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A Comprehensive Mutational Analysis of the Arabidopsis
Resistance Protein RPW8.2 Reveals Key Amino Acids for
Defense Activation and Protein Targeting
W
Wenming Wang,a,b,1 Yi Zhang,b,1 Yingqiang Wen,b,c Robert Berkey,b Xianfeng Ma,a,b Zhiyong Pan,b,d
Dipti Bendigeri,b Harlan King,b Qiong Zhang,b and Shunyuan Xiaob,e,2
a Rice
Research Institute, Sichuan Agricultural University, Chengdu 611130, China
for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850
c State Key Laboratory of Crop Stress Biology for Arid Areas and College of Horticulture, Northwest A&F University, Yangling, Shaanxi
712100, China
d National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
e Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland 20742
b Institute
ORCID ID: 0000-0003-1348-4879 (S.X.).
The Arabidopsis thaliana RESISTANCE TO POWDERY MILDEW8.2 (RPW8.2) protein is specifically targeted to the extrahaustorial
membrane (EHM) encasing the haustorium, or fungal feeding structure, where RPW8.2 activates broad-spectrum resistance
against powdery mildew pathogens. How RPW8.2 activates defenses at a precise subcellular locale is not known. Here, we
report a comprehensive mutational analysis in which more than 100 RPW8.2 mutants were functionally evaluated for their
defense and trafficking properties. We show that three amino acid residues (i.e., threonine-64, valine-68, and aspartic acid-112)
are critical for RPW8.2-mediated cell death and resistance to powdery mildew (Golovinomyces cichoracearum UCSC1). Also, we
reveal that two arginine (R)– or lysine (K)–enriched short motifs (i.e., R/K-R/K-x-R/K) make up the likely core EHM-targeting
signals, which, together with the N-terminal transmembrane domain, define a minimal sequence of 60 amino acids that is
necessary and sufficient for EHM localization. In addition, some RPW8.2 mutants localize to the nucleus and/or to a potentially
novel membrane that wraps around plastids or plastid-derived stromules. Results from this study not only reveal critical amino
acid elements in RPW8.2 that enable haustorium-targeted trafficking and defense, but also provide evidence for the existence of
a specific, EHM-oriented membrane trafficking pathway in leaf epidermal cells invaded by powdery mildew.
INTRODUCTION
Despite their diverse origins, many fungal and oomycete pathogens,
including those that cause powdery mildew, rust, and downy mildew in plants, have evolved a similar invasive strategy: They brutally
break the host cell wall, develop a feeding structure (the haustorium)
inside the host cell, and steal nutrients from plants (SchulzeLefert and Panstruga, 2003; O’Connell and Panstruga, 2006).
This haustorium-dependent invasive strategy probably reflects
an advanced form of parasitism and causes many important
widespread crop diseases. Understanding naturally evolved plant
defense mechanisms constitutes a crucial step toward utilization
and engineering of effective resistance in crop plants to fight
against haustorium-forming pathogens.
For a haustorium-forming pathogen to colonize a host plant, it
has to overcome at least two spatiotemporally distinct defense
barriers. The first barrier is the host cell wall, which keeps
nonadapted pathogens out. One common conserved defense
1 These
authors contributed equally to this work.
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Shunyuan Xiao (xiao@
umd.edu).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.117226
2 Address
response at the cell wall is the synthesis and deposition of
callose (b-1,3-glucan) and other chemicals with potential antimicrobial properties, at the site of penetration, forming a papilla
to fortify the breached cell wall (Israel et al., 1980; ThordalChristensen et al., 1997). Several distinct (but likely coordinated)
mechanisms contribute to this defense layer. These include the
PENETRATION RESISTANCE1 (PEN1)–dependent vesicle trafficking pathway (Collins et al., 2003; Assaad et al., 2004; Kwon
et al., 2008) and PEN3-dependent transport apparatus (Stein et al.,
2006) for the delivery of host materials to the penetration site (Lipka
et al., 2005; Humphry et al., 2010). Not surprisingly, actin and microtubule cytoskeletons also contribute to penetration resistance
(Yun et al., 2003; Hardham et al., 2007; Miklis et al., 2007).
An adapted pathogen, by definition, can penetrate the papillafortified cell wall and develop a functional haustorium inside the
invaded cell. However, the host also launches active defenses to
intercept this infection step. One defense is the hypersensitive
response (HR), which is rapid production of reactive oxygen species (e.g., hydrogen peroxide [H2O2]), and subsequent collapse of
the invaded cell (Xiao et al., 2001, 2003). Another defense strategy
is the encasement of the haustorium complex by a cell wall–like
structure, which conceivably constrains the haustorium while
keeping the host cell alive (Meyer et al., 2009; Wang et al., 2009;
Wen et al., 2011). The callosic haustorial encasement is similar
to the papilla in structure and may be extended from the papilla by
rim growth (Meyer et al., 2009). Haustorial invasion from poorly
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adapted pathogens may be halted by this induced intracellular
defense barrier (Hückelhoven and Panstruga, 2011; Wen et al.,
2011). Well-adapted pathogens suppress the formation of the
haustorial encasement, thereby establishing functional haustoria
and subsequent colonization. Under selection pressure, surviving
host plants must have evolved additional defense mechanisms to
constrain haustoria, resulting in postpenetration resistance against
aggressive pathogens.
The molecular basis of these spatiotemporal host defenses
may be attributable to the two major well-known immune branches:
pathogen-associated molecular pattern–triggered immunity (PTI)
and effector-triggered immunity (ETI) (Chisholm et al., 2006; Jones
and Dangl, 2006). PTI is conceivably the major contributor to
penetration resistance, and the host plant may use both PTI and
ETI to mount effective postpenetration resistance.
Genetically defined host resistance (R) genes that confer
postpenetration resistance to well-adapted haustorium-forming
pathogens include those encoding nucleotide binding site (NB)
leucine-rich repeat (LRR) immune receptors that are involved in
ETI (Lawrence et al., 1995; Parker et al., 1997; Halterman et al.,
2001; Song et al., 2003; Yahiaoui et al., 2004; Periyannan et al.,
2013; Saintenac et al., 2013) and several others encoding nonNB-LRR proteins that may or may not be involved in ETI. The
latter category includes REACTION TO PUCCINIA GRAMINIS1
(Rpg1), a receptor-like kinase from barley (Hordeum vulgare;
Brueggeman et al., 2002); LEAF RUST RESISTANCE34 (Lr34),
an ATP-binding cassette transporter from wheat (Triticum aestivum; Krattinger et al., 2009); and RESISTANCE TO POWDERY
MILDEW8.1 (RPW8.1) and RPW8.2, two homologous proteins of
unknown biochemical function from Arabidopsis thaliana (Xiao
et al., 2001).
