阿拉伯芥中LecRK-V.2和LecRK-VII.1蛋白質在處理細菌鞭毛蛋白後與辨識鞭毛蛋白受體FLS2和其共同受體BAK1的蛋白質複合體形成之分析

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國立臺灣大學生命科學學院植物科學研究所碩士論文

Institute of Plant Biology College of Life Science National Taiwan University

Master Thesis

阿拉伯芥中 LecRK-V.2 和 LecRK-VII.1 蛋白質在處理細菌鞭毛蛋白後與辨識鞭毛蛋白受體 FLS2 和其共同

受體 BAK1 的蛋白質複合體形成之分析 Analysis of LecRK-V.2 and LecRK-VII.1 complex formation with FLS2 and BAK1 upon flagellin perception

in Arabidopsis 麥賢偉 Hsien Wei Mai

指導教授：金洛仁博士 Advisor: Laurent Zimmerli, Ph.D.

中華民國 105 年 7 月

July, 2016

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摘要

在阿拉伯芥植物中有許多 L 型態凝集素受體激酶(LecRKs)參與病原菌辨識機制中引起細胞內對抗生物性跟非生物性的逆境防禦機制。此種受體激酶有細胞外的豆科植物凝集素區域，穿膜區域和細胞內的絲氨酸/蘇氨酸激酶區域。在我們實驗室先前研究發現 LecRK-V.2 和 LecRK-VII.1 都參與在病原菌防禦機制當中。此兩基因突變株比起野生

型 Col-0 阿拉伯芥對抗 Pseudomonas syringae pv. tomato DC3000 (Pst

DC3000)此病的能力來得弱；然而，此兩基因過量表現轉殖株則相較

野生型對抗 Pst DC3000 的抗性更強，並且我們發現此兩基因突變株在

氣孔的免疫機制上有明顯的缺陷。在此，我們利用阿拉伯芥原生質體

過量表現此兩基因蛋白質再利用共免疫沉澱法去偵測兩蛋白質對於鞭

毛蛋白辨識受體 FLS2 和其複合體蛋白質 BAK1 是否有與此免疫複合

體產生結合的情形。我們也在酵母菌雙雜合系統實驗發現 LecRK-V.2

和 LecRK-VII.1 蛋白質可以透過內部激酶區域與 FLS2 及 BAK1 蛋白

質內部激酶區域產生結合的現象。為了更加瞭解此兩基因參與的植物

抗病菌的防禦機制調控，在此研究，我們發現此兩基因參與在細菌鞭

毛蛋白引起植物體免疫防禦機制的 FLS2-BAK1 複合體當中。

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關鍵字：

阿拉伯芥、LecRK-V.2、LecRK-VII.1 、病原菌辨識受體、防禦

機制、FLS2、BAK1

。

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Abstract

In Arabidopsis, several L-type lectin receptor kinases (LecRKs) were found to

function as or together with pattern-recognition receptors (PRRs) in recognition of

stress signals and initiation of plant defense responses towards abiotic and biotic

stresses. LecRKs possess an extracellular legume-lectin domain, a transmembrane

domain and an intracellular serine/threonine kinase domain. Our previous analyses

show that LecRK-V.2 and LecRK-VII.1 are involved in the pattern-triggered immunity

(PTI) defense response. Both lecrk-V.2 and lecrk-VII.1 mutant plants are indeed more

susceptible to bacteria Pseudomonas syringae pv. tomato DC3000 than Col-0

wild-type plants. LecRK-V.2 and LecRK-VII.1 overexpression lines demonstrate

increased resistance to Pst DC3000. In addition, both lecrk-V.2 and lecrk-VII.1 mutants

are defective in stomatal immunity. Here, we show by co-immunoprecipitation in

Arabidopsis protoplast that LecRK-V.2 and LecRK-VII.1 associated with the PRR

FLS2 and the co-receptor BAK1 before and after perception of the bacterial

pathogen-associated molecular pattern (PAMP) flagellin. Moreover, LecRK-V.2 and

LecRK-VII.1 could interact with FLS2 and BAK1 through their kinase domains when

analyzed by the in vitro yeast-two-hybrid assay. In this study, I clarified the role of

LecRK-V.2 and LecRK-VII.1 in the FLS2-BAK1 complex upon flagellin perception.

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Keywords:FLS2、LecRK-V.2、LecRK-VII.1、BAK1、stomatal closure、Pst DC3000、

PTI.

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Contents

摘要 ... II

Abstract ... IV

Contents ... VI

Introduction ... 1

Material and Methods ... 9

Biological Material and Plant Growth Conditions ... 9

Co-immunoprecipitation Assay in Arabidopsis Protoplast. ... 9

Yeast Two-Hybrid Assays ... 11

Results ... 14

LecRK-V.2 and LecRK-VII.1 associate with FLS2 in vivo ... 14

LecRK-VII.1 could directly interact with FLS2 through their kinase domain, but LecRK-V.2 could not. ... 15

LecRK-V.2 and LecRK-VII.1 associate with BAK1 in vivo ... 16

In vitro, BAK1 can directly interact with LecRK-V.2 and LecRK-VII.1 through their kinase domain ... 17

In vitro and in vivo association analyses of LecRK-V.2 and LecRK-VII.1 ... 18 Each LecRKs may not affect the association of FLS2 with the other LecRK20

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Discussion ... 22

Conclusions and Future Perspectives ... 27

Figures ... 30

Figure 1：FLS2 associate with LecRK-V.2 and LecRK-VII.1 when analyzed

by co-immunoprecipitation in Arabidopsis protoplasts. ... 30

Figure 2：In vitro FLS2 direct interaction with LecRK-VII.1 kinase domains

but not LecRK-V.2. ... 32

Figure 3：BAK1 associates with LecRK-V.2 and LecRK-VII.1 when analyzed

by co-immunoprecipitation in Arabidopsis protoplasts. ... 33

Figure 4：BAK1 can directly interact with LecRK-V.2 and LecRK-VII.1

through its kinase domain. ... 35

Figure 5： Co-immunoprecipitation analyses of LecRK-V.2 association with

LecRK-VII.1 in Arabidopsis protoplasts. ... 36

Figure 6： LecRK-V.2 cannot directly interact with LecRK-VII.1 through

their kinase domain when analyzed with the yeast two hybrid assay. ... 38

Figure 7： FLS2 associates with BAK1 and LecRK-VII.1 in the lecrk-V.2

mutant background. ... 39

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Figure 8： FLS2 associates with BAK1 and LecRK-V.2 in the lecrk-VII.1

mutant background. ... 40

Figure 9：Proposed model ... 41

Tables ... 43

Table 1 ：Primers for kinase domain constructs ... 43

Table 2 ：List of abbreviations ... 44

Supplementary Figures ... 45

Supplemental Figure 1： LecRK-V.2 and LecRK-VII.1 are necessary for the resistance against to Pst.DC3000. ... 45

