6
A Proposal for Plant Pathology 692 (JUNHUI
ZHOU)
Phytophthora infestans effector AVR1 targets host proteins and
manipulates plant immunity
1. STATEMENT OF THE
RESEARCH PROBLEM AND GENERAL
BACKGROUND
The oomycetes are filamentous eukaryotic microorganisms that cause a great
number of devastating diseases in host plants, and one of the most notorious is the
late blight of potato. Caused by Phytophthora infestans, Late blight of potato had
resulted in death of millions people in 1840s (Vleeshouwers et al., 2011).
Oomycetes secrete hundreds of effector proteins to promote infection in host plants,
which target two distinct compartments in the host plant: apoplast and cytoplasm
(Morgan and Kamoun, 2007; Lamour et al., 2009). Cytoplasmic effectors are
translocated into the plant cell, which are first identified by their avirulence (Avr)
function and their ability to cause hypersensitive response (HR) (Morgan and
Kamoun, 2007). Several characterized domains, such as RXLR (Rehmany et al., 2005;
Whisson et al., 2007; Dou et al., 2008; Bozkurt et alk., 2012), LXLFLAK (Haas et al.,
2009; Schornack dt al., 2010) and CHXC (Kemen et al., 2011) are shown to be
required for translocation into plant cytoplasm in cytoplasmic effectors. RXLR class of
oomycete effectors, include a conserved RXLR motif in the N-terminus follows the
signal peptide, are characterized in Peronosporales clade of oomycete effectors
(Phytophthora and downy mildews) that secreting through haustoria (Le´ vesque et
al., 2010; Bozkurt et al., 2012).
In host plants, these RXLR effectors are recognized by resistance (R) proteins, mainly
from NBS-LRR group (Rehmany et al., 2005; Bouwmeester et al., 2011). RXLR motif in
oomycete effectors is usually followed by another conserved domain dEER motif
(Govers and Gijzen, 2006).Study shows that the mutations on either RXLR or dEER
motifs result in host resistance protein R3a fails to detect P.infestans effectors Avr3a,
as well as fails to trigger Hypersensitive Response (HR) (Whisson et al., 2007).
In the past one hundred years, at least 11 late blight race-specific resistance(R) genes
from Solanum demissum had been identified, which suggests there are 11
corresponding virulence(VIR) and avirulence (AVR) genes in P.infestans (Lee et al.,
2001). However, until now, the modes of action of P.infestans effectors mediated
virulence are still poorly understood. Among all 11 AVR genes, AVR3a is first
identified to target and stabilize host E3 ubiquitin ligase CMPG1 (Bos et al., 2010;
Gilroy et al., 2011). CMPG1 is required for infestin 1(INF1)-triggered cell death (ICD)
(Gonzalez-Lamothe et al., 2006; Bos et al., 2006). Thereafter, AVR3a targets and
modifies the normal activity of CMPG1 leads to attenuation of cell death during
biotrophic phase of infection. Among the two alleles of AVR3a (Avr3a KI and Avr3aEM),
only Avr3aKI can activate R3a (ETI) or trigger virulence function (Armstrong et al.,
2005; Huang et al., 2005). A recent report also shows that AVR3a forms homodimers
in its RxLR translocation motif (Wawra et al., 2012). Avr3b from Phytophthora sojae
was identified to serve as an ADP-ribose/NADH pyrophosphorylase. Transient
expression of Avr3b decrease reactive oxygen species (ROX) accumulation, therefore,
increase susceptibility in Nicotiana benthamiana. Even though, mutation on
C-terminal Nudix motif only abolishes the virulence activity of Avr3b, but not
avirulence activity (Dong et al., 2011). A very recent report indicates that AVR2 is
physically interacts with a putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) in
host plant Solanum demissum. BSL1 was shown to mediate the recognition of AVR2
by R2 as well as triggering resistance to P. infestans, probably by forming a ternary
complex with R2 and AVR2 (Saunders et al., 2012). This pattern confers indirectly
recognition of AVR effectors by NBS-LRR R proteins.
Another group indicates a direct recognition of effectors by R proteins, Chen et al
shown that P. infestans effector AVRblb1 (IPI-O) is directly interacts with coilded-coil
(CC) domain of RB (Rpi-blb1) and affects RB functions. Four amino acids (82, 86, 129,
and 135) within IPI-O are crucial for its interaction with RB CC domain, and L129
determines the elicitation of hypersensitive response (HR) in planta (Chen et al.,
2012).
P. infestans effector AVRblb2 is a multi-gene family with at least seven duplicates in
the genome of P. infestans strain T30-4 (Haas et al., 2009), which also includes
N-terminal signal peptide and RXLR domain, and the amino acid position 69 plays a
key role in activating the cognate host resistance protein Rpi-blb2 (Oh et al., 2009).
Recently, AVRblb2 is identified to target a papain-like cysteine protease (PLCPs) C14
and prevent it from secreting into the apoplast, therefore, target to PLCP might
suggest a new strategy for pathogens to counter host plant defense proteases
(Bozkurt et al., 2012).
2. HYPOTHESES AND OBJECTIVES
Among all identified AVR proteins which involve in race-specific resistance, only
AvR3a-meidated virulence function (Bos et al., 2010) and AVR2-meidated avirulence
function (Saunders et al., 2012) are identified. Next, we explore to elucidate the
virulence function of P. infestans effector AVR1. Though R1 gene is the first cloned
late blight R gene from S.demissum (Ballvora et al., 2002) and avr1 gene was already
being isolated (van der Lee et al., 2001; Tyler et al 2009), the mode of action of
AVR-mediated virulence is still unclear (Stassen and Ackerveken, et al, 2011). AVR1
also belongs to RXLR type oomycete effectors with the modular structure including a
signal peptide and a conserved RXLR motif in its N-termini (Vivianne et al., 2011) and
a effector domain in C-termini carrying biochemical activity (Win et al., 2007).