Unlike most NB-LRR R proteins that trigger race-specific resistance, RPW8.1 and RPW8.2 confer broad-spectrum resistance
in Arabidopsis to powdery mildew pathogens (Xiao et al., 1997,
2001). However, RPW8.1 and RPW8.2 engage the same salicylic
acid–dependent signaling pathway used by NB-LRR R proteins
for defense activation (Xiao et al., 2003, 2005). To understand how
RPW8.2 confers broad-spectrum resistance using the same
conserved pathway recruited for basal resistance (largely PTI) and
ETI, we recently investigated the defense responses in plants
expressing RPW8.2 at the subcellular level and found that RPW8.2
apparently activates haustorium-targeted defenses that include
H2O2 accumulation in the host cell–haustorium interface and
diffusion into the haustorium as well as formation of the callosic
haustorial encasement, explaining the broad-spectrum nature of
RPW8.2-mediated mildew resistance (Wang et al., 2009, 2010).
To understand how RPW8.2 could activate such highly targeted
defense responses, we examined the subcellular localization of
RPW8.2 and found that RPW8.2 is specifically targeted to the
extrahaustorial membrane (EHM) where H2O2 accumulates at
high concentrations and the callosic haustorial encasement forms
(Wang et al., 2009). These observations indicate that RPW8.2 is
able to activate salicylic acid–dependent signaling to redeploy
postpenetration resistance that has been suppressed by adapted
powdery mildew pathogens at a precise subcellular locale. These
observations also raise an interesting question as to how
these two functional aspects (i.e., defense and targeting) are integrated in RPW8.2. Moreover, the identification of RPW8.2 as an
EHM-specific resident protein suggests that a haustorium-oriented
membrane/protein trafficking pathway may be activated during EHM
biogenesis and that RPW8.2 may contain an EHM-targeting signal
that is recognized by the cellular trafficking machinery for sorting the
RPW8.2-containing vesicles to the EHM.
In this study, we conducted a comprehensive mutational
analysis of RPW8.2 using multiple approaches and identified
critical sites for regulation of RPW8.2’s defense function and
targeting to the EHM. Additionally, we found that several RPW8.2
mutant proteins localize to a potentially novel membrane surrounding plastids and/or plastid-derived stromules.
RESULTS
Natural Mutation-Guided Mutagenesis of RPW8.2 Reveals
Three Amino Acids Critical for Induction of Cell Death and
Disease Resistance
A previous study on intraspecific polymorphism at RPW8.2
showed that compared with the functional RPW8.2 allele from
accession Ms-0 (designated R82Ms-0), Thr-64 to Ser (T64S),
Asp-116 to Gly (D116G), and Thr-161 to Lys (T161K) substitutions are present in alleles from most accessions that are
susceptible to powdery mildew (Figure 5 in Orgil et al., 2007).
This implies that Thr-64, Asp-116, and/or Thr-161 in R82Ms-0
may contribute to the defense function of RPW8.2. By contrast,
the R82Bg-1 allele contains the highest number (13) of nonsynonymous substitutions, including those resulting in T64S,
D116G, and T161K amino acid replacements; however, plants of
accession Bg-1 plants showed an intermediate infection phenotype with more extensive powdery mildew–induced cell death
(Orgil et al., 2007; Wang at al., 2009). This suggests that some
amino acid substitutions in R82Bg-1 may offset the phenotypic
effect produced by T64S, D116G, and T161K. To assess the
functional impact of the three common substitutions and others
found in R82Bg-1, we conducted site-directed mutagenesis using R82Ms-0 as template and generated RPW8.2 mutants whose
protein products contain one of the T64S, D116G, T161K, E59K,
and RRR90-92KKK substitutions. The rationale for the RRR9092KKK mutation is that the LRRR motif in the mammalian potassium channel is a 14-3-3 binding site for promoting its cell
surface expression (Yuan et al., 2003); therefore, it is possible
that 89-92LRRR in RPW8.2 may serve a similar role because
RPW8.2 has been shown to interact with 14-3-3 lambda (Yang
et al., 2009).
Unless otherwise indicated, for making these five and many
other RPW8.2 mutant constructs described in this study (see
Figure 1 for a schematic illustration), we made in-frame translational fusion of yellow fluorescent protein (YFP) to the C terminus of the protein encoded by each RPW8.2 mutant. The DNA
constructs were placed under control of the promoter of R82Ms-0
and introduced into accession Col-gl (Columbia-0 [Col-0]
carrying the glabrous mutation). Col-gl lacks RPW8.1 and
RPW8.2 and is susceptible to the adapted powdery mildew
isolate Golovinomyces cichoracearum UCSC1 (Xiao et al., 2001)
and thus is ideal for functional evaluation of the RPW8.2 mutant
constructs. We generated at least 30 independent T1 transgenic
Mutational Analysis of RPW8.2
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Figure 1. Schematic Illustration of RPW8.2 Mutants Constructed and Tested.
RPW8.2 mutant genes were in-frame fused with YFP and tested in Arabidopsis by stable expression. For site-specific mutagenesis, only the functionally
important mutants are highlighted above the schematic RPW8.2 sequence; the remaining mutants are indicated by gray bars underneath. For NAAIRS
replacement, five of the 29 constructs show significant reduction in EHM targeting (gray bar). For deletion analysis, constructs in black show EHM
localization; constructs in gray show no or little EHM localization. Internal deletions are shown by dashed line flanked by two dark circles. The two
shaded regions delimited by the mutational analyses are critical for EHM localization of RPW8.2. Stars indicate the functionally informative breaking
points. The black arrow indicates the minimal sequence construct in the bottom containing 60 amino acids. TMS-R82 andTMA-R82, RPW8.2-YFP
constructs in which the TMD is replaced by that of SYP122 or ACBP2. WT, wild type.
lines for each DNA construct and examined (1) the frequency of
lines displaying spontaneous HR-like cell death (SHL) and
powdery mildew–induced cell death (HR) and resistance and
(2) the subcellular localization of the mutant proteins. As
shown in Figure 2 and Table 1, very few (2.4%) T1 transgenic
plants expressing YFP-tagged R82Ms-0 developed SHL before
inoculation, while about a quarter (25.4%) displayed apparent
resistance (disease reaction score < 1 to 2; Xiao et al., 2005) and
HR as small lesions visible to the naked eye. In comparison,
none of the T1 lines expressing YFP-tagged RPW8.2 containing
E59K (designated R82E59K), R82 RRR90-92KKK, or R82T161K exhibited SHL, while the percentage of lines showing resistance
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Figure 2. Cell Death and Disease Reaction Phenotypes of Site-Directed RPW8.2 Mutants.
(A) Representative leaves of Col-gl lines transgenic for 35S:YFP or PRPW8.2:RPW8.2-YFP (line R2Y4) as control. Plants were inoculated with Gc UCSC1
and pictures taken at 7 d postinoculation (dpi).
(B) to (O) Representative mildew-infected leaves at 7 or 10 d postinoculation or uninfected plants expressing the indicated YFP-tagged RPW8.2
mutants from the RPW8.2 promoter in comparison with Col-gl or Col-gl transgenic for 35S:YFP. Arrowheads indicate spontaneous HR-like cell death
lesions; arrows indicate big necrotic lesions resulting from merge of small lesions.
and HR was only slightly reduced to 22.4, 21.5, and 21.3%,
respectively. In addition, confocal laser scanning microscopy
showed that the three mutant proteins exhibited typical EHMspecific localization (see Supplemental Figures 1A to 1C online).