Supplemental Figure 2： LecRK-V.2 and LecRK-VII.1 are necessary for stomatal immunity. ... 46

Supplemental Figure 3：The PTI apoplastic responses and regulations in lecrk-V.2 and lecrk-VII.1 mutant lines. ... 47

Supplemental Figure 4：LecRK-V.2-GFP and LecRK-VII.1 –GFP fusion proteins localized on the membrane in Arabidopsis mesophyll protoplast. 49 References... 52

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Introduction

Pathogen-triggered plant defense immunity

Plant growth and reproduction are affected by many pathogens known as

plant-associated microbes. Mainly, two pathways are typical of the innate immune

responses that plants activate upon infection with plant-associated microbes. The first

branch is triggered by the recognition of highly conserved pathogen /microbe-associated

molecular patterns (PAMPs/MAMPs) by host cell transmembrane proteins that function

as pattern recognition receptors (PRRs), which in turn, activate

PAMP/MAMP-triggered immunity (PTI/MTI). The second branch recognizes type III

effectors inside the plant cell via nucleotide-binding site-leucine-rich repeat (NB-LRR)

resistance (R) proteins. This leads to the activation of the effector-triggered immunity

(ETI) (Jones and Dangl 2006).

Membrane receptors recognize microbe-associated molecular patterns in plants

Membrane-localized receptors which are is also called PRRs can recognize the

conserved molecules characteristic of an entire group of microbes known as MAMPs.

MAMPs include bacterial lipopolysaccharides (LPS), peptidoglycans (PGN), fungal

chitin, quorum sensing factors, flagellin, and even danger signals (damage-associated

molecular patterns; DAMPs) which are released by plants during microbial infection

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(Monaghan and Zipfel 2012). Arabidopsis flagellin-sensing 2 (FLS2) is one of the

best-characterized PRR in plants. It encodes a leucine-rich repeat-receptor-like kinases

(LRR-RLK) that recognizes bacterial flagellin or its active peptide derivative flg22

(Gomez-Gomez and Boller 2000). Upon flg22 perception, FLS2 instantaneously

interacts with another LRR-RLK, the brassinosteroid (BR) insensitive 1-associated

kinase 1 (BAK1) (Chinchilla et al. 2007, Heese et al. 2007). Botrytis-induced kinase 1

(BIK1), a plasma membrane-localized receptor-like cytoplasmic kinase (RLCK), is

rapidly phosphorylated upon flg22 perception in an FLS2- and BAK1-dependent

manner (Lu et al. 2010, Zhang et al. 2010). BIK1 functions as a kinase substrate of

BAK1 and forms a complex with FLS2 and BAK1 in transducing flagellin signaling

(Lu, Wu, Gao, Zhang, Shan and He 2010).

Flagellin triggers plant defense responses

After exposure to flg22 or other MAMPs, the plant cell undergoes a rapid activation

of the MAP kinase (MPK) cascade (Nuhse et al. 2000), a burst of reactive oxygen

species (ROS) controlled by the NADPH oxidase RbohD (Blume et al. 2000, Nuhse et

al. 2007), and a transient influx of calcium ion from the apoplast (Blume, Nurnberger,

Nass and Scheel 2000, Lecourieux et al. 2002), with the latter being essential for the

ROS production. The plasma-membrane-associated kinase BIK1, which is a direct

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substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon

PAMP perception (Kadota et al. 2014). BIK1 phosphorylates in different residues of

calcium-dependent protein kinases. Both MAMP-induced BIK1 activation and

BIK1-mediated phosphorylation of RBOHD are calcium independent. Importantly,

phosphorylation of these residues is critical for the MAMP-induced ROS burst and

antibacterial immunity (Kadota, Sklenar, Derbyshire, Stransfeld, Asai, Ntoukakis,

Jones, Shirasu, Menke, Jones and Zipfel 2014, Li et al. 2014).

Lectin receptor kinases are involved in plant defense innate responses

Arabidopsis possesses many receptor-like kinases (RLKs), and one class of RLKs is

the lectin receptor kinase which contain an extracellular lectin domain that are

predicted to bind to carbohydrates, a transmembrane domain, and an intracellular

kinase domain. Lectin receptor kinases are classified by their extracellular lectin

motifs. The first group is called G-type lectins because it contains a GNA-related

motif. Beside lectin GNA-related motif, the extracellular domains of this group also

contain cysteine-rich EGF-like (epidermal growth factor) and PAN

(plasminogen-apple-nematode) motifs that both function in protein homodimerization

(Naithani et al. 2007, Van Damme et al. 2007). G-type lectins group contain around 40

RLK genes in Arabidopsis. But the function of G-type lectins is unknown. The second

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group includes calcium-dependent lectins which are also abbreviated as C-type lectins,

and this group is mostly found in mammalian cells but only one gene which encoding a

C-type lectins protein in Arabidopsis has been found until now (Bouwmeester and

Govers 2009, Cambi et al. 2005). The third group regroups lectin proteins with a

legume-like or L-type lectin domain in their extracellular domains. Proteins in this

group are also called LecRKs. There are 45 genes encoding L-type lectin receptor

kinases in plants. LecRKs may be involved in the binding of potential

ligands-oligosaccharide. LecRKs may also recognize small hydrophobic ligands, such

as PAMPs and plant hormones (Andre et al. 2005, Bouwmeester and Govers 2009,

Van Damme, Nakamura-Tsuruta, Smith, Ongenaert, Winter, Rouge, Goldstein, Mo,

Kominami, Culerrier, Barre, Hirabayashi and Peumans 2007).

LecRKs are involved in many biotic and abiotic stress

LecRKs are involved in both biotic and abiotic stress (Vaid et al. 2013). In abiotic

stress, LecRK-I.3 gene is differentially up-regulated during salt stress (He et al. 2004).