Besides, avr1, just like other P. infestans Avr genes avr3a, avr2, avr4 etc., is highly
induced in the biotrophic phase of infection (Vivianne et al., 2011). AVR1 resides in a
repeat-rich and expanded region in P. infestans , with only one allele in P. infestans
strain T30-4 (Vleeshouwers et al., 2011).Segregation ratios for vir/avir in potato lines
carrying the R1 is around 1:1 in the progeny of P. infestans (Lee et al., 2001). AVR1
confers avirulence function when it is recognized by R1, in other case, it also triggers
virulence function in these plants without R1, probably by recognizing other host
proteins. To elucidate the molecular process underlying virulence for host plants, we
will identify these AVR1 interacting proteins (AVR1-IP) that mediate AVR1-triggered
suppression of host defense. In this study, our groups will focus on determining the
following research objectives:
Objective 1. Screen the interaction proteins of AVR1 (AVR1-IP) in potato cDNA
library and identify the interaction model of AVR1 and AVR1-IP. Working hypothesis:
yeast-two-hybrid (Y2H) will be applied to screen AVR1 interacting proteins (AVR1-IP);
The C-terminus of AVR1 interacts with AVR1-IP and manipulates host resistance.
Objective 2. Biochemical activity analyses of AVR1 and AVR1-IP. Working hypothesis:
AVR1-IP may serve as a positive regulator or a negative regulator for host immunity;
biologic activities of AVR1-IP are manipulated upon recognition by AVR1; however,
AVR1-IP may not involve in R1-AVR1 recognition.
Objective 3. Investigate the effects of dimerization on full activity of AVR1. Working
hypothesis: pathogen effectors AVR1 may form dimers for its full activity; the
homo-dimerization of AVR1might be important to its translocation activity.
Objective 4. Demonstrate the pathogenesis mechanism of AVR1 in regulating host
protein activities and manipulating host defense. Working hypothesis: AVR1-IP
knockdown and overexpressed potato lines are infiltrated with wild type P. infestans
strain and AVR1 mutated strain to investigate the pathogenesis mechanism of AVR1.
3. RATIONALE AND SIGNIFICANCE
P.infestants translocates AVR effectors into plant cells, the resistant host plants
express corresponding R proteins to recognize AVR effectors and trigger defense
response; in other case, AVR effectors will manipulate host protein activities and
suppress host defense. For example, AVR3a, targets host defense-related E3 ubiquitin
ligase CMPG1 through its C-terminus effector domain, leads to suppressing
INF1-triggered hypersensitive response (HR). Another effector AVRblb2, which
confers avirulence function when it was recognized by R protein Rpi-blb2, but
counters host defense papain-like cysteine protease (PLCPs) in absence of Rpi-blb2.
AVR1 shares the modular structure with other AVR effectors in P. infestans, with a
C-terminal effector domain to confer biochemical activity. We anticipate that
C-terminus of AVR1 is also responsible for suppression of host defense, in doing so; it
may target a series of host defense-related proteins (ubiquitin ligases, kinases,
phosphatases etc.) and manipulate their activities. The elucidation of mode of action
of AVR3a-mediated suppression of host defense is significant for people to better
understand the effectors’ virulence function, as well as provide new insights into the
mechanisms by which P. infestans invades host plants, leading to development of
better strategies to control P. infestans in potato.
4. PRELIMINARY RESULTS
4.1 AVR1 gene isolation. AVR1 (627nt) is isolated by PCR amplification from the
Phytophthora infestans strain T30-4. It encodes a 208 aa polypeptide, with a modular
structure includes N-terminal signal peptide domain, RxLR motif (RQLR), dEER motif
(DEAR) and C-terminal effector domain.
4.2 Structure and expression pattern of Phytophthora Avr gene products.
Fi g 1. Fea tures of cha racteri zed Phytophthora Avr gene products . The figure depi cts AVR1, AVR2, AVR3a , AVR4,
AVRblb1, AVRbl b2, and AVRvnt1. The domain s tructure of P. infestans AVR proteins shows a typi cal RXLR effector
modular s tructure wi th N-terminal (signal peptide) domain, RXLR moti f, and C-terminal effector domain. The
N-terminal domain functions in secreti on and host transloca tion whereas the va riable C-terminal domain ca rries
the effector bi ochemi cal a cti vi ty. Expression in pota to panels illustra te a ti me course expression pattern of the Avr
genes during infecti on of pota to [2–5 days pos t infection (dpi)], wi th the y-a xis showing gene induction. Ea ch of
the Avr genes is ma ximall y induced at 2 dpi in potato during the ea rl y phase of the disease. Genome envi ronment
hea t maps a re two-di mensional plots of 5 and 3 intergeni c distance for all P. infestans genes. The Avr genes reside
in the gene sparse regi ons (upper right corner of the map) wi th longer distance to thei r nei ghboring genes .