These results suggest that these substitutions do not significantly alter the function of RPW8.2. By contrast, we observed
a high frequency of SHL (37.1%) and HR (51.8%) in T1 lines
expressing R82T64S (Figures 2F and 2G, Table 1). This result
suggests that either Thr-64 or Ser-64 may be phosphorylated
and there is a functional consequence of this phosphorylation.
Sequence analysis with NetPhos 2.0 (http://www.cbs.dtu.dk/
services/NetPhos/) suggested that Thr-64 (score = 0.950) but
not Ser-64 (score = 0.185) is likely to be phosphorylated. To test
if this is the case, we made two additional mutants, R82T64E, in
which Thr is changed to a phosphomimetic Glu, and R82T64A, in
which Thr is changed to Ala. T1 lines transgenic for R82T64E
Mutational Analysis of RPW8.2
showed a slightly higher rate of SHL (10.9%), but the percentage
of plants that showed HR and resistance (21.2%) was comparable to that of the R82Ms-0 T1 population (Figure 2H, Table 1).
By contrast, the rates and severity of SHL (46.7%) and HR
(53.3%) in the R82T64A T1 lines were comparable to those of the
R82T64S T1 lines (Figures 2I and 2J, Table 1). Taken together,
these results suggest that phosphorylation of RPW8.2 at Thr-64
may indeed play a critical role in preventing inappropriate cell
death in the absence of powdery mildew pathogens.
Next, we examined the phenotypic effect of the D116G mutation. Out of 121 T1 lines transgenic for R82D116G, none had
SHL and only three lines (2.5%) showed weak HR and moderate
resistance to powdery mildew, and the remaining lines lacked
SHL or HR and were susceptible (Figure 2E, Table 1). Confocal
microscopy showed that R82D116G was expressed in the majority of these T1 lines in which the mutant protein was also
specifically targeted to the EHM (see Supplemental Figure 1D
online), excluding the possibility that this phenotype is due to
the lack of expression or mislocalization of R82D116G. These
results indicate that Asp-116 is a critical residue for the defense
function of RPW8.2 and reinforce the notion that EHM targeting
of RPW8.2 is controlled by mechanisms most likely independent of those governing RPW8.2’s defense function (Wang
et al., 2009).
To examine if there is interaction between the T64S, D116G,
T161K, and E59K mutations, we constructed five double-site
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RPW8.2 mutants. T1 lines transgenic for R82E59K/T161K were
phenotypically similar to the respective single-site mutant lines
and to the R82Ms-0 wild-type control lines (Table 1). Not surprisingly, T1 lines transgenic for R82T64S/T161K showed enhanced SHL and HR similar to those seen in R82T64S lines, and
T1 lines transgenic for R82E59K/D116G or R82D116G/T161K were
phenotypically similar to those transgenic for R82D116G. Interestingly, none of the T1 lines transgenic for R82T64S/D116G
developed SHL and only 16.2% of them exhibited delayed (;1
d) HR and moderate resistance (Figure 2K; disease reaction
score 2), suggesting that D116G can largely suppress T64Smediated SHL and constitutive defense.
To identify additional amino acid substitutions likely responsible for the pronounced SHL and fungus-induced massive
HR cell death phenotype of the Bg-1 accession (Orgil et al.,
2007; Wang et al., 2009), we constructed RPW8.2 mutants
carrying multiple-site substitutions (Table 1) by overlapping PCR
using a fragment from R82Bg-1 that contains the desired mutations and an overlapping fragment(s) from R82Ms-0 for the rest.
While T1 lines transgenic for R82H19Q/S45T/V50I/Q52K/E59K showed
phenotypes similar to those of the R82Ms-0 wild-type control
lines, adding T64S and F56L to this mutant construct resulted in
enhanced SHL and HR in the T1 lines comparable to that seen in
T1 plants transgenic for R82T64S (Table 1). This suggests that
these six mutations (i.e., H19Q, S45T, V50I, Q52K, F56L, and
E59K) do not have significant contribution to the pronounced
Table 1. Cell Death and Resistance Phenotypes of Col-gl T1 Lines Transgenic for Mutant RPW8.2-YFP Constructs
Before Inoculation
7 d Postinoculation
Construct
SHL Lines/Total
SHL (%)
R+HR Lines/Totala
R+HR (%)
*R82Ms-0
3/124
0/68
0/116
0/121
0/111
43/116
21/45
13/119
12/46
33/66
0/37
1/87
0/43
1/73
3/87
1/64
5/90
53/84
3/87
8/88
26/82
54/89
42/88
14/33
2.4
0.0
0.0
0.0
0.0
37.1
46.7
10.9
26.2
50.0
0.0
1.1
0.0
1.4
3.4
1.6
5.6
63.1
3.4
9.1
31.7
60.7
47.7
42.4
28/110
11/49
20/93
3/121
19/89
59/114
24/45
22/104
21/46
34/66
6/37
2/87
0/43
1/73
16/87
6/64
20/90
50/78
18/87
19/88
29/76
69/89
45/85
17/33
25.4
22.4
21.5
2.5
21.3
51.8
53.3
21.2
45.7
51.5
16.2
2.3
0.0
1.4
18.4
9.4
22.2
64.1
20.7
21.6
38.2
77.5
51.1
51.5
R82E59K
R82R90-92K
*R82D116G
R82T161K
*R82T64S
*R82T64A
*R82T64E
R82T64S/R90-92K
R82T64S/T116K
*R82T64S/D116G
R82E59K/D116G
R82R90-92K/D116G
R82D116G/T161K
R82E59K/T161K
R82R90-92K/T161K
R82H19Q/S45T/V50I/Q52K/E59K
R82H19Q/S45T/V50I/Q52K/F56L/E59K/T64S
R82T64S/K70E/E77V/L89Q/D116G
R82T64S/K70E/E77V/D116G/T161K
R82E59K/T64S/V68F/K70E/E77V/D116G/T161K
R82E59K/T64S/V68F/K70E/E77V/L89Q/D116G
*R82V68F
*R82V68F/D116G
Asterisks indicate constructs whose phenotypes were confirmed with the T2 or T3 generation derived from three to five independent T1 lines. Note,
those having SHL in most cases also show R+HR phenotypes.
a
R+HR, resistance (disease reaction score <1 to 2; Xiao et al., 2005) and its associated HR induced by powdery mildew.