The LecRK-V.1 functions as a positive regulator in responsive to abscisic acid (ABA)

and is also involved in osmotic and salt stress (Deng et al. 2009). In biotic stress,

LecRK-VI.2 provides resistance against the hemibiotrophic bacteria Pseudomonas

syringae and the necrotrophic bacteria Pectobacterium carotovorum (Singh et al.

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2012). The Arabidopsis LecRK-V.5 gene was found to reverse the stomatal closure

induced by bacterial infection and relieve the plant from CO2 deficiency (Arnaud et al.

2012, Desclos-Theveniau et al. 2012). LecRK-IX.1 and LecRK-IX.2 are functional

analogs in the resistance regulation of Phytophthora infection and are involved in the

plant cell death in Arabidopsis (Wang et al. 2015). LecRK-I.9 is involved in the

binding of extracellular ATP and signaling of ATP-induced calcium response,

mitogen-activated protein kinase activation, and gene expression (Cao et al. 2014,

Choi et al. 2014). Hence, LecRKs are known to be involved in the plant defense

response, signaling, and even in extracellular ligand binding.

LecRK-V.2 and LecRK-VII.1 are related to flg22-triggered defense responses

T-DNA insertion mutant lines of LecRKs with high expression levels under biotic

stress from previous microarray studies (Bouwmeester and Govers 2009) were ordered

from the Arabidopsis Biological Resource Center (ABRC). Afterward, T-DNA

insertion mutant plants were dipped in a solution of hemibiotrophic Pseudomonas

syringae pv. tomato DC3000 (Pst DC3000) bacteria and symptoms were evaluated 3

days later. From this screening, two lecrk mutants, namely leck-V.2 and lecrk-VII.1

demonstrated increased susceptibility to Pst DC3000 bacteria (Supplemental Figure 1).

Lecrk-V.2 and lecrk-VII.1 showed defective stomatal closure compare to wild-type

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Col-0 after infection with Pst DC3000 (Supplemental Figure 2). But lecrk-V.2 and

lecrk-VII.1 T-DNA insertion mutant line showed no difference compare with wild-type

Col-0 in the other PTI pathway, such as the production of extracellular ROS, the

deposition of callose, the activation of MAP kinase (MPK) cascades, and expression of

defense-related genes (Supplemental Figure 3). The transient expression in

Arabidopsis protoplast to clarify the localization of LecRK-V.2 and LecRK-VII.1

proteins. LecRK-V.2 and LecRK-VII.1 proteins were indeed localized on the plasma

membrane of mesophyll Arabidopsis protoplast (Supplemental Figure 4). Previous

experiments showed that LecRK-V.2 and LecRK-VII.1 only interact with FLS2 in the

presence of flg22 by performing bimolecular fluorescence complementation (BiFC)

assay (Supplemental Figure 5).

LecRK-V.2 and LecRK-VII.1 are involved in FLS2-BAK1 complex

In order to clarify the roles of LecRK-V.2 and LecRK-VII.1 in the flagellin-triggered

immunity response, my works analyzed the relationship among LecRK-V.2,

LecRK-VII.1, FLS2, and BAK1. Towards this goal, the relationships of association

among these proteins were performed by the co-immunoprecipitation (Co-IP) of

proteins in Arabidopsis protoplasts. Both LecRKs associated with the FLS2-BAK1

complex with and without the perception of flagellin. LecRK-V.2 cannot interact with

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FLS2, but LecRK-VII.1 can interact with FLS2 directly through their kinase domains

by performing the yeast two-hybrid. Both LecRKs can interacted with the BAK, but

LecRK-V.2 cannot interact with LecRK-VII.1 through their kinase domains. It is

wondered that if both LecRKs will affect each other. Hence, I tested the association

between FLS2, BAK1, and LecRK-VII.1 in lecrk-V.2 mutant protoplasts by

performing the Co-IP assay. It was found that LecRK-V.2 didn’t affect the association

between FLS2, BAK1, and LecRK-VII.1, respectively. Further, I tested the association

between FLS2, BAK1, and LecRK-V.2 in lecrk-VII.1 mutant protoplasts by

performing the Co-IP assay. The results indicated that the LecRK-VII.1 didn’t affect

the association between FLS2, BAK1, and LecRK-V.2, respectively either. So we

proposed that LecRK may affects the interaction between FLS2 and the other LecRK,

but they are still in the FLS2-BAK1 complex before and after the treatment with flg22.

In this study, we found that LecRK-V.2 and LecRK-VII.1 associate with FLS2 and

BAK1 before and after the treatment of flg22. FLS2 can interact with LecRK-VII.1

through their intracellular kinase domain but FLS2 cannot interact with LecRK-V.2.

BAK1 can interact with LecRK-V.2 and LecRK-VII.1 through their intracellular

kinase domain. LecRK-V.2 cannot interact with LecRK-VII.1 through their

intracellular kinase domain.

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Material and Methods

Biological Material and Plant Growth Conditions

Arabidopsis thaliana ecotype Col-0 plants were grown in commercial potting soil

and perlite (ratio-3：2) at 22–24°C day and 17–19°C night temperature under short day

(9 hours under the light / 15 hours under the dark) condition of light illumination

period. The lighting was supplied at an intensity of ∼100μE m⁻²s⁻¹ by fluorescence

white light tubes. T-DNA insertion mutant lecrk-V.2 and lecrk-VII.1 lines were

obtained from the Arabidopsis Biological Resource Center (ABRC) (the T-DNA

insertion site).

Co-immunoprecipitation Assay in Arabidopsis Protoplast.

For Co-IP assay, we constructed the plasmid of FLS2-HA, BAK1-HA,

LecRK-V.2-HA, BAK1-GFP, LecRK-V.2-GFP, LecRK-VII.1-GFP, GFP-empty vector,

RCI2B-GFP. FLS2, BAK1, and LecRK-V.2 PCR amplified products were cloned into

pCR8 TOPO TA cloning vector (Invitrogen) and then cloned into pEG100 vector by

the ph.D student, Shweta Bagul, and the postdoctoral researcher, Mathieu Gauthier.

BAK1-GFP, LecRK-V.2-GFP, LecRK-VII.1-GFP, and RCI2B-GFP constructs were

PCR amplified from wide type Col-0 cDNAs, and PCR products were then cloned into

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pCR8 TOPO. Then, these plasmids were finally recombined into the

Gateway-compatible expression vectors pEarlyGate103 (ordered from ABRC).