(Vi vi anne et a l., 2011)
4.3 AVR1 proteins 3-D structure prediction and functional analysis
BY employing on-line protein structure and function predictions platform I-TASSER
(http://zhanglab.ccmb.med.umich.edu/I-TASSER/), we predicted the 3D structure of
AVR1, which shares relatively higher similarity with another P. infestans effector
protein AVR3a. The Top 1 Model predicted by I-TASSER was shown here:
Fi g 2. 3-D s tructure of AVR1
5. EXPERIMENTAL PLANS AND EXPECTED RESULTS
5.1. Objective 1. Screen the interaction proteins of AVR1 (AVR1-IP) in
potato cDNA library and assay the interaction model of AVR1 and
AVR1-IP
5.1.1.INTRODUCTION . AVR1 from Phytophthora infestans strain T30-4 encodes a
modular structure protein, with a conserved RxLR motif (RQLR) and a conserved
dEER motif (EDAR) in its N-termini, and an effector domain in C-termini dedicated to
virulence function. By doing so, AVR1 targets certain host proteins and modulates the
host immune system. The identification of the AVR1 target proteins is pivotal for
revealing the mode of action of AVR1-mediated suppression of host defense.
5.1.2.EXPERIMENTAL DESIGN
STUDY #1. Yeast-two-hybrid (Y2H) screen the AVR1 host targets (AVR1-IP) in Solanum
tuberosum cDNA library. Potato (Solanum tuberosum L) cultivar Bintjeto is a
Netherland variety that is highly susceptible to late blight (Phytophthora infestan).
We will construct the cDNA library from Bintjeto following the instruction of yeast
two-hybrid Library Construction kit (Clontech Inc.). AVR1 is cloned from P. infestans
strain T30-4 and ligated in GAL4-BD vector PGBK T7. Then, we use PGBK T7-AVR1 as
bait to screen the Bintjeto cDNA library (fused with PGAL4-2.1 vector) in yeast strain
AH109, and confirm the positive colons by X-GAL stain. The positive yeast clones are
transformed into E.coli strain X-blue and sequenced by chain-termination sequencing
method. The candidate AVR1 interacting proteins (AVR1-IP) are decided through
bioinformatic analysis.
STUDY #2. Confirm the interaction between AVR1 and AVR1-IP through GST
pull-down assay in vitro. GST-AVR1 fusion proteins are purified through glutathione
agarose. The AVR1-IP proteins fused with maltose-binding protein (MBP) tag and
3*FLAG tag are purified by using amylose columns. The GST pull down assay is
performed to detect the pull-down products of AVR1 through anti-FLAG antibody.
The interaction between AVR1 and AVR1-IP will be examined in vitro.
STUDY #3. Confirm the interaction between AVR1 and AVR1-IP through Bimolecular
Fluorescence Complementation (BiFC) assay in vivo, as well as investigate the
homodimers formations in AVR1 and AVR1-IP. We will design primers to amplify
AVR1 and AVR1-IP fused with different epitope tag (AVR1-3*MYC; AVR1-IP-3*FLAG)
and TAG stop codon, then AVR1-3*MYC; AVR1-IP-3*FLAG are ligated into BiFC vector
PXY105 (NYFP) and PXY106 (CYFP). The interaction between AVR1 and AVR1-IP will
be examined by co-infiltration of PXY105-AVR1-3*MYC and PXY106-(AVR1-IP)-3*FLAG
in N.benthamiana through Agrobactrium-mediated transformation. To determine the
homodimers formation in AVR1 and AVR1-IP, we co-infiltrate PXY105-AVR1-3*MYC/
PXY106-AVR1-3*MYC pair and PXY105-(AVR1-IP)-3*FLAG/ PXY106-(AVR1-IP)-3*FLAG
pair in N.benthamiana. The validation of BiFC florescence is determined by
immunoblotting with specific primary antibodies (α-MYC for AVR1, α-FLAG for
AVR1-IP). The localizations of AVR1 and AVR1-IP are also indicated in the BiFC assay.
STUDY #4. AVR1-IP structure and function predictions. On line protein structure and
function predictions platform I-TASSER (http://zhanglab.ccmb.med.umich.edu/
I-TASSER/) is applied to predict the 3D structure and functional domains of AVR1-IP.
STUDY #5. Determine the interacting domains in AVR1 and AVR1-IP that are
responsible for AVR1 and AVR1-IP interaction. We will use deletion assay combine
with Y2H to determine the interaction domains in AVR1 and AVR1-IP. The N-terminal
fragment including signal peptide (SP), RxLR motif and dEER motif and the C-terminal
fragment including effector domains are cloned and ligated into PGBK T7, different
AVR1-IP fragments with deletion on each predicted motif are ligated into GAL4-AD
vector PGAD T7. The minimal interacting motifs on these two proteins will be
determined by co-transforming each AVR1 fragments and each AVR1-IP fragments
5.1.3. EXPECTED OUTCOMES.
By using Y2H assay, we expect to screen a group of AVR1 interacting proteins
(AVR1-IP), including these defense-related host proteins, based on previous reports
on race-specific AVR effectors (Bos et al., 2010; Chen et al., 2012). The physical
interaction between AVR1 and AVR1-IP will be determined by GST pull-down assay
and BiFC assay.
Besides, we expect that AVR1 may also involve in homodermization in vivo following
previous research (Wawra et al., 2012). Structure predictions by I-TASSER and BLAST
search may suggest the possible activities of AVR1-IP. Based on the previous study on
AVR3a (Armstrong et al., 2005) and AVR2 (Saunders et al., 2012), we expect that
AVR1 is also localized in cytoplasm and the C-terminus of AVR1 is responsible for the
interaction between AVR1 and AVR1-IP.
5.1.4. ANTICIPATED PROBLEMS AND ALTERNATIVE STRATEGIES.
By using Y2H to screen the host targets suffers certain problems, especially for these
low abundance proteins or these unstable proteins. For these issues, we may apply
another approach called planta co-IP followed by liquid chromatography-tandem
mass spectrometry (LC-MS/MS) (Win et al., 2011). We will fuse FLAG epitope tag at
N-termini of AVR1 instead of signal peptide and RxLR domain, and then perform
co-IP in N.benthamiana with transient expression of this construct. The candidate
host target proteins of AVR1 will be further confirmed by GST pull down assay.