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cell death phenotype of Bg-1 plants. We then made two RPW8.2
mutant constructs that contain T64S and D116G plus K70E/
E77V/L89Q or K70E/E77V/T161K. T1 lines transgenic for either
of these two constructs showed more frequent HR than
R82T64S/D116G T1 lines but less frequent HR than R82Ms-0 T1
plants (Table 1), implying that K70E, E77V, and L89Q or T161K
mutations may partially contribute to the more extensive HR
found in plants expressing R82Bg-1 (Wang et al., 2009). Furthermore, we made two RPW8.2 mutants carrying seven substitutions, E59K/T64S/V68F/K70E/E77V/D116G/T161K or E59K/
T64S/V68F/K70E/E77V/L89Q/D116G. T1 plants transgenic for
either construct (the latter in particular) showed much more
extensive SHL (31.7% for the first and 60.7% for the second)
and HR (38.2 and 77.5%) than T1 transgenic lines of R82Ms-0
(Table 1). Based on these observations, we reasoned that (1)
L89Q in combination with other six mutations may have a more
pronounced cell death–inducing effect on R82Bg-1 than does
T161K in the same context; and (2) V68F must account for
a major increase of the pro-cell death activity of the two RPW8.2
mutants. To test the latter speculation, we made the R82V68F
single-site mutant and found that T1 lines transgenic for this
construct indeed developed more severe and more frequent
SHL (47.7%) and HR (51.1%) (Table 1; Figures 2L and 2M). Interestingly, mildew-induced cell death in these plants was not
apparently associated with resistance as indicated by moderate
fungal mass on the infected leaves (Figure 2M). This phenotype
is reminiscent of the extensive necrotic cell death of mildewinfected Bg-1 plants (Wang et al., 2009). To see if D116G can
suppress V68F-mediated cell death, we made the R82V68F/D116G
double-site mutant. Interestingly, the cell death and disease
phenotypes of this mutant were similar to those of the R82V68F
single-site mutant (Figures 2N and 2O), indicating that V68F is
epistatic to D116G. All of the above-described RPW8.2 mutants
with site-directed mutations, when detectable in invaded cells,
exhibited normal EHM localization (see Supplemental Figure 1
online), indicating that none of these mutations significantly affects RPW8.2 trafficking properties.
The N Terminus of RPW8.2 Is Required for Protein Stability
and Contributes to EHM Targeting Specificity
To search for domains or motifs in RPW8.2 responsible for the
EHM-specific targeting, we conducted systematic and comprehensive mutational analyses. We first evaluated the role of
the N-terminal domain of RPW8.2 in EHM targeting. In a previous study, we found that two RPW8.2 mutant proteins truncated for either the N-terminal 22 or 30 amino acids were
expressed at extremely low levels and localized as a few puncta
in an unknown compartment (Wang et al., 2010). Because the
N-terminal 22 amino acids are predicted to be a transmembrane
domain (TMD) (Xiao et al., 2001), it was no surprise that removal
of the entire TMD resulted in protein instability and mislocalization.
However, whether the TMD serves as a signal peptide and/or
whether specific residues in the TMD contribute to RPW8.2’s
EHM targeting is not known. To this end, we made a series of
N-terminal deletion mutants (Figure 1) and evaluated their
defense function and subcellular localization in stable transgenic lines. Surprisingly, we found that mutant R82Δ5-12, in
which amino acids 5 to12 (VAAGGALG) were deleted, largely
retained normal EHM-specific localization in ;65% of the invaded cells. However, in the remaining invaded cells, most of
the mutant protein was detected as variable-sized spots or
patches around the haustorial neck and/or penetration site, although some signal was still detected as small puncta at the
EHM or diffused into the EHM (Figures 3A and 3B). Strikingly, in
some invaded cells, we detected YFP signal as numerous small
puncta in a fireworks-like distribution of 10 to 30 µm in diameter
radiating from the penetration site where the signal was more
concentrated (Figure 3C). In some cases, such a fireworks-like
distribution was confined by a weak propidium iodide (PI)–
positive ring (see Supplemental Figure 2A online). Intriguingly, in
the same infection site, the mutant protein was also found in the
EHM (inset in Figure 3C; see Supplemental Movie 1 online).
From a side view, most of the small puncta were arranged at the
same horizontal level around the fungal penetration site, and
intensive signal was also observed at the haustorial neck (indicated by an arrow in Figure 3C). This raised a question as to in
what compartment the fireworks-like domain resides. The localization patterns for R82Δ5-12 were essentially replicated in the
R82Δ5-14 mutant in which amino acids 5 to 14 were deleted
(Figure 3D; see Supplemental Figure 2B online), with the latter
having slightly less frequent (;50%) normal EHM-specific localization (inset in Figure 3D). In some rare cases, R82Δ5-14 was
only found in the haustorial neck region (see Supplemental
Figure 2C online), which is the portion of the EHM that may be
first synthesized. This observation implies that this subdomain
of the EHM is the preferred destination of R82Δ5-14 and/or the
overall level of R82Δ5-14 accumulation is too low to allow signal
of R82Δ5-14 detectable in the EHM encasing the main body of the
haustorium. To further characterize the fireworks-like domain,
we introduced a plasma membrane (PM) marker PIP2A-mCherry
(Nelson et al., 2007) into Col-gl plants expressing R82Ms-0,
R82Δ5-12, or R82Δ5-14 for colocalization analysis. As expected,
R82Ms-0 was located at the EHM encasing the haustorial complex. However, the membrane compartment marked by PIP2AmCherry could not reach into the surrounding region of the
haustorium such that a hollow was observed (see Supplemental
Figure 2D online). Conversely, puncta containing R82Δ5-12 and
R82Δ5-14 were able to localize and/or move through this domain
to reach the EHM (Figures 3C and 3D; see Supplemental Figure
2 online). These observations support the formation of a special
fungal penetration-perturbed membrane domain around the
penetration site where R82Δ5-12 and R82Δ5-14 are localized.
We then made one more amino acid deletion to make
R82Δ5-15 in which amino acids 5 to 15 (VAAGGALGLAL) were
removed. Imaging analysis of multiple T1 lines transgenic for
R82Δ5-15 showed that this mutant protein completely lost its
EHM targeting since it was only seen as randomly distributed
puncta unrelated to the EHM (Figure 3E). This suggests that
removal of amino acids 5 to 15 may lead to a complete loss of
membrane anchorage of R82Δ5-15 or Leu at 15 is essential for
EHM targeting. Finally, we made and functionally evaluated
another mutant R82Δ5-18 in which amino acids 5 to 18 (VAAGGALGLALSVL) were deleted. Interestingly, R82Δ5-18 was found
to be exclusively nuclear localized (Figure 3F). This observation
suggests that (1) 15-LSVL-18 in the predicted TMD is required
Mutational Analysis of RPW8.2
7 of 20
for EHM localization of RPW8.2, and (2) RPW8.2 may contain
a nuclear localization signal (NLS) that could render nuclear localization to R82Δ5-18.