GFP-empty vector construct was modified from pEarlyGate103 with removing

gateway cassette by using XhoI restriction enzyme. Plasmids of FLS2-HA and

BAK1-GFP or FLS2-HA and LecRK-V.2-GFP, or FLS2-HA and LecRK-VII.1-GFP or

FLS2-HA and GFP-empty vector, or FLS2-HA and RCI2B-GFP were co-transformed

into Arabidopsis protoplasts by a polyethylene glycol method as described previously

(Yoo et al. 2007).

We separated 150 μL protoplasts from a total volume of 500 μL of total

protoplasts for running of input proteins. The rest of total proteins were extracted in an

extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl,10 mM DTT, 10 mM EDTA

pH8.0, 1 mM Na3VO4, 10% glycerol, and 1% IGEPAL CA-630 [Sigma-Aldrich]) with

protease inhibitor cocktail (Roche). The solution was incubated with gentle shaking at

4 °C for at least 30 minutes and then centrifuged in the speed of 14,000 g for 15 min at

4°C. The supernatant was incubated with 15 μL GFP-Trap○^R _MA (ChromoTek,

GmbH) for 4 hours to 16 hours at 4°C, and GFP-Trap was diluted with dilution buffer

(10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% EDTA,pH8.0) before the incubation

with the supernatant. After the incubation at 4°C, the beads were centrifuged and

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washed four times with the washing buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl,

0.5% IGEPAL CA-630 [Sigma-Aldrich]). Proteins were separated by 10% SDS–PAGE

gel and then transferred to a polyvinylidine fluoride membrane (ImmobilonP;

Millipore). GFP and HA fusion proteins were detected by immunoblotting with

anti-GFP and anti-HA primary antibodies, respectively.

Yeast Two-Hybrid Assays

Yeast two-hybrid assays were performed as described in the manual of Matchmaker

Gold Yeast Two-Hybrid Systems (Clontech) and the user manual of Yeast Protocol

Handbook (Clontech). Briefly, we amplified the kinase domains of FLS2, BAK1,

LecRK-V.2, and LecRK-VII.1 fragments from pCR8-FLS2, pCR8-BAK1,

pCR8-LecRK-V.2, and pCR8-LecRK-VII.1 by using the kinase domain forward and

reverse primers (primers are listed in S1 Table). The PCR amplified products were

subcloned into pGBKT7 and pGADT7 vectors to construct different bait and prey

constructs (primers are listed in S1 Table). Then, different pairs of bait and prey

constructs were co-transformed into yeast AH109 strain by lithium acetate (LiAc)

method. From Clontech Yeast Protocol Handbook, they used and suggested users to

adopt the LiAc method (Ito et al. 1983), which then modified into much more simple

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and more reproducible protocols (Gietz et al. 1992, Hill et al. 1991, Schiestl and Gietz

1989). In this LiAc transformation method, yeast competent cells were cultured in

small amounts form the colonies of streaked YPD plates in fresh and incubated for

12-16 hours with shaking (180–200 rpm) in 28°C. The next day, we transferred enough

of the overnight culture into the fresh medium and incubated at 28°C for 3–5 hours

with shaking (180–200 rpm) to produce an OD600 concentration of yeast cultures of 0.4

– 1.0. The yeast cultures were then centrifuged at 1000~1500 g for 5 minutes in 4°C

and washed with sterilized water for 2 times and suspended in the LiAc solution. The

transformed DNA were prepared by mixture of 200 ng plasmid DNA per each

reaction, and the mixture of DNA and yeast was mixed with Polyethylene glycol

(PEG) and the appropriate amount of LiAc and then incubated at 28°C for half an hour.

After the incubation, the heat shock method, which allows the DNA to enter the cells,

was used for 15 minutes. The yeast cells were then centrifuged at 500 g for 5 minutes

and then resuspended into the LiAc solution. The yeast cells were then plated on the

synthetic dropout (SD) medium, or DDO medium (minimal media double dropouts,

SD medium with-Leu/-Trp), to select for transformants containing the introduced

plasmids for 3 days. Transformed colonies were dropped onto TDO medium (Triple

dropout medium: SD/–His/–Leu/–Trp), TDO/3-AT medium (SD/–His/–Leu/–Trp

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supplemented with 3-amino-1,2,4-triazole from Avocado Lot No. H7734B), QDO

medium (minimal media quadruple dropouts, SD medium with-Leu/-Trp/-Ade/-His),

and QDO/X medium (SD medium with-Leu/-Trp/-Ade/-His) containing 4 mg mL^-1

X-α-Gal to test for possible interactions between FLS2 and LecRK-V.2 or FLS2 and

LecRK-VII.1 or BAK1 and LecRK-V.2 or BAK1 and LecRK-VII.1 according to their

growth status.

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Results

LecRK-V.2 and LecRK-VII.1 associate with FLS2 in vivo

Former members of the Zimmerli laboratory found that both lecrk-V.2 and

lecrk-VII.1 mutant plants are more susceptible to Pst DC3000 than Col-0 wild-type,

while LecRK-V.2 and LecRK-VII.1 overexpression lines are more resistant. We thus

speculated that LecRK-V.2 and LecRK-VII.1 are involved in the flagellin-triggered

immunity defense response. Flagellin is a bacterial MAMP that can be recognized by

the plant PRR, FLS2. We thus asked whether LecRK-V.2 and LecRK-VII.1 associate

with FLS2. Toward this goal, I performed Co-IP analyses in Arabidopsis protoplasts.

In this assay, the plasmid of FLS2-HA was co-transformed with BAK1-GFP,

LecRK-V.2-GFP, LecRK-VII.1-GFP, the GFP-empty vector, or RCI2B-GFP.

Immunoprecipitation was conducted with GFP-Trap beads to immunoprecipitate either

BAK1, LecRK-V.2, LecRK-VII.1, the GFP-empty vector, or RCI2B proteins and

western blot analyses were performed with anti-GFP and anti-HA antibodies. Using

this approach, I showed that BAK1 associates with FLS2 after the treatment of flg22,

but not before the treatment of flg22, confirming previous studies (Chinchilla et al.

2006, Chinchilla, Zipfel, Robatzek, Kemmerling, Nurnberger, Jones, Felix and Boller

2007), and thus suggesting that our experimental conditions were appropriate (Figure

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1). By contrast, LecRK-V.2 and LecRK-VII.1 could associate with FLS2 before and

after treatment with flg22 (Figure 1). These results suggest that LecRK-V.2 and

LecRK-VII.1 are already in the FLS2 complex even before treatment with flg22. Upon

perception of flg22, FLS2 will recruit BAK1 into the complex and still associate with

LecRK-V.2 and LecRK-VII.1. These data suggest that LecRK-V.2 and LecRK-VII.1

are part of the FLS2 protein complex that are involved in the flg22-triggered immunity

response.