Besides, the probably low affinity between AVR1 and AVR1-IP in BiFC assay may
hinder us to examine the homodimerization of AVR1 or AVR1-IP. In this case, we may
apply other methods like Fluorescence resonance energy transfer (FRET) (Pollok et al.,
1999).
5.2 Objective 2. Biochemical activity analyses of AVR1 and AVR1-IP
5.2.1 INTRODUCTION. AVR1 secreting into plant cells is dependent on the
N-terminal RXLR leader, and the C-terminus effector domain carries biochemical
activities. We propose that C-terminus of AVR1 will interact with AVR1-IP and
mediate the virulence function. To determine the model of interaction between
AVR1 and AVR1-IP as well as AVR1-mediated virulence, we apply PCR-based
site-directed mutagenesis to mutate these key residues in AVR1 that mediate
AVR1/AVR1-IP interaction and trigger virulence.
5.2.2. EXPERIMENTAL DESIGN
STUDY #1. Virulence assay of P. infestans effector AVR1. We will express resistance
protein R1 (35S-R1) and effector protein AVR1 (35S-AVR1) in N. benthamiana
through Agrobacterium-mediated transformation to detect the effect of our AVR1
protein. To demonstrate the virulence function of AVR1, we will examine whether
AVR1 will suppress the cell death induced by PtoY207D (Rathjen et al., 1999), elicitin
INF1 (Kamoun et al., 2003), CRN2 (Torto et al., 2003), and AvrBs3 (Bonas et al. 1991)
in N.benthamiana. We first infiltrate 35S-AVR1 in N.benthamiana with 35S-GFP as
control, after 1 day post inoculation (dpi), the infiltration sites are challenged with
Agrobacterium tumefaciens strains EHA105 transformed with 35S-PtoY207D,
35S-INF1, 35S-CRN2 and 35S-AvrBs3, the cell death symptom are scored from 2dpi to
5dpi. We propose that AVR1 might attenuate the HR response caused by certain HR
inducers, thereafter, AVR1-HRD (HR down-regulation).
STUDY #2. Mutate candidate residues within AVR1 which are responsible for
AVR1/AVR1-IP interaction and AVR1-mediated virulence. Based on deletion assay and
I-TASSER server prediction, we predict these candidate residues (67, 119, 151) and
mutate them through PCR-based site-directed mutagenesis. The interactions
between each AVR1 point mutations and AVR1-IP are tested by Y2H assay. These
double-residue mutants are also examined. To test the effects of each point mutation
on virulence function of AVR1, we co-express these AVR1 mutants and the specific
AVR1-HRD (HR inducer that shown to be suppressed by AVR1 in triggering HR) in
N.benthamiana, and score the extent of cell death from 2 dpi to 6dpi.
STUDY #3. Determine the extent to which AVR1-IP is involved in AVR1/AVR1-IP
recognition. First of all, we will examine the biochemical activities of AVR1-IP. Then
we will perform VIGS of AVR1-IP in N.benthamiana to examine the roles of AVR1-IP in
AVR1-mediated suppression of host defense. An N-terminal fragment specific for
AVR1-IP is cloned and expressed in tobacco rattle virues (TRV) VIGS-based silencing
system (Ratcliff et al., 1999). The effect of gene silence induced by TRV/AVR1-IP is
assayed through monitoring the decrease of AVR1-IP transcripts but not of its
orthologs (AVR2, AVR3a, AVR3b, AVR4). To examine whether AVR1-mediated
suppression of cell death is dependent on AVR1-IP or not, the N.benthamiana
expressing TRV/AVR1-IP constructs are co-infiltrate with 35S-AVR1 and the specific
AVR1-HRD, with TRV-GFP plants as control. To examine whether AVR1-IP silence will
affect other AVR effectors-mediated virulence, we co-express the effector protein
35S-AVR3a and the 35S-INF1 (INF1-triggered HR was strongly suppressed by AVR3a,
depends on host protein CMPG1) (Bos et al., 2010) in TRV/AVR1-IP plants and the
TRV/GFP control plants.
STUDY #4. Determine whether AVR1-IP is involved in AVR1 recognition by R1. First,
we will examine if AVR1-IP interacts with host resistance protein R1 through Y2H
assay by co-transformation of PGBK T7/AVR1-IP and PGAD T7/R1 in yeast strain
AH109. Second, we will examine if the recognition of AVR1 by R1 is attenuated in
AVR1-IP silenced N.benthamiana plants (TRV/AVR1-IP as described in STUDY #3). For
this purpose, we co-express 35S-AVR1 and 35S-R1 in TRV/AVR1-IP plants and
TRV/GFP plants (control). After 2 to 6 days post infiltration, we observe and score the
development of HR caused by AVR1-R1 recognition.
5.2.3. EXPECTED OUTCOMES
In study #1, we expect AVR1 may suppress the cell death caused by one or more than
one HR inducers, which displays the virulence function of AVR1 after translocation
into plant cells. In STUDY #2, we expect that at least two residues are key amino acids
that are responsible for virulence function of AVR1, based on the previous reports
(Bos et al., 2006; Bos et al., 2010). In STUDY #3, , we propose that AVR1-IP may
function as a kinase, an E3 ubiquitin ligase or a phosphatase etc., as described in
prior research (Bos et al., 2010; Saunders et al., 2012). And if AVR-IP is a positive
regulator for host immunity, we expect that the suppression of HR will be enhanced
in TRV/AVR1-IP plants, and vice versa. However, the AVR1-IP silenced plants do not
attenuate the suppression of HR caused by AVR3a (AVR3a mediated virulence is
dependent on CMPG1), which suggests the specific interaction between AVR1 and
AVR1-IP. In STUDY #4, we expect that AVR1-IP doesn’t direct interact with R1 in Y2H
assay, as well AVR1-IP silence doesn’t attenuate the R1-mediated resistance, which
suggests that AVR1-IP is not involved in R1/AVR1-mediated defense response, in
other words, AVR1-IP is not a “guardee” mediating recognition of AVR1 by R1.