Our above observations seem to support a certain role for
amino acids 5 to 14 (VAAGGALGLA) in RPW8.2’s EHM-specific
localization. However, because these 12 amino acids are part of
the predicted TMD, the compromised EHM-specific localization
of R82Δ5-12 and R82Δ5-14 may be simply due to a defect of these
mutant proteins in membrane insertion. To test this speculation,
we replaced the TMD (amino acids 2 to 18; IAEVAAGGALGLALSVLH) of RPW8.2 by a TMD from validated PM-localized
proteins. We selected the TMD (285-WTCFAILLLLIIVVLIVVFT304) of SYP122, a syntaxin localized to the PM but not found
in the EHM (Assaad et al., 2004), and the N-terminal TMD
(7-LAQSVILGLIFSYLLAKLISIVV-29) of the PM-localized ACBP2,
which has been demonstrated to enable PM localization of YFP
(Li and Chye, 2003). We obtained multiple lines transgenic for
either TMDSYP122- R82Δ2-18 or TMDACBP2-R82Δ2-18 and found
that these two fusion proteins were distributed as diffuse signal
or small puncta aligning the cell wall and peripheral to or at the
EHM (see Supplemental Figure 3 online). However, we rarely
detected strong and diffuse YFP signal at the EHM from these
two TMD-replaced fusion proteins, and none of the transgenic
lines expressing these two constructs showed resistance to Gc
UCSC1.
The above observations, together with our previous results
(Wang et al., 2010), indicate that the N terminus (1 to 22 amino
acids) of RPW8.2 comprises a special TMD or more likely
a signal peptide that is not only important for RPW8.2’s membrane anchorage but may also contribute to its EHM targeting
efficiency and protein stability and, consequently, its defense
function. Meanwhile, these results also suggest that the C-terminal
portion (amino acids 23 to 174) of RPW8.2 may contain an
EHM targeting signal(s) that probably determines RPW8.2’s
EHM-specific localization.
The C Terminus of RPW8.2 Ensures Efficient EHM Targeting
by Suppressing Nuclear Localization
Figure 3. The N-Terminal TMD Is Required for EHM-Specific Localization of RPW8.2.
Col-gl transgenic lines expressing each of the four N-terminal deletion
RPW8.2 mutants tagged with YFP were inoculated with Gc UCSC1.
Infected leaves were collected at 2 d postinoculation, stained with PI,
and subjected to confocal microscopy. The YFP signal is pseudo-colored
in green and PI-stained structures in red. Images are representative Z-stack
projections of 15 to 65 optical sections. h, haustorium; n, nucleus; p, penetration site. Bars = 10 mm.
(A) Mutant R82Δ5-12 in some cells showed reduced EHM localization as
reflected by small puncta and aggregates at or peripheral to the EHM,
particularly at the haustorial neck region (arrow).
(B) R82Δ5-12 as protein aggregate (arrow) was seen around the penetration site (top panel) and/or surrounding the haustorial neck (low panel).
(C) R82Δ5-12 was found as small puncta in a fireworks-like domain centering in the penetration and as small puncta or diffuse signal at the EHM
in the same cell (inset). Note that when viewed horizontally, the R82Δ5-12–
labeled fireworks-like domain seemed to be a thin layer aligning to the
To search for a putative EHM targeting signal, we conducted
a systematic deletion analysis from the C-terminal end of
RPW8.2. Previously, we found that 12 RPW8.2 alleles contain
a single base pair indel that results in frame shift and a truncation of 28 to 34 amino acids at the C termini of the deduced
proteins (Orgil et al., 2007). However, the functional consequence of these truncations is not known. As an entry point for
our C-terminal deletion analysis, we first made R82Δ138-174,
which encodes a mutant RPW8.2 lacking the C-terminal 37
cell wall (bottom panel). The haustorial neck region (arrow) was also labeled by R82Δ5-12.
(D) Mutant R82Δ5-14 was also found in the fireworks-like domain in some
cells, while it was also found at the EHM in other cells (inset).
(E) Mutant R82Δ5-15 was found in puncta not apparently associated with
the EHM, indicating it is incapable of EHM targeting.
(F) Mutant R82Δ5-18 was exclusively found as varied sized puncta in the
nucleus.
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The Plant Cell
amino acids. T1 lines transgenic for R82Δ138-174 displayed cell
death and resistance phenotypes similar to those of T1 lines
transgenic for R82Ms-0 (see Supplemental Figure 4A online).
However, to our surprise, while this fusion protein exhibited
normal EHM localization in some cells, it was also found in the
nucleus in other cells (Figures 4A and 4B). The nuclear localization was evidenced by the presence of YFP puncta of varied
sizes in nuclei stained by both PI (Figure 4C) and 49,6-diamidino2-phenylindole (Figure 4H). We thus speculated that the
C-terminal 37 amino acids of RPW8.2 may suppress RPW8.2’s
nuclear localization to ensure efficient EHM targeting. To obtain further evidence for this, we made nine additional C-terminal
truncation mutants (i.e., R82 D120-174, R82 D116-174, R82 D115-174,
R82 D113-174, R82 D112-174, R82 D111-174, R82 D110-174, R82 D109-174, and
R82 D88-174) in which the C-terminal 55, 59, 60, 62, 63, 64, 65, 66,
or 87 amino acids were respectively truncated (Figure 1; see
Supplemental Table 1 online). For the first five mutants, we found
that, as the size of the truncation increases (from 55 to 63 amino
acids), the respective mutant proteins exhibited decreased EHM
localization while gaining more nuclear localization (Figures 4E
and 4F). Starting from R82Δ111-174, the remaining four mutant
proteins completely lost EHM targeting and became exclusively
nuclear localized (Figures 4G and 4H). All mutant proteins failed
to activate cell death and disease resistance (see Supplemental
Figure 4B online). These observations, when added to earlier results, indicate that (1) amino acids 1 to 111 in RPW8.2 must
contain an EHM targeting signal; (2) Leu-111 is absolutely critical
for EHM targeting, as removing it in the R82Δ111-174 mutant
abolished EHM targeting, whereas keeping it in R82Δ112-174 achieved EHM targeting, albeit at a low frequency; (3) there must be
at least one NLS in the N-terminal half (amino acids 1 to 110) of
RPW8.2; and (4) the C-terminal tail (amino acids 138 to 174) of
RPW8.2 suppresses nuclear localization of RPW8.2.
We noted that the YFP signal in the nucleus representing the
above-mentioned C-terminally truncated RPW8.2 proteins was
often detected in multiple patterns ranging from faint homogenous distribution to small puncta (<0.1 mm) and to large aggregates (up to 1 mm) in different cells (see Supplemental Figure
5 online), suggesting a dynamic nature for the localization.
Intriguingly, we noticed that R82Δ120-174 was also found in
stromule-like membranes that wrap around and connect individual plastids (Figure 5A), in addition to EHM and nuclear
localization. Stromules are defined as stroma-filled, dynamic
tubular structures extending out from the envelope membrane
of different plastid types (Köhler et al., 1997; Waters et al.,
2004). This unexpected novel localization was further signified
by our observations that additional RPW8.2 mutant proteins
were also found in the stromule-like membrane structures (see
later text).