LecRK-VII.1 could directly interact with FLS2 through their kinase domain, but

LecRK-V.2 could not.

To further understand the mechanism and interaction between FLS2, LecRK-V.2, and

LecRK-VII.1, I used the yeast two-hybrid assay with both LecRK proteins as baits. The

kinase domain of FLS2 containing amino acids 219-1173, the kinase domain of

LecRK-V.2 containing amino acids 299-656, and the kinase domain of LecRK-VII.1

containing amino acids 309-686 were used to perform the yeast two-hybrid assay.

LecRK-V.2 could not directly interact with FLS2 through its kinase domain in yeast

two-hybrid assay (Figure 2). However, the kinase domain of LecRK-VII.1 could

interact with the kinase domain of FLS2 when analyzed with the yeast two-hybrid

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system (Figure 2). PGBKT7-53 that encodes the murine p53 and pGADT7-T that

encodes the SV40 large T-antigen were used as positive control, because they are

known to interact in the yeast two-hybrid assay (Li and Fields 1993, Mokdad-Gargouri

et al. 2001). pGBKT7-Lam (which encodes the lamin proteins) and pGADT7-T were

used as negative controls (Li and Fields 1993).

LecRK-V.2 and LecRK-VII.1 associate with BAK1 in vivo

BAK1 is a membrane protein and is the co-receptor of FLS2 to sense the MAMP

flagellin (Chinchilla, Bauer, Regenass, Boller and Felix 2006, Chinchilla, Zipfel,

Robatzek, Kemmerling, Nurnberger, Jones, Felix and Boller 2007). During perception

of flagellin, BAK1 is recruited to the FLS2 complex and associate with FLS2 (Sun et al.

2013b). I therefore tested the interaction between BAK1 and LecRK-V.2 or

LecRK-VII.1 with the Co-IP approach in Arabidopsis protoplasts. In this assay, the

plasmid of BAK1-HA was co-transformed with LecRK-V.2-GFP, or LecRK-VII.1-GFP,

or the GFP-empty vector, or RCI2B-GFP. Immunoprecipitation was conducted with

GFP-Trap beads to immunoprecipitate LecRK-V.2, LecRK-VII.1, GFP-empty vector, or

RCI2B proteins and immunoblot analyses were performed with anti-GFP and anti-HA

antibodies. BAK1 associated with both LecRK-V.2 and LecRK-VII.1 before and after

(26)

flg22 treatment (Figure 3). As a negative control, I analyzed the association of BAK1

with RCI2B-GFP or only GFP-empty vector by immunoprecipitating equal amounts of

RCI2B-GFP or GFP-empty vector with GFP-Trap beads and by analyzing the presence

of BAK1-HAusing anti-HA immunoblotting. BAK1 could not be detected, suggesting

that they do not associate with GFP at the plasma membrane. Taken together, these data

suggested that BAK1 already associated with LecRK-V.2 and LecRK-VII.1 before

perception of flg22. After the treatment of flg22, BAK1 still associated with LecRK-V.2

and LecRK-VII.1. LecRK-V.2 and LecRK-VII.1 may be involved in the flg22-triggered

innate immunity response through association with the FLS2/BAK1 complex.

In vitro, BAK1 can directly interact with LecRK-V.2 and LecRK-VII.1 through

their kinase domain

Co-IP assays in Arabidopsis protoplast suggested that BAK1, LecRK-V.2, and

LecRK-VII.1 are in the same protein complex before and after treatment with flg22. To

further analyze the interaction mechanism between BAK1, LecRK-V.2, and

LecRK-VII.1, The yeast two-hybrid assay was used with the kinase domain of BAK1 as

bait. To perform the yeast two-hybrid assay, the clones that contain the kinase domain

of BAK1 from the amino acid 158 to 663, the kinase domain of LecRK-V.2 from the

(27)

amino acid 299 to 656, and the kinase domain of LecRK-VII.1 from the amino acid 309

to 686 were used. BAK1 was found to directly interact with both LecRK-V.2 and

LecRK-VII.1 through their kinase domains when analyzed with the yeast two-hybrid

assay (Figure 4). As with previous results, pGBKT7-53 and pGADT7-T were used as

positive control, because they interact in the yeast two-hybrid assay (Li and Fields 1993,

Mokdad-Gargouri, Belhadj and Gargouri 2001). The negative control was performed by

using pGBKT7-Lam (which encodes lamin proteins) and pGADT7-T (Li and Fields

1993).

In vitro and in vivo association analyses of LecRK-V.2 and LecRK-VII.1

LecRK-V.2 and LecRK-VII.1 are involved in the FLS2/BAK1 complex

independently of flagellin. Next, I focused on elucidating the association and the

interaction between LecRK-V.2 and LecRK-VII.1. First, co-transient expression in

Arabidopsis protoplasts and Co-IP of proteins were used. In this assay,

LecRK-V.2-HA was co-transformed with LecRK-VII.1-GFP, the GFP-empty vector,

and RCI2B-GFP. Immunoprecipitation was conducted with GFP-Trap beads to

immunoprecipitate the GFP-tagged proteins and immunoblot analyses were performed

with anti-GFP and anti-HA antibodies. LecRK-V.2 could associate with LecRK-VII.1

(28)

before and after flg22 treatment (Figure 5). As a negative control, I analyzed the

association of LecRK-V.2 with RCI2B-GF or the GFP-empty vector by

immunoprecipitating equal amounts of RCI2B-GFP and the GFP-empty vector with

GFP-Trap beads and by analyzing the presence of BAK1-HA using anti-HA

immunoblotting. LecRK-V.2 could not be detected, suggesting that LecRK-V.2 does

not associate non-selectively with GFP at the plasma membrane. Second, in order to

know whether LecRK-V.2 can directly interact with LecRK-VII.1, the yeast

two-hybrid assay was performed with LecRK-V.2 proteins as baits. To perform this

assay, the clones containing the kinase domain of LecRK-V.2 from the amino acid 299

to 656, and the kinase domain of LecRK-VII.1 from the amino acid 309 to 686 were

used. In this assay, pGBKT7-53 and pGADT7-T were used as positive control (Li and

Fields 1993, Mokdad-Gargouri, Belhadj and Gargouri 2001). A negative control was

performed by using pGBKT7-Lam and pGADT7-T (Li and Fields 1993). LecRK-V.2

did not directly interact with LecRK-VII.1 through its kinase domain when analyzed

with the yeast two-hybrid assay (Figure 6). Taken together these data suggest that

LecRK-V.2 associate with LecRK-VII.1, but they cannot directly interact with each

other through their kinase domain.