5.2.4 ANTICIPATED PROBLEMS AND ALTERNATIVE STRATEGIES.
AVR1 serves to suppress host defense but not always suppress HR, so we may
employ some other strategies to detect the virulence function, such as by assaying P.
infestans population in potato plants. Besides, if the VIGS of AVR1-IP doesn’t lead to
specific decrease in AVR1-IP transcripts, we may create knock down transgenic plants
through Agrobacterium-mediated transformation of PANDA/AVR1-IP. The effects of
AVR1-IP silence on AVR1-mediated virulence will be further examined.
5.3 Objective 3. Investigate the effects of dimerization on full activity
of AVR1
5.3.1 Introduction. Dimerization is widely found and plays an important role in
biology including pathogenesis of pathogen effector in planta (Marianayagam et al.,
2004; Deshaies et al., 2009); Recent report shown that AVR3a self-associated with
each other through these amino acids surrounding the RxLR translocation region
(Wawra et al., 2012), suggests that dimerization on RxLR motif might be a common
feature for these AVR effectors. In this study, we will examine if this dimerization on
RxLR motif is required for translocation activities of AVR1.
5.3.2. EXPERIMENTAL DESIGN
STUDY #1. Examine if single residue mutation on RxLR motif will affect translocation
of AVR1 into host cells. Previous study shows that single mutation on RxLR motif will
abolish the translocation activity in AVR3a (Whisson et al., 2007). We will next test if
it is also applicable for AVR1. A series of mutants on RxLR motif are created by PCRbased mutagenesis (RQLR->KQLR, RKLR, RQMR, RQLI, KKMI) (Whisson et al., 2007),
then we will fuse the native RQLR motif and each of these RxLR motif mutants with
gusA gene (GUS gene). GUS was chosen because it is only active in plant cells but not
in apoplast. After that, we will infect potato cultivar Bintje (R gene free) with P.
infestans RGUS9 stably expressing these six constructs. The GUS distribution will be
assayed two days after infection through light microscopy, which indicates
translocation activity of AVR1.
STUDY #2. Test the role of dimerization on AVR1 translocation. Previous study shows
that replacement of all four residues in RxLR motif will abolish the self-association
ability within AVR effector (Whisson et al., 2007; Wawra et al., 2012), we next
explore to investigate whether the abolishment of translocation activity due to single
residue mutation in RQLR motif will still carry self-association activity in AVR1. For
this purpose, we ligate each of these RxLR mutated AVR1 protein (KQLR, RKLR, RQMR,
RQLI, KKMI) and native AVR1 into GAL4-AD yeast vector PGAD T7, then test their
interactions with GAL4-BD construct PGBK T7/AVR1 through Y2H assay. The
self-association activity will be examined by four drop-out growth assay and X-GAL
assay.
5.3.3 EXPECTED OUTCOMES
In STUDY #1, we expect that the mutations on each residue in RxLR motif will abolish
the translocation activity of AVR1, as shown by GUS activity. And native RxLR
construct exhibits GUS activity which lies in these plant cells in contact with
haustorium. In STUDY #2, we expect that the abolishment of translocation activity
due to single residue mutation remains abolished self-association ability in AVR1,
which suggests that dimerization may correlate with RxLR-mediated translocation.
5.3.4 ANTICIPATED PROBLEMS AND ALTERNATIVE STRATEGIES
To better visualize the AVR1 translocation, we may employ another strategy by
examining if AVR1 could be recognized in R1-background potato lines. Since
recognition of AVR1 by R1 is independent of the RxLR motifs , the cell death in R1
-background potato plants suggests AVR1 is translocated into plant cells (Whisson et
al., 2007).
5.4.
Objective 4. Demonstrate the pathogenesis mechanism of AVR1
in regulating host protein activities and manipulating host defense
5.4.1 INTRODUCTION. This objective of our study is to determine the mode of
action of AVR1-triggered suppression of host defense as described in objective 2 and
3. For this purpose, we will apply RNAi to create AVR1-IP knockdown potato Bintjeto
(R gene free) lines. Also the AVR1-IP overexpressed Bintjeto lines are acquired by
Agrobacterium-mediated transformation of 35S-AVR1-IP. P. infestans wild type strain
T30-4 and AVR1 mutation strain T30-4△AVR1 are employed to assay the virulence
function of AVR1.
5.4.2. EXPERIMENTAL DESIGN
STUDY #1. Construct AVR1-mutated P. infestans strain. We create AVR1-mutated P.
infestans T30-4 strain by the overlap PCR and replacement of gene coding sequence
with selection marker gene (Song et al., 2010). The effects of mutation on AVR1
(designed T30-4△AVR1) will be test by inoculating with R1-background potato plants.
STUDY #2. AVR1-IP knockdown Bintjeto lines construction and virulence analysis. The
AVR1-IP knockdown vector is constructed by ligating the C-terminus of AVR1-IP into
RNAi vector PANDA through Gateway technology kit (Sigma), then PANDA/AVR1-IP is
transformed into Bintjeto plants through Agrobacterium-mediated transformation.
AVR1-IP transcript levels in silenced plants are measured by quantitative RT-PCR.