Two Internal Regions of RPW8.2 Are Required for EHM
Targeting, and RPW8.2 May Be Targeted to the
Peristromule Membrane
Based on our N- and C-terminal deletion analyses, it seemed
that neither the TMD at the N terminus nor the C-terminal portion
(amino acids 112 to 174) of RPW8.2 encodes the EHM targeting
signal, although both are required for efficient EHM targeting. To
Figure 4. The C-Terminal Portion Is Required for Suppression of Nuclear
Localization of RPW8.2.
YFP signal from tagged RPW8.2 mutant proteins is pseudo-colored
green, PI-stained structures are red, and DAPI-stained nuclei are blue.
Images are representative Z-stack projections of 15 to 45 optical sections. h, haustorium; n, nucleus. Bars = 10 mm.
(A) Nuclear localization of R82Δ138-174.
(B) EHM localization of R82Δ138-174.
(C) Epidermal cells expressing YFP alone as control to show that both
nuclei and the haustorium were stained red by PI.
(D) EHM and nuclear localization of R82D120-174.
(E) EHM and nuclear localization of R82Δ113-174.
(F) Nuclear localization and weak EHM targeting of R82D112-174.
(G) Exclusive nuclear localization of R82D111-174.
(H) Nuclear localization of R82D110-174. Note both the haustorium and
nucleus were stained blue by DAPI.
(I) Nuclear localization of R82D88-174.
Mutational Analysis of RPW8.2
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Figure 5. RPW8.2 Variants Are Targeted to the PSM.
YFP signal from tagged RPW8.2 mutant proteins is pseudo-colored green, PI-stained structures are red, and autofluorescent chloroplasts or plastids
are blue. Images are representative Z-stack projections of 15 to 45 optical sections. h, haustorium. Bars = 10 mm.
(A) R82Δ120-174 was observed in the PSM tightly associated with and connecting individual plastids (asterisks). Note that there are bulges or lumps close
to the encased plastids or in the middle of the membrane strand.
(B) and (C) R82Δ43-97 was found in the PSM as puncta at or peripheral to the EHM.
(C) R82Δ43-97 was targeted at the EHM as shown by diffuse YFP signal in the EHM.
(D) to (G) Localization of mutant proteins R82Δ43-97 (D), R82Δ43-115 (E), R82Δ43-135 (F), and R82Δ43-141 (G) in epidermal cells containing a haustorium.
Note the big dots/patches (arrows) in some cells.
(H) R82Δ65-93 was found in ring structures (asterisks) associated with chloroplasts in mesophyll cells.
(I) R82Δ65-93+Δ138-174 was occasionally found both in a PSM (asterisks) and the EHM and the tubules that connect these two compartments.
(J) R82Δ65-93+Δ138-174 was observed in the PSM similarly as R82Δ120-174. The bulged portion is indicated by arrows.
(K) and (L) Colocalization analysis of YFP-tagged R82Δ65-93+Δ138-174 expressed from the RPW8.2 promoter and a stromule marker (a plastid-targeting
[PT] signal in fusion with mCherry; Nelson et al., 2007) expressed from the 35S promoter in stable Arabidopsis Col-0 plants. While some R82Δ65-93+Δ138-174
signal seemed to colocalize with PT-mCherry in thin stromule strands (arrows in [K]), most of it was found in the periphery of PT-mCherry labeled plastids or
stromule protrusions (arrows in [L]).
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identify the EHM targeting signal, we made 11 internal deletion
constructs in which we kept the N-terminal 1 to 42 or 1 to 64
amino acids intact and combined it with different sized C-tails
(Figure 1). We first removed amino acids 43 to 93 to make the
R82Δ43-93 mutant. This mutant protein retained EHM-specific
localization, although the expression level was generally low as
reflected by weak YFP signal largely in punctate and occasionally diffuse signal at the EHM (Figures 5B and 5C). This indicates that amino acids 43 to 93 are not essential for EHM
targeting, although they may contribute to protein stability. We
then made and evaluated four additional mutants R82Δ43-97,
R82Δ43-115, R82Δ43-135, and R82Δ43-141 (Figure 1). As shown by
representative images in Figures 5D to 5G, all these four mutant
proteins showed rare and poor EHM localization and were found
in small puncta to big patches (2 to 3 µm) in the cytoplasm.
These observations, together with those from earlier deletion
analyses, suggested that (1) the region from amino acids 94 to
111 contributes to the EHM targeting specificity and (2) either
amino acids 1 to 42 (the N terminus) or amino acids 135 to 174
(the C terminus) contribute to the residual EHM targeting observed with the above four mutant proteins in comparison with
R82Δ43-93. We thus subsequently made R82Δ65-93 to see if
a longer N-terminal portion could improve the EHM targeting
specificity. We failed to notice any obvious improvement in EHM
targeting specificity and efficiency for R82Δ65-93. Instead, we
occasionally found this mutant protein in ring structures surrounding plastids in epidermal cells (Figure 5H), similar to the
localization pattern of R82Δ120-174. Next, we further deleted the
C-terminal 37 residues (amino acids 138 to 174) using R82Δ65-93
as template to make R82Δ65-93+Δ138-174 in order to examine if
removal of the C terminus compromises EHM targeting in
R82Δ65-93. As shown in Figure 5I, R82Δ65-93+Δ138-174 was still
occasionally found in the EHM, suggesting that the EHM targeting specificity in R82Δ43-93 (and other three mutants) is not
conferred by the C-terminal 37 amino acids. Combining the
results from all the deletion analyses described thus far, we inferred that two regions (i.e., the N terminus [amino acids 1 to 42]
and particularly an internal region [amino acids 94 to 111]) are
mainly responsible for EHM-specific localization (Figure 1).
Interestingly, R82Δ65-93+Δ138-174 was more frequently found in
the stromule-like membrane structures that tightly wrap around
and connect individual plastids in uninfected epidermal cells in
some T1 lines that had high levels of constitutive expression
(Figure 5J). This localization pattern was observed for R82Δ120-174
(Figure 5A), and in both cases, there were big bright spots indicative of high-level protein accumulation at one spot of the rings
or in the strands bridging two different plastids (Figures 5A and
5J). Most strikingly, we observed that in some haustoriuminvaded cells R82Δ65-93+Δ138-174-labeled stromule-like membrane
strands apparently connected plastids and the haustoria
(Figure 5I).
Stromules are tubular protrusions of the plastid envelope
membrane, and to our knowledge, proteins found in stromules
are all plastid-targeted proteins, although a myosin motor
protein has been shown to be associated with stromules in
Nicotiana benthamiana (Sattarzadeh et al., 2009). Our above
findings raised a question as to whether some RPW8.2 mutant
proteins are truly targeted to stromules. Thus, we examined if
R82Δ65-93+Δ138-174 colocalizes with a canonical stromule marker
protein. By coexpressing a stromule marker protein (i.e., a plastid targeting signal in fusion with mCherry) (Nelson et al., 2007) in
the same background, we found that R82Δ65-93+Δ138-174 was
localized at the periphery of, but not exactly colocalized with, the
stromule marker (Figures 5K and 5L). Taken together, our observations that several RPW8.2 mutant proteins were targeted
to a membrane wrapping around plastids and peripheral to
stromules suggest the existence of a novel interfacial membrane
between the cytoplasm and plastids or plastid-derived stromules. We tentatively named this novel membrane the peristromule membrane (PSM). Given that several RPW8.2 mutant
proteins were found in the PSM and the EHM (Figure 5), we
speculate that the PSM may share certain common membrane
characteristics with the EHM.