(29)

Each LecRKs may not affect the association of FLS2 with the other LecRK

To test whether one LecRK affects the other LecRK association with FLS2, the

co-transient expression assay in Arabidopsis protoplasts of Col-0, lecrk-V.2, and

lecrk-VII.1 T-DNA insertion mutant plants was performed. In this Co-IP assay, the

FLS2-HA construct was used to co-transform with BAK1-GFP, LecRK-V.2-GFP,

LecRK-VII.1-GFP, or the GFP-empty vector. Immunoprecipitation was conducted

with GFP-Trap beads to immunoprecipitate the GFP-tagged proteins and immunoblot

analyses were performed with anti-GFP and anti-HA antibodies. In lecrk-V.2 mutant

protoplasts, the association between FLS2 and LecRK-VII.1 was still observed (Figure

7). Similarly, the association between FLS2 and LecRK-V.2 was still observed in the

lecrk-VII.1 mutant background (Figure 8). Together these data suggest that FLS2 and

LecRK-V.2 or LecRK-VII.1 are still in the same complex before and after treatment

with flg22 even when one of the LecRK is missing. These data suggested that the

association of FLS2 and the LecRKs is independent from the presence of the other

LecRK. From the data of BiFC, it shows that FLS2 cannot interact directly with

LecRK-VII.1 in the lecrk-V.2 mutant protoplasts and vice-versa. We proposed that one

LecRK affects the direct interaction between FLS2 and the other LecRK, but they are

still in the same FLS2 PRR-complex.

(30)

(31)

Discussion

LecRK-V.2 and LecRK-VII.1 associate with FLS2

FLS2 and BAK1 interact and form an immune-complex to induce the

flagellin-triggered immunity pathway upon perception of flagellin (Chinchilla, Zipfel,

Robatzek, Kemmerling, Nurnberger, Jones, Felix and Boller 2007). Previous studies

from the Zimmerli laboratory showed that LecRK-V.2 and LecRK-VII.1 are involved in

the resistance against Pst DC3000 and are also involved in the regulation of stomatal

immunity (Supplemental Figure 1 and 2). We hypothesized that LecRK-V.2 and

LecRK-VII.1 participate in the FLS2 PRR complex to modulate the flagellin-triggered

immunity responses. First, we focused on the flagellin receptor, FLS2. Because

LecRK-V.2 and LecRK-VII.1 are membrane-localized proteins, they may affect

flagellin-triggered immunity pathway by interacting with FLS2. From previous BiFC

data, it was shown that FLS2 can interact with LecRK-V.2 and LecRK-VII.1 only after

treatment with flg22 but no interactions were observed prior to flg22 treatment. By

contrast, this work shows that FLS2 associates with LecRK-V.2 and LecRK-II.1 before

and after treatment with flg22 when analyzed by Co-IP in Arabidopsis protoplast. It is

known that BAK1 interacts with FLS2 in a ligand-dependent manner (Chinchilla,

Zipfel, Robatzek, Kemmerling, Nurnberger, Jones, Felix and Boller 2007). We propose

(32)

that LecRK-V.2 and LecRK-VII.1 are part of the FLS2 receptor complex already

before flg22 elicitation, but direct interact with FLS2 is flg22 dependent. It is known

that BIK1 interacts with FLS2, but dissociate in the presence of flagellin and BIK1 will

be phosphorylated after flg22 treatment (REF needed). After the dissociation from the

FLS2 complex, BIK1 will directly phosphorylate NADPH oxidase RbohD at specific

sites in a calcium-independent manner to enhance ROS generation (Kadota, Sklenar,

Derbyshire, Stransfeld, Asai, Ntoukakis, Jones, Shirasu, Menke, Jones and Zipfel

2014, Laluk et al. 2011, Li, Li, Yu, Zhou, Liang, Liu, Cai, Gao, Zhang, Wang, Chen

and Zhou 2014). Our data showed that LecRK-V.2 and LecRK-VII.1 can interact with

FLS2 through their kinase domain. LecRK-V.2 and LecRK-VII.1 may act like BIK1

and have the modification, such as phosphorylation, with FLS2, and then affect the

NADPH oxidase, ion-pumps or stomatal membrane proteins, such as Open Stomata 1

(OST1) (Montillet et al. 2013, Shang et al. 2016), to regulate stomatal immunity.

BAK1 associates and interacts through kinase domain with LecRK-V.2 and

LecRK-VII.1

BAK1 is another important receptor in Arabidopsis. Notably, BAK1 is involved in

hormone signaling and also in MTI (Chinchilla et al. 2009, Heese, Hann,

Gimenez-Ibanez, Jones, He, Li, Schroeder, Peck and Rathjen 2007). BAK1 interacts

(33)

with FLS2 after the perception of flg22 (Chinchilla, Bauer, Regenass, Boller and Felix

2006, Chinchilla, Zipfel, Robatzek, Kemmerling, Nurnberger, Jones, Felix and Boller

2007, Sun, Li, Macho, Han, Hu, Zipfel, Zhou and Chai 2013b). Previous BiFC data

suggest that BAK1 can interact with LecRK-V.2 and LecRK-VII.1 independently of

flg22 treatment. By Co-IP however, BAK1 associates with LecRK-V.2 and

LecRK-VII.1 before and after elicitation with flg22. BAK1 is known to interact with

BRI1 in the presence of brassinosteroids (BRs) and dissociates upon flagellin

perception (Kim et al. 2013, Sun et al. 2013a). Both LecRKs interact with BAK1 prior

to and after treatment with flg22, so both LecRKs are not only important for the

flagellin-sensing receptor, FLS2, but also important for the flagellin-sensing

co-receptor, BAK1. BAK1 unidirectionally phosphorylates BIR2 and BAK1 and BIR2

interactions are kinase-activity dependent (Blaum et al. 2014, Halter et al. 2014). From

our yeast two-hybrid results, BAK1 can directly interact with LecRK-V.2 and

LecRK-VII.1 through its kinase domain. BAK1 and both LecRKs may thus

phosphorylate each others and regulate the downstream signals during perception of

flg22. Further studies and researches are needed to clarify this point.