Then PANDA/AVR1-IP lines and wild type lines will be challenged with P. infestans
strain T30-4 and mutant strain T30-4△AVR1, the virulence level of P. infestans is
scored by measuring P. infestans populations per area.
STUDY #3. AVR1-IP overexpressed lines construction and virulence analysis. AvR1-IP
overexpressed lines are constructed through Agrobacterium-mediated
transformation of 35S-AVR1-IP into Bintjeto. Then transgenic lines and wild type line
will be inoculated with T30-4 strain and T30-4△AVR1 strain, the levels of virulence
are measured by the population of P .infestans.
5.4.3 EXPECTED OUTCOMES
We expect that AVR1-IP silenced Bintjeto lines and AVR1-IP over-expressed Bintjeto
lines show opposite phenotype upon infiltration by T30-4. However, upon
T30-4△AVR1 infiltration, there are no significant difference between transgenic
plants and wild type plants. These assays on transgenic potato plants demonstrate
that AVR1-mediated suppression of host defense is dependent on host AVR1-IP
proteins.
5.4.4. ANTICIPATED PROBLEMS AND ALTERNATIVE STRATEGIES
Construct transgenic
Bintjeto plants
through Agrobacterium-mediated
transformation may suffers certain problems like low transformation efficiency. To
deal with this problem, we may employ microparticle bombardment method (Taylor
et al., 2004).
TIMETABLE OF THE WORK PLAN
Objective
Year 1
Year 2
Year 3
Objective 1 .Screen the interaction
proteins of AVR1 (AVR1-IP) and assay the
interaction model of AVR1 and AVR1-IP
Objective 2. Biochemical activity
analyses of AVR1 and AVR1-IP
Objective 3.Investigate the effects of
dimerization on full activity of AVR1
Objective4.
Demonstrate
the
pathogenesis mechanism of AVR1 in
regulating host protein activities and
manipulating host immunity
Dec. 2012—Jul. 2013:
1. Y2H screen AVR1 interacting proteins (AVR1-IP), decide candidate genes for
AvR1-IP.
2. Confirm the interaction between AVR1 and AVR1-IP through the pull-down assay
and the BiFC assay.
Aug. 2013-Oct. 2014:
1. AVR1 and AVR1-IP interacting domain detection
2. AVR1 virulence assay
3. AVR1-IP biochemical activities analysis
4. Construct AVR1-IP over expression transgenic plants and know-down transgenic
plants
Jun. 2014-Jan. 2015:
1. Examine the effects of dimerization on AVR1 translocation
2. Construct AVR1-mutated P. infestans strain
Sep. 2013-Dec. 2015:
1. Knockdown and overexpressed AVR1-IP transgenic plants assay
2. Investigate the model of action of AVR1 on AVR1-IP
3. Demonstrate the pathogenesis mechanism of AvR1 on manipulate the host
immunity.
4. Publications
RELEVANT BIBLIOGRAPHY
Armstrong, M.R., Whisson, S.C., Pritchard, L., Bos, J.I.B., Kamoun, S., Beynon, J.L., and
Birch, P.R.J. (2005). An ancestral oomycete locus contains late blight avirulence gene
Avr3a, encoding a protein that is recognized in the host cytoplasm. Proceedings of
the National Academy of Sciences of the United States 102, 7766-7771.
Ballvora, A., Ercolano, M.R., Wei, J., Meksem, K., Bormann, C.A., Oberhagemann, P.,
Salamini, F., and Gebhard, C. (2002). The R1 gene for potato resistance to late blight
(Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant
resistance genes. The Plant Journal 30, 361-371.
Bonas, U., Schulte, R., Fenselau, S., Minsavage, G.V., Staskawicz, B.J., and Stall, R.E.
(1991). Isolation of a gene cluster from Xanthomonas campestris pv. vesicatoria that
determines pathogenicity and the hypersensitive response on pepper and tomato.
Molecular Plant-Microbe Interactions 4, 81-88.
Bos, J.I.B., Kanneganti, T.-D., Young, C., Cakir, C., Huitema, E., Win, J., Armstrong, M.R.,
Birch, P.R.J., and Kamoun, S. (2006). The C-terminal half of Phytophthora infestans
RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and
suppress INF1-induced cell death in Nicotiana benthamiana. The Plant Journal 48,
165-176.
Bos, J.I.B., Armstrong, M.R., Gilroy, E.M., Boevink, P.C., Hein, I., Whisson, S.C.,
Kamoun, S., and Birch, P.R.J. (2010). Phytophthora infestans effector AVR3a is
essential for virulence and manipulates plant immunity by stabilizing host E3 ligase
CMPG1. Proceedings of the National Academy of Sciences of the United States 107,
9909-9914.
Bouwmeester, K., Meijer, H.J.G., and Govers, F. (2011). At the frontier: RXLR effectors
crossing the Phytophthora-host interface. Frontiers in Plant Science 2, 1-8.
Bozkurt, T.O., Schornack, S., Win, J., Shindo, T., Ilyas, M., Hoorn, R.A.L.v.d., and
Kamoun, S. (2012). Phytophthora infestans effector AVRblb2 prevents secretion of a
plant immune protease at the haustorial interface. Proceedings of the National
Academy of Sciences of the United States 108, 20832-20837.
Bozkurt, T.O., Schornack, S., Banfield, M.J., and Kamoun, S. (2012). Oomycetes,
effectors, and all that jazz. Current Opinion in Plant Biology 15, 483-492.
Chen, Y., Liu, Z., and Halterman, D.A. (2012). Molecular Determinants of Resistance
Activation and Suppression by Phytophthora infestans Effector IPI-O. PLOS Pathogens
8, e1002595.
