI1 there will be no binding site for coronatine and plants do not respond to it.  Thus, JA signaling pathway is required for coronatine mediated amino acid transport.
Mutation of ProT3 reduces coronatine mediated susceptibility
ProT3 is one of the AA transporter genes that is up-regulated by coronatine. I tested bacterial infection in a ProT3 loss-of-function mutant to determine if the loss of AA export function of this gene would affect the ability of the mutant to suppress coronatine mediated bacterial growth. I found that ProT3 mutant is resistant to bacterial infection than WT plants (Fig3(A)). The prot3 expression is restricted to epidermal cells of the leaves and mediates proline and glycine betaine transport across the plasma membrane in plants43. Proline antagonizes plant defenses by interfering with the γ-aminobutyrate-mediated degradation of bacterial quorum-sensing signals that normally increase pathogen spread44. I hypothesize that failure to accumulate proline in ProT3 mutant caused increased resistance to bacterial infections.
Mutation of AAP6 increases coronatine mediated susceptibility
AAP6 is one of the AA transporter genes that are down-regulated by coronatine. I found that AAP6 loss-in-function mutant is more susceptible to bacterial infection than WT plants (Fig3(B)). AAP6 is mainly expressed in roots, leaves, and xylem parenchyma. AAP6 is known to involved in xylem-phloem transfer for long-distance transport of amino acids in Arabidopsis45. I hypothesize that in AAP6 mutant there is reduced phloem loading of amino acids, leading to an increased amino acid concentration in the apoplast. This increased amino acid supports the growth of bacteria during infection. Thus, there is a correlation between amino acid concentration in the apoplast and bacterial susceptibility.
SA is required for coronatine mediated amino acid transport
To determine the role of SA in coronatine mediated AA transport in plants, I tested the SA signaling deficient mutant sid2. This sid2 mutant fails to accumulate salicylic acid. I observed that there is no change in amino acid concentration in exudates collected from sid2 mutant treated with mock and coronatine. This suggests that SA is required for coronatine mediated AA transport in plants or SA/JA crosstalk is required for this process.
Research Designs and Methods
In this research, I will use bioinformatics, plant physiology, genetics and molecular biology to determine how AA/s transport contributes to coronatine mediated susceptibility. I will start with examining AA/S transport in JA signaling deficient mutant to determine the role of JA signaling pathway in coronatine mediated nutrients transport. I will screen transporter genes that participate in the coronatine mediated transport via transcriptional analysis. I will also investigate the role of such transporter gene in plant defense. The candidate genes will be further studied using molecular and cellular biology method and genetics.
AIM I. Determining if coronatine regulates apoplastic AA/S transport via JA signaling pathway

  1. Determine the amino acid concentration of AA and sugars in seedlings exudates and apoplast of leaves elicited with coronatine

For AA/S quantification in exudates, I will use 10-day old seedlings grown in liquid MS culture. 10-day old seedlings will be elicited with mock and coronatine treatment separately and exudates will be collected after 24 hours. For adult leaves, 6-weeks old plants leaves will be infiltrated with buffer containing 1M sorbitol, 40mg/ml of the Lucifer Yellow fluorescent dye (LYCH). This fluorescent dye does not permeate the PM and allows for the correction of the dilution of the solutes that extracted from the apoplast. Infiltrated leaves will be cut and centrifuged at 700g for 10 minutes to obtain the apoplastic wash fluid(AWF). The concentration of AA and glucose in these samples will be assessed by using kits from BioVision. If there is no difference in apoplastic AA/S concentration between mock and coronatine treated seedlings, that would indicate that regulation of the concentration of AA/S in the apoplast is not mediated by coronatine.