LecRK-V.2 cannot interact with LecRK-VII.1

(34)

Because LecRK-V.2 and LecRK-VII.1 are both membrane-localized proteins and

they associate or interact with FLS2 and BAK1, we asked whether LecRKV-2 and

LecRK-VII.1 associate or directly interact together. From previous protein-protein

interaction experiments, we knew that LecRK-V.2 and LecRK-VII.1 associate with

FLS2 and BAK1 independently of flg22 perception. We thus expected that both

LecRKs will associate together prior to and after treatment with flg22. By in vivo

Co-IP assay, LecRK-V.2 was shown to associate with LecRK-VII.1. It indicates that

both LecRKs are involved in the same FLS2 PRR complex. The kinase domains

interaction of membrane proteins may involve in the phosphorylated modification

(Blaum, Mazzotta, Noldeke, Halter, Madlung, Kemmerling and Stehle 2014, Halter,

Imkampe, Mazzotta, Wierzba, Postel, Bucherl, Kiefer, Stahl, Chinchilla, Wang,

Nurnberger, Zipfel, Clouse, Borst, Boeren, de Vries, Tax and Kemmerling 2014,

Kadota, Sklenar, Derbyshire, Stransfeld, Asai, Ntoukakis, Jones, Shirasu, Menke,

Jones and Zipfel 2014, Li, Li, Yu, Zhou, Liang, Liu, Cai, Gao, Zhang, Wang, Chen and

Zhou 2014). But LecRK-V.2 cannot interact directly through their kinase domain with

LecRK-VII.1 by performing in vitro yeast two-hybrid, suggesting that both LecRKs

may not phosphorylate each other.

LecRK-V.2 and LecRK-VII.1 are involved in FLS2-BAK1 complex

(35)

In summary, LecRK-V.2 and LecRK-VII.1 affect the PTI pathway such as the

stomatal closure by interacting with FLS2 or BAK1 upon perception of flg22.

LecRK-V.2 and LecRK-VII.1 may have the modification with FLS2 and BAK1 and

then affect the downstream stomatal closure and the resistance against Pst DC3000

bacteria and even the perception of flg22. In this thesis works, I clarified the

relationships between FLS2, BAK1, LecRK-V.2, and LecRK-VII.1. These new data

helped us to unravel the complex model of LecRK-V.2 and LecRK-VII.1 involvement

in the FLS2/BAK1 immune-complex.

There is proposed model to conclude our results (Figure 9). LecRK-V.2 and

LecRK-VII.1 interact with BAK1 but not FLS2 before the treatment of flg22. But both

LecRKs associate with FLS2 and BAK1 before the treatment of flg22. Upon

perception of flg22, FLS2 recruits LecRK-V.2, LecRK-VII.1, and BAK1. The

involvement of both LecRKs and FLS2/BAK1 complex may play the key role to

regulate stomatal immunity when the perception of flg22.

(36)

Conclusions and Future Perspectives

LecRKs contain extracellular lectin domain, transmembrane domain, and

intracellular kinase domain (Bouwmeester and Govers 2009, Senchou et al. 2004). The

extracellular lectin domain is suggested to be critical for monosaccharide binding or

ligand binding and then trigger the downstream signals (Vaid, Macovei and Tuteja

2013). LecRK-I.9 can bind to extracellular ATP and is required for the ATP-induced

calcium response, mitogen-activated protein kinase activation, and gene expression

(Cao, Tanaka, Nguyen and Stacey 2014, Choi, Tanaka, Cao, Qi, Qiu, Liang, Lee and

Stacey 2014). From previous studies, the extracellular domain of BAK1 and FLS2

interactions can proceed independently of intracellular domain interactions upon the

perception of flg22 (Koller and Bent 2014). The extracellular domains of FLS2 and

BAK1 are important for defense signaling activation independently of intracellular

kinase domain. So we proposed that the extracellular domain of LecRK-V.2 and

LecRK-VII.1 may play the key role in the perception of flg22 together with FLS2. We

can try to isolate only both LecRKs kinase domains and check their sensing of flg22

will be affected or not.

Upon perception of flg22, BIK1 is phosphorylated and dissociates from the

FLS2/BAK1 complex. Then BIK1 phosphorylates the NADPH oxidase, RbohD, at

(37)

specific sites in a calcium-independent manner to enhance ROS generation to trigger

plant immunity against pathogens (Kadota, Sklenar, Derbyshire, Stransfeld, Asai,

Ntoukakis, Jones, Shirasu, Menke, Jones and Zipfel 2014, Laluk, Luo, Chai, Dhawan,

Lai and Mengiste 2011, Li, Li, Yu, Zhou, Liang, Liu, Cai, Gao, Zhang, Wang, Chen

and Zhou 2014). Besides, BAK1 can unidirectionally phosphorylate BIR2 and

negatively regulate MTI (Blaum, Mazzotta, Noldeke, Halter, Madlung, Kemmerling

and Stehle 2014, Halter, Imkampe, Mazzotta, Wierzba, Postel, Bucherl, Kiefer, Stahl,

Chinchilla, Wang, Nurnberger, Zipfel, Clouse, Borst, Boeren, de Vries, Tax and

Kemmerling 2014). It was found that both LecRKs can directly interact with FLS2

after the treatment of flg22. Both LecRKs can interact with BAK1, and both LecRKs

were involved in stomatal immunity. Hence, both LecRKs may be involved in the

phosphorylated modification between FLS2, BAK1, and BIK1, and then affect

stomatal closure. It would be interesting to perform the kinase assay to clarify the

phosphorylated modification between FLS2, BAK1, and both LecRKs. This should

help to understand the functional relationships between members of the FLS2 complex

during stomatal immunity.

(38)

(39)

Figures

Figure 1：FLS2 associate with LecRK-V.2 and LecRK-VII.1 when analyzed by

co-immunoprecipitation in Arabidopsis protoplasts.