Two Short Motifs Enriched in Basic Residues Are Essential
for EHM Targeting
To further characterize the EHM targeting signals in the two
regions delimited by the deletion analyses described above, we
conducted a NAAIRS scan across the entire RPW8.2 protein
sequence. NAAIRS is a six–amino acid (Asn-Ala-Ala-Ile-Arg-Ser)
segment frequently found in both a-helices and b-sheets, and
replacement of six consecutive residues with NAAIRS is thus
believed to minimize gross disruptions in secondary structures
(Wilson et al., 1985). NAAIRS scanning has been used successfully to identify critical elements in the Arabidopsis SNI1
protein (Mosher et al., 2006) and the potato (Solanum tuberosum)
resistance protein Rx (Rairdan et al., 2008). To this end, we
made 29 NAAIRS replacement mutants covering almost all
amino acids of RPW8.2 (Figures 1 and 6A). Each of the 29
NAAIRS mutants was fused with YFP at the C terminus and
expressed in Col-gl plants from the RPW8.2 promoter (see
Supplemental Table 1 online). Among these NAAIRS mutants,
three showed significant reduction in EHM targeting. The first
mutant R82N20-25, in which amino acids 20 to 25 (EAVKRA) were
replaced by NAAIRS, was rarely (<5% infected epidermal cells)
found in the EHM (Figure 6B). Instead, the mutant protein was
more frequently found in ring structures peripheral to chloroplasts in mesophyll cells (Figure 6C), which is reminiscent of
the localization pattern of RPW8.1 (Wang et al., 2007). The
second mutant R82N26-31, in which amino acids 26 to 31
(KDRSVT) were replaced by NAAIRS, showed even less obvious
EHM targeting, as reflected by the formation of various sized
puncta in or peripheral to the EHM (Figure 6E), or randomly
distributed in the invaded cell (Figure 6D). The third mutant,
R82N95-100, in which amino acids 95 to 100 (RKKFRY) were
replaced with NAAIRS, showed similar localization patterns as
R82N26-31, with puncta ranging from being barely visible to as
big as 2 to 3 µm in diameter docked around the haustorium,
likely incapable of fusing into the EHM (Figures 6F and 6G). T1
lines (>30) transgenic for each of the three mutants were all
susceptible, indicating that these mutant proteins are also defective in defense activation. Interestingly, these three NAAIRS
replacements were located in two small regions, amino acids 20
to 31 and amino acid 95 to 100, each of which respectively falls
into the two bigger regions, amino acids 1 to 42 and amino acids
Mutational Analysis of RPW8.2
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Figure 6. NAAIRS Scanning Identified Short Amino Acid Motifs in RPW8.2 Important for EHM Targeting.
YFP signal from tagged RPW8.2 mutant proteins is pseudo-colored green, PI-stained structures are red, and autofluorescent chloroplasts or plastids
are blue. Images are representative Z-stack projections of 15 to 45 optical sections. h, haustorium; p, penetration site. Bars = 10 mm.
(A) The RPW8.2 protein sequence and positions of the 29 NAAIRS replacement and potential functional motifs and residues. Short lines underneath the
amino acid sequences indicate positions of NAAIRS replacements; green lines indicated mutants with more or less normal EHM localization; red lines
indicate mutants largely defective in EHM targeting; black lines indicates mutants without detectable protein accumulation. Two R/K-R/K-x-R/K motifs
critical for EHM targeting are shaded pink; two putative NLSs are shaded green; two putative NESs are shaded purple.
(B) The NAAIRS replacement mutant R82N20-25 was only rarely observed in the EHM. In most cases, it was found as varied sized puncta peripheral to
the haustorium.
(C) R82N20-25 was occasionally observed in ring structures surrounding chloroplasts in the mesophyll cells.
(D) and (E) R82N26-31 is largely defective in EHM targeting, forming protein aggregates (arrows) unrelated to the haustorium (D) and/or small puncta at or
peripheral to the EHM (E).
(F) and (G) R82N95-100 is largely defective in EHM targeting, forming big protein aggregates (arrows) and small puncta at or peripheral to the EHM.
(H) R82N20-25+N95-100 is defective in EHM targeting, forming small puncta in the cytoplasm and protein aggregates (arrows) at the penetration site where
the papilla was stained red by PI.
(I) An optical section from (H) with bright field showing the presence of the haustorium. Arrows indicate protein aggregates.
(J) R82N20-25+N95-100 was occasionally observed in ring structures surrounding chloroplasts in the mesophyll cells.
(K) R82N26-31+N95-100 is defective in EHM targeting, forming small puncta in the cytoplasm and around the haustorium, and big aggregates (arrow) at the
penetration site.
(L) An optical section from (K) with bright field showing the presence of the haustorium.
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94 to 115, defined as largely responsible for EHM targeting by
our deletion analyses (Figure 1). In addition to these three mutants,
three other mutants (i.e., R82N8-19, R82N32-37, and R82N89-94) also
showed loss of (for R82N8-19) or obvious reduction (the remaining
two) in EHM targeting. Apart from R82N8-19 in which 12 amino
acids in the TMD were replaced by NAAIRS (which likely affected
the TMD properties), the remaining two had the replacements
close to the two regions amino acids 20 to 31 or amino acids 95 to
100 most critical for EHM targeting, suggesting the neighboring
residues may also be important for efficient EHM targeting.
All these six mutants were nonfunctional in defense activation,
as none of the T1 lines transgenic for each of these constructs
displayed SHL, HR, or resistance to powdery mildew (see
Supplemental Table 1 online).
The remaining 23 NAAIRS mutants showed a range of
phenotypes both in terms of cell death activation and EHM
targeting (see Supplemental Table 1 online). For example, T1
seedlings transgenic for R82N137-142 (ISTKLD replaced by
NAAIRS) and R82 N143-148 (KIMPQP replaced by NAAIRS)
started to develop massive SHL in rosette leaves when they
were 2 to 3 weeks old and died before bolting (see Supplemental
Figures 4C and 4D online). This observation suggested that
the two mutant proteins have higher capacity to trigger inappropriate cell death at early stages of plant development.
Unfortunately, we were unable to detect any YFP signal in
seedlings transgenic for these two mutant constructs. Similarly,
we also failed to detect YFP signal in mutant R82N83-88 (VEENAE
replaced by NAAIRS); however, we found that 10 out of 32 of the
T1 plants transgenic for this construct developed SHL (see
Supplemental Figure 4E online) and showed resistance to
powdery mildew. R82N38-43 also failed to accumulate to a detectable level and Col-gl lines transgenic for this mutant construct were also susceptible. The remaining 19 NAAIRS mutants
did not activate SHL. However, some were able to induce normal HR and resistance to powdery mildew (see Supplemental
Table 1 online). Not surprisingly, all of these NAAIRS mutants
showed normal EHM targeting, even though there was variation
in YFP signal intensity, most likely reflecting varied protein stability among these mutant proteins (see Supplemental Table 1
online).