Chisholm, S.T., Coaker, G., Day, B., and Staskawicz, B.J. (2006). Host-microbe
interactions: Shaping the evolution of the plant immune response. Cell 124, 803-814.
Deshaies, R.J., and Joazeiro, C.A.P. (2009). RING Domain E3 Ubiquitin Ligases. Annual
Review of Biochemistry 78, 399-434.
Dong, S., Yin, W., Kong, G., Yang, X., Tyler, B.M., and Wang, Y. (2011). Phytophthora
sojae Avirulence Effector Avr3b is a Secreted NADH and ADP-ribose
Pyrophosphorylase that Modulates Plant Immunity. PLOS Pathogens 7.
Dou, D., Kale, S.D., Wang, X., Jiang, R.H.Y., Bruce, N.A., Arredondo, F.D., Zhang, X., and
Tyler, B.M. (2008). RXLR-mediated entry of Phytophthora sojae effector Avr1b into
soybean cells does not require pathogen-encoded machinery. The Plant Cell 20,
1930-1947.
Gilroy, E.M., Taylor, R.M., Hein, I., Boevink, P., Sadanandom, A., and J.Birch, P.R.
(2011). CMPG1-dependent cell death follows perception of diverse pathogen elicitors
at the host plasma membrane and is suppressed by Phytophthora infestans RXLR
effector AVR3a. New Phytologist 190, 653-666.
Govers, F., and Gijzen, M. (2006). Phytophthora Genomics: The Plant Destroyers'
Genome Decoded. Molecular Plant-Microbe Interactions 19, 1295-1301.
Haas, B.J., Kamoun, S., Zody, M.C., Jiang, R.H.Y., Govers, F., Birch, P.R.J., Whisson, S.C.,
Judelson, H.S., and Nusbaum, C. (2009). Genome sequence and analysis of the Irish
potato famine pathogen Phytophthora infestans. Nature 461, 393-398.
Huang, S., Vossen, E.A.G.v.d., Kuang, H., Vleeshouwers, V.G.A.A., Zhang, N., Borm,
T.J.A., Eck, H.J.v., Baker, B., Jacobsen, E., and Visser, R.G.F. (2005). Comparative
genomics enabled the isolation of the R3a late blight resistance gene in potato. The
Plant Journal 42, 251-261.
Kale, S.D., Gu, B., Capelluto, D.G.S., Dou, D., Feldman, E., Rumore, A., Arredondo, F.D.,
Hanlon, R., Fudal, I., Rouxel, T., Lawrence, C.B., Shan, W., and Tyler, B.M. (2010).
External
lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and
animal host cells. Cell 142, 284-295.
Kemen, E., Gardiner, A., Schultz-Larsen, T., Kemen, A.C., Balmuth, A.L.,
Robert-Seilaniantz, A., Bailey, K., Holub, E., Studholme, D.J., MacLean, D., and Jones,
J.D.G. (2011). Gene gain and loss during evolution of obligate parasitism in the
white rust pathogen of Arabidopsis thaliana. PLoS Biology 9, e1001094.
Lamour, K., and Kamoun, S. (2009). Oomycete Genetics and Genomics: Diversity,
Interactions and Research Tools. (Wiley-Blackwell).
Lee, T.v.d., Robold, A., Testa, A., Klooster, J.W.v.t., and Govers, F. (2001). Mapping of
avirulence genes in Phytophthora infestans with amplified fragment length
polymorphism markers by bulk segregant analysis. Genetics 157, 3949-3956.
Lévesque, C.A., Brouwer, H., Cano, L., Hamilton, J.P., Holt, C., Huitema, E., Raffaele, S.,
Robideau, G.P., Thines, M., Win, J., Zerillo, M.M., Tyler, B.M., Vries, R.P.D., Kamoun, S.,
Yandell, M., Tisserat, N., and Buell, C.R. (2010). Genome sequence of the
necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity
mechanisms and effector repertoire Genome Biology 11, R73.
lez-Lamothe, R., Tsitsigiannis, D.I., Ludwig, A.A., Panicot, M., Shirasu, K., and Jones,
J.D.G. (2006). The U -Box Protein CMPG1 Is Required for Efficient Activation of
Defense Mechanisms Triggered by Multiple Resistance Genes in Tobacco and Tomato.
The Plant Cell 18, 1067-1083.
Marianayagam, N.J., Sunde, M., and Matthews, J.M. (2004). The power of two:
protein dimerization in biology. Trends in Biochemical Sciences 29.
Morgan, W., and Kamoun, S. (2007). RXLR effectors of plant pathogenic oomycetes.
Current Opinion in Microbiology 10, 332-338.
Oh, S.-K., Young, C., Lee, M., Oliva, R., Bozkurt, T.O., Cano, L.M., Win, J., Bos, J.I.B., Liu,
H.-Y., Damme, M.v., Morgan, W., Choi, D., Vossen, E.A.G.V.d., Vleeshouwers, V.G.A.A.,
and Kamouna, S. (2009). In Planta Expression Screens of Phytophthora infestans RXLR
Effectors Reveal Diverse Phenotypes, Including Activation of the Solanum
bulbocastanum Disease Resistance Protein Rpi-blb2. The Plant Cell 21, 2928-2947.
Pollok, B.A., and Heim, R. (1999). Using GFP in FRET-based applications. Trends in Cell
Biology 9, 57-60.
Rathjen, J.P., Chang, J.H., Staskawicz, B.J., and Michelmore, R.W. (1999).
Constitutively active Pto induces a Prf-dependent hypersensitive response in the
absence of avrPto. The EMBO Journal 18, 3232-3240.