  1. Determine if coronatine regulates AA/S concentration via JA signaling pathway

It has been established that coronatine regulates most of its activity via a COI1-MYC pathway. To understand the role of JA signaling pathway in coronatine mediated amino acid transport, I will generate mutants that are defective in JA signaling pathway (coi1, myc2, med25). After that, I will perform AA/S quantification as explained in aim 1.1. Also, I will check the expression of JA signaling marker genes (PDF1.2) in the coronatine treated seedlings/leaves. If there is a significant difference in apoplastic AA/S concentration between mock and coronatine treated seedlings, that would indicate that coronatine doesn’t regulate AA/S in apoplast via JA signaling pathway.
AIM II. Identify the transporters that execute the changes in AA/S concentration after the activation of JA signaling pathway
2.1 Identify AA/S transporter genes that are differentially expressed when elicited with coronatine
The changes in AA/S concentration in the apoplast of leaves elicited with coronatine must be linked to changes in the activity of the AA/S transporters. Transporter genes that respond to coronatine are regulated via JA signaling pathway. Candidate genes that respond to coronatine are listed in Table. To confirm the candidate genes that are activated by coronatine in JA signaling pathway-dependent manner, I will profile the transcription of those genes in JA pathway deficient lines (coi1, myc2, andnac-3). For that, I will treat 10 days old WT and mutant seedlings with coronatine or water, and extract RNA from them after 3 hours. RNA samples will be analyzed using Nanostring hybridization methods (nCounter Analysis, Nanostring Inc) to check gene expression. Then I will analyze the fold change between coronatine and mock treated in two genotypes. I will compare the list and pick up the candidate genes that are induced by coronatine in JA signaling manner.
2.2 Generate loss-of-function and gain-of-function mutant plant of candidate genes
Loss-of-function and gain-of-function is a great tool to study the role of genes in specific processes. For the genes that are upregulated, the loss-of-function mutant will be particularly informative. The bacterial pathogen Agrobacterium tumefaciens is used to obtain mutant Arabidopsis plants. The random insertion of a bacterial DNA, Ti plasmid (T-DNA), in a plant coding region of gene eliminates gene function. Arabidopsis mutant lines for the selected candidate genes can be ordered from TAIR seeds stock repository ( T-DNA insertions in the lines will be mapped via PCR to confirm genome location. Also, qRT-PCR will be performed to confirm homozygous plants that no longer have wild-type gene. Other strategies will be used to generate mutant lines. The first strategy includes the use of CRISPR-Cas9 to a target gene of interest and any potential gene that may contribute to a redundant function46. The second strategy includes the production of transgenic lines that express micro RNAs to knock down the candidate gene47.
To study genes that are downregulated, gain-of-function will be used. To generate gain-of-function lines, I will use two strategies. First, I will clone coding sequence (CDS) of a candidate gene in an expression vector under the control of Cauliflower Mosaic Virus 35S sequence.  Secondly, I will clone CDS of candidate gene under the control of Dexamethasone inducible promoter. As overexpression of some genes may lead to inhibition of plant growth, the second strategy will be more advantageous. Constructs will be confirmed via sequencing and then transformed to plant genome via Agrobacterium-mediated transformation of flower buds.  Over-expression line is expected to have opposite phenotype than loss-of-function if it showed any.
2.3 Test infection phenotype in of loss- and gain-of-function mutants
To test the role of these genes in coronatine mediated susceptibility in plants, I will do infection assay of mutant plants. Mutants that show the statistically significant difference in bacterial titer as compared to WT when infected with Pseudomonas syringae ES4326 will be prioritized for the functional analysis described in 2.5. If knocking down any single gene is not enough to observe susceptibility/resistance I will generate double or triple mutants by crossing single mutants and test their phenotype. According to my preliminary data.
2.4 Measure AA/S concentration in apoplast and exudates of transporter mutants
I will examine if there is any correlation between AA/S concentration in the apoplast/exudates and bacterial susceptibility mediated by coronatine from 2.3. This will determine if there is any role between nutrients availability in apoplast and coronatine mediated plant susceptibility in transporter mutants. For this, I will use protocol same as aim 1.1.
2.5 Functional analysis of candidate AA/S transporters
Functional analysis will characterize the function of the candidate genes in AA/S transport and how they regulate AA/S transport when treated with coronatine.
2.5.1 Characterize the transport activity and kinetics of the candidate genes in the heterologous system
I will test the uptake kinetics of the candidate transporter genes in both Arabidopsis seedlings and protoplasts. However, in these experiments, there will be endogenous uptake and secretion by another transporter present in the plant cells. A complementary method to study uptake is to express candidate genes in the yeast defective for uptake of AA/S (strain 22∆8AA deficient in AA uptake and EBY4000 deficient in sugar uptake) or Xenopus oocytes. Also, I will use media of different pH and CCCP to test if the transport mechanism is dependent on H+ gradient. I will also test secretion kinetics with Xenopus oocytes transformed with constructs expression transporter genes. Radiolabeled AA or sugar will be co-injected into oocytes with the mRNA of transporter genes. The amount of radioactivity retained in the cell over time can determine the Vmax and K0.5 value of transporter in transporting solute. It has been observed that AA/S transporters selectively transport some types of AA or sugar. For AA transporters, I will test the kinetics of them transporting all AA found in plants. For sugar transporters, kinetics of transporting glucose, lactose, fructose, and sucrose will be determined. These studies will characterize the working mechanism and substrate selectivity of the transporters.
2.5.2 Define subcellular localization of the transporters
To understand the contribution of the transport function of the transporter genes it is necessary to determine the subcellular localization. To determine the subcellular localization of candidate transporter proteins, I will transform WT protoplasts to overexpress the candidate transporter proteins fused with a fluorescent protein (GFP/mCherry) reporter to visualize the subcellular localization of protein by confocal microscopy. If the protein is an AA/S transporter, it should localize at the plasma membrane or at the tonoplast (the vacuolar membrane). PM marker m-rk CD3-1007 or pm-rb CD3-1008 (available on, which encode the PM protein aquaporin AtPIP2A fused to mCherry will be also transfected in protoplast as a control. Co-localization of both markers will confirm that the transporter is localized at the plasma membrane. To test if transporters are expressed on the tonoplast, the same strategy will be used by co-expressing the tonoplast integral protein-1 vac-rb CD3-976 with a fluorescent protein.
2.5.3 Determine tissue-specific expression of transporters
The tissue-specific expression patterns of the transporter gene are informative for predicting its physiological function in plants. I will use GUS (β-glucuronidase)-reporter system to determine it. The GUS reporter gene will be expressed in the plants under the control of the promoter of candidate transporter genes. When the substrate X-Gluc is provided, GUS expressed in the plants will cleave it and produce a blue precipitate in the tissue where GUS is expressed. To test the expression pattern of the candidate genes during coronatine elicitation, I will culture WT seedlings in liquid MS media and elicit them with coronatine. I will collect samples at different time points and examine the expression pattern. This will provide spatial-temporal information of expression of the candidate genes during elicitation.
AIM III. Investigate the role of SA in coronatine mediated AA/S transport in plants
To investigate the role of SA/JA crosstalk in coronatine mediated AA/S transport I will measure AA/S concentration in liquid exudates and apoplast of SA deficient mutants (sid2, nahG). Then I will also measure the AA/S concentration in the liquid exudates of coronatine treated nac-3 mutant which is deficient in JA mediated SA suppression.
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