Because none of three single NAAIRS replacements in the
two defined regions amino acids 20 to 31 and amino acids 95
to 100 completely abolished the EHM targeting of RPW8.2, it is
likely that the these two regions may additively contribute to
EHM targeting. To test this, we made double NAAIRS replacement mutants, R82N20-25+N95-100 and R82N26-31+N95-100,
and examined their subcellular localization in stable transgenic
T1 plants. As anticipated, these two double mutant proteins
were mostly found in puncta unrelated to the EHM in the invaded cells, with bigger patches close to the haustorial neck
region (Figures 6H to 6L), indicating a complete loss of EHM
targeting. Occasionally, in some invaded cells, the mutant
proteins were seen as punctate or diffuse signals in the proximity of haustoria (see Supplemental Figures 6A and 6B online).
In addition, similar to R82N20-25 (Figure 6C), R82N20-25+N95-100
was also found to form punctate rings surrounding chloroplasts in mesophyll cells (Figure 6J). Collectively, these data
further suggest that the two regions, amino acids 20 to 31 and
amino acids 95 to 100, indeed comprise two small regions
critical for EHM targeting of RPW8.2.
R/K-R/K-X-R/K Represents the Core EHM Targeting Signal
A common feature of the two regions required for EHM targeting
is that they both are enriched in basic residues, each containing
four Arg/Lys residues. We thus decided to assess the importance of the basic residues and a few others in this motif using
site-directed substitution with Ala. Among eight single-site
mutations (i.e., R24A, K26A, D27A, R28A, R95A, K96A, K97A,
and F98A), only R28A and F98A resulted in the formation of
protein aggregates as shown by various-sized puncta at or
peripheral to the EHM (Figures 7A and 7B), indicating that Arg28 and Phe-98 are important for vesicle docking to or fusion with
the EHM, the remaining six mutations did not grossly affect EHM
localization of the respective mutant proteins (see Supplemental
Figures 6C and 6D and Supplemental Table 1 online). In addition, we made 15 single- or multiple-site Ala substitutions at
residues from 100 to 127. Except for Y100A, which did not have
detectable expression, and L111A, which significantly reduced
EHM targeting (thus agreeing with results from the C-terminal
deletion analysis; Figure 4), the remaining 13 mutations did not
significantly affect RPW8.2’s EHM targeting and defense function (see Supplemental Table 1 online). Next, we made three
two-site mutations with a single site in each of these two regions. Mutant R82R24A+R95A, in which Arg-24 and Arg-95 were
both replaced by Ala, formed big protein aggregates at or
around the EHM, implying a defect in fusion of R82R24A+R95A
vesicles with the EHM (Figure 7C). Mutant R82R24A+K97A, in
which Arg-24 and Lys-97 were replaced by Ala, was partially
compromised in its ability to localize to the EHM. However, it
seemed to be restricted to the portion of the EHM that wraps the
haustorial neck with weak signal forming a ring around the
penetration site (Figure 7D). Most significantly, mutant R82K26A
+R95A, in which Lys-26 and Arg-95 were replaced by Ala, largely
failed in targeting to the EHM, as evidenced by randomly distributed puncta in the haustorium-invaded cells (Figure 7E), indicating that these two basic residues together are essential for
EHM targeting. Close examination of these two regions identified a common R/K-R/K-x-R/K motif (shaded in pink in Figure
6A). Based on the above results, we propose that the R/K-R/K-xR/K–containing motifs in amino acids 20 to 30 and amino
acids 95 to 100 probably comprise the core EHM targeting
signals in RPW8.2. We tentatively named amino acids 20 to 30
EHM-TARGETING SIGNAL1 (ETS1) and amino acid 95 to 100
ETS2.
Defining the Minimum Amino Acid Sequence in RPW8.2
for EHM Targeting
Our data from deletion/truncation, NAAIRS replacement, and
site-specific mutational analyses indicate that the putative TMD
or signal peptide, ETS1, and ETS2 are necessary for efficient
targeting of RPW8.2 to the EHM. To determine if these three
domains/motifs are sufficient for EHM targeting, we made
R82Δ43-93+Δ113-174 that contains amino acids 1 to 42 (the TMD
and ETS1) and amino acids 95 to 112 (ETS2) (60 amino acids in
Mutational Analysis of RPW8.2
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Figure 7. Site-Directed Mutagenesis at the Two R/K-R/K-x-R/K–Containing Motifs Provided Further Evidence for Their Role in EHM Targeting.
YFP signal from tagged RPW8.2 mutant proteins is pseudo-colored green, and PI-stained structures are red. Images are representative Z-stack
projections of 15 to 45 optical sections. h, haustorium. Bars = 10 mm.
(A) R82R28A is largely defective in EHM targeting, forming small puncta and big aggregates (arrows) at or peripheral to the EHM.
(B) R82F98A is largely defective in EHM targeting, forming small puncta and patches at or peripheral to the EHM.
(C) R82R24A+R95A is largely defective in EHM targeting, forming small puncta and big aggregates (arrows) at or peripheral to the EHM.
(D) R82R24A+K97A is also largely defective in EHM targeting. YFP signal was mainly detected in the portion of the EHM around the haustorial neck (arrow).
Weak YFP signal was also observed in a ring structure surrounding the penetration site and possibly in nearby PM (asterisks).
(E) R82K26A+R95A is defective in EHM targeting as shown by formation of varied sized puncta in the cytoplasm unrelated to the EHM.
total; Figure 1) using R82Δ43-93+Δ138-174 as template. All transgenic lines (>24) expressing this construct were as susceptible
as Col-0 wild type, indicating this mutant protein is not functional in defense. However, this protein was clearly detected as
diffuse and punctate signal at the EHM (Figures 8A to 8C). We
thus conclude that the 60 amino acids containing the putative
TMD or signal peptide and two ETSs are both necessary and
sufficient for EHM localization. Interestingly, YFP signal from this
fusion protein was also found around the nucleus (Figure 8B)
and in the PSM connecting plastids with the nucleus (Figure 8D)
in the same epidermal cells. In some epidermal cells, YFP signal
was also found in punctate ring structures surrounding plastids in uninfected epidermal cells (Figure 8E). These features of
subcellular localization of R82Δ43-93+Δ113-174 are similar to several RPW8.2 mutants, such as R82Δ65-93+Δ138-174 (Figures 5H to
5L), which collectively suggests that the EHM and the PSM may
share certain common characteristics that enable localization of
several RPW8.2 mutant proteins.
DISCUSSION
The RPW8.2 plant R protein activates defense in a specific
subcellular locale: the host-pathogen interface (Wang et al.,
2009). In this study, we conducted a comprehensive mutational