Rehmany, A.P., Gordon, A., Rose, L.E., Allen, R.L., Armstrong, M.R., Whisson, S.C.,
Kamoun, S., Tyler, B.M., Birch, P.R.J., and Beynon, J.L. (2005). Differential Recognition
of Highly Divergent Downy Mildew Avirulence Gene Alleles by RPP1 Resistance Genes
from Two Arabidopsis Lines The Plant Cell 17, 1839-1850.
Saunders, D.G.O., Breen, S., Win, J., Schornack, S., Hein, I., Bozkurt, T.O., Champouret,
N., Vleeshouwers, V.G.A.A., Birch, P.R.J., Gilroy, E.M., and Kamoun, S. (2012). Host
Protein BSL1 Associates with Phytophthora infestans RXLR Effector AVR2 and the
Solanum demissum Immune Receptor R2 to Mediate Disease resistance. The Plant
Cell, 10.1105/tpc.1112.099861.
Schornack S, v.D.M., Bozkurt TO, Cano LM, Smoker M, Thines M, Gaulin E, Kamoun S,
Huitema E. (2010). Ancient class of translocated oomycete effectors targets
the host nucleus Proceedings of the National Academy of Sciences of the United
States 107, 17421-17426.
Stassen, J.H., and Ackerveken, G.V.d. (2011). How do oomycete effectors interfere
with plant life? Current Opinion in plant biology 14, 407-414.
Taylor, N.J., and Fauquet, C.M. (2004). Microparticle Bombardment as a Tool in Plant
Science and Agricultural Biotechnology. DNA and Cell Biology 21, 963-977.
Torto, T.A., Li, S., Styer, A., Huitema, E., Testa, A., Gow, N.A.R., West, P.v., and Kamoun,
S. (2003). EST Mining and Functional Expression Assays Identify Extracellular Effector
Proteins From the Plant Pathogen Phytophthora. Genome Research 13, 1675-1685.
Vleeshouwers, V.G.A.A., Raffaele, S., Vossen, J.H., Champouret, N., Oliva, R., Segretin,
M.E., Rietman, H., Cano, L.M., Lokossou, A., Kessel, G., Pel, M.A., and Kamoun, S.
(2011). Understanding and Exploiting Late Blight Resistance in the Age of Effectors.
Annual Review of Phytopathology 49, 507-531.
Wawra, S., Agacan, M., Boddey, J.A., Davidson, I., Gachon, C.M.M., Zanda, M.,
Grouffaud, S., Whisson, S.C., Birch, P.R.J., Porter, A.J., and West, P.v. (2012). The
avirulence protein 3a (AVR3a) from the potato pathogen Phytophthora
infestans, forms homodimers through its predicted translocation region and does
not specifically bind phospholipids In The Journal of Biological Chemistry. In press.
Whisson, S.C., Boevink, P.C., Moleleki, L., Avrova, A.O., Morales, J.G., Gilroy, E.M.,
Armstrong, M.R., Grouffaud, S., West, P.v., Chapman, S., Hein, I., Toth, I.K., Pritchard,
L., and Birch, P.R.J. (2007). A translocation signal for delivery of oomycete effector
proteins into host plant cells. Nature 450, 115-118.
Win, J., Morgan, W., Bos, J., Krasileva, K.V., Cano, L.M., Chaparro-Garcia, A., Ammar,
R., Staskawicz, B.J., and Kamoun, S. (2007). Adaptive Evolution Has Targeted the
C-Terminal Domain of the RXLR Effectors of Plant Pathogenic Oomycetes. The Plant
Cell 19, 2349-2369.
Win, J., Kamoun, S., and Jones, A.M.E. (2011). Purification of Effector–Target Protein
Complexes via Transient Expression in Nicotiana benthamiana. Methods in Molecular
Biology 712, 181-184.
Project Budget Summary
1. Salaries and Social Benefits
2. Non-expendable Equipment
3. Operating Expenses
4. Travel
Total Direct Costs
5. Overhead Expenses (20%)
Annual Totals
1. Salaries and Social Benefits
2. Non-expendable Equipment
3. Operating Expenses
4. Travel
Total Direct Costs
5. Overhead Expenses
Project Totals
(In US dollars)
First Year
76000
0
33700
0
109700
21940
121640
Project Totals
228600
0
93700
3500
325800
65160
390960
Second Year
76200
0
25900
500
102600
20520
123120
Third Year
76400
0
34100
3000
113500
22700
136200
BUDGET DETAILS
A. Personnel
Name (last, first)
Role
%
time
Post Doc
Graduate Student
Technician
Total salaries
Post Doc
Graduate Student
Technician
100
100
20
Salaries
(in US$)
1st year
45000
23000
8000
76000
Salaries
(in US$)
2nd year
45000
23000
8200
76200
Salaries
(in US$)
3rd year
45000
23000
8400
76400
B. Operating expenses (in US$)
Item
Requested sums
1st year
Plant growing supplies
2000
Chemicals,
Molecular 15000
biology reagents
DNA
primers
and 10000
sequencing
Glass and plastic ware
2500
Services for publication
0
Tuition
4200
Total
supplies
and 33700
materials
Requested sums
2nd year
2000
15000
Requested sums
3rd year
2000
20000
2000
2000
2500
0
4400
25900
2500
3000
4600
34100
Requested sums
1st year
76000
0
Requested sums
2nd year
76200
0
Requested sums
3rd year
76400
0
33700
0
109700
21940
121640
25900
500
102600
20520
123120
34100
3000
113500
22700
136200
C. Budget summary (in US$)
Item
Personnel
Non-expendable
equipment
Operating expenses
travel
Sub total
Overhead (20%)
Grand total