FLS2-HA was co-transformed with BAK1-GFP, LecRK-V.2-GFP, LecRK-VII.1-GFP,

GFP empty vector, and RCI2B-GFP into Arabidopsis protoplasts. Total proteins

(Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads followed by

immunoblot analysis with anti-HA to detect FLS2-HA. EV-GFP and RCI2B-GFP were

(40)

used as negative control to confirm that FLS2-HA did not stick to GFP beads or

associate with GFP at the plasma membrane. The protoplasts were treated with or

without 1 μM flg22 for 10 minutes. Experiments were repeated three times with the

Tables

Table 1 ：Primers for kinase domain constructs

Gene Primers Sequence (5’ - 3’)

FLS2 FLS2 KD Fp CACCTTACCGGATTTGGAT

FLS2 FLS2 KD Rp CTAAACTTCTCGATCCTCGTTACG

BAK1 BAK1 KD Fp CACCCCTACACCGCCAT

BAK1 BAK1 KD Rp TTATCTTGGACCCGAGGGG

LecRK-V.2 LecRK-V.2-KD Fp GGATCCTTGAAGAGGAAGAAGTT

LecRK-V.2 LecRK-V.2-KD Rp CTCGAGTTAGCGTCCACTAGAGA

LecRK-VII.1 LecRK-VII.1-KD Fp GGATCCAGAAAGAGATTAGAGAGG

LecRK-VII.1 LecRK-VII.1-KD Rp CTCGAGTCACCTCCCTTCTAAAA

(53)

Table 2 ：List of abbreviations

Abbreviation Full name

PAMPs/MAMPs Pathogen/microbe-associated molecular patterns

PRRs Pattern recognition receptors

PTI/MTI PAMPs/MAMPs-triggered immunity

LPS Lipopolysaccharides

PGN Peptidoglycans

FLS2 Flagellin-sensing 2

BAK1 Brassinosteroid insensitive 1-associated kinase 1

BIK1 Botrytis-induced kinase 1

RLKs Receptor-lie kinase

Pst DC3000 Pseudomonas syringae pv. tomato DC3000

BiFC Bimolecular fluorescence complementation

Co-IP Co-immunoprecipitation

LiAc Lithium acetate

PEG Polyethylene glycol

SD medium Synthetic dropout medium

(54)

3-AT 3-amino-1,2,4-triazole

Supplementary Figures

Supplemental Figure 1： LecRK-V.2 and LecRK-VII.1 are necessary for the

resistance against to Pst.DC3000.

(A) The disease symptoms were observed 2-3 days after dip-inoculation with Pst

DC3000. (B) The bacterial growth were assessed 2-3 days after dip-inoculation with 5

x 10⁶ cfu•ml^-1 Pst DC3000 in Col-0 and lecrk mutants. Bacterial titers were evaluated

at 3d after inoculation (dpi). Values are the means ± SD of three biological replicates

(n = 9). Asterisks indicate a significant difference to Col-0 wild-type control based on

a t test (P <0.01). This experiments was repeated by Ph.D student, Shweta Bagul and

the other former members in the Zimmerli laboratory.

(55)

Supplemental Figure 2： LecRK-V.2 and LecRK-VII.1 are necessary for stomatal

immunity.

(A and B) The stomata aperture from the epidermal peels of Col-0, lecrk-V.2, and

lecrk-VII.1 mutant lines. Epidermal peels were exposed to MES buffer (Mock) or 5 x

10⁶ cfu•mL^-1 Pst DC3000 for 1.5 or 3 hours. (C) The stomatal aperture from the

epidermal peels of Col-0, lecrk-V.2, and lecrk-VII.1 mutant lines after 3 hours of

incubation in flg22 with 0 (Mock), 0.5, 1 or 5 μM, respectively.

(56)

Supplemental Figure 3：The PTI apoplastic responses and regulations in lecrk-V.2

and lecrk-VII.1 mutant lines.

(A and B) The reactive oxygen species productions in Arabidopsis leaves of Col-0,

lecrk-V.2 and lecrk-VII.1 mutant lines after treatment with 1 μM flg22 as relative light

units (RLU). The bak1-4 mutant line is used as negative control. Values are the means

± SE (n = 6). (C) Callose deposition in Col-0, lecrk-V.2 and lecrk-VII.1 mutant leaves

9 hours after infiltration with 10 mM MgSO4 (Mock) or 1 x 10⁸ cfu•mL^-1 Pst DC3000

hrcC. Data are means ± SD of callose deposits per square millimeter (n = 9).

Differences among Col-0, lecrk-V.2 and lecrk-VII.1 mutant were not significantly

different base on a t test (P < 0.01). Bar = 400μm. (D) FRK1 expression levels in

(57)

10 mM MgSO4 (Mock) or 1 x 10⁸ cfu•mL^-1 Pst DC3000 hrcC (h). Transcripts levels

determined by qRT-PCR and normalized to both EF-1 and UBQ10 were compared to

Col-0 mock at 45 minutes (defined value of 1). Three independent biological replicated

performed in triplicate were average ± SD (n = 9). Differences among Col-0,

lecrk-V.2 and lecrk-VII.1 mutant were not significantly different base on a t test (P <

0.01).

(58)

Supplemental Figure 4：LecRK-V.2-GFP and LecRK-VII.1 –GFP fusion proteins

localized on the membrane in Arabidopsis mesophyll protoplast.

LecRK-V.2-GFP and LecRK-VII.1–GFP fusion proteins were driven by the

cauliflower mosaic virus 35S promoter and transiently expressed in Arabidopsis

mesophyll protoplast. (A and D) GFP fluorescence channel. (B and E) Plasma

membrane marker pm-rk CD3-1007-mCherry fluorescence localization. (C and F)

Overlapping images of the GFP, plasma membrane marker and chlorophyll (red)

autofluorescence. (G) Protoplast expressing the GFP vector only as a GFP control.

(59)

Supplemental Figure 5：LecRK-V.2 and LecRK-VII.1 interact with FLS2 only

after the treatment with flg22.

In vivo, the interactions between FLS2 and LecRK-V.2 or FLS2 and LecRK-VII.1 are

determined by performing bimolecular fluorescence complementation (BiFC) analyses.

LecRK-V.2-GFP and LecRK-VII.1–GFP fusion proteins were under the control of the

cauliflower mosaic virus (CaMV) 35S promoter. FLS2-YFP^N and BAK1-YFP^C,

FLS2-YFP^N and LecRK-V.2-YFP^C, FLS2-YFP^N and LecRK-VII.1-YFP^C were

transiently co-transformed into Arabidopsis protoplast and with (+) or without (-) the

treatment of 1μM flg22 for 10 minutes. The red and yellow signals are

autofluorescence and YFP, respectively. There were the image of bright field and the

merge which is combined the autofluorescence, YFP, and bright field image.

(60)

(61)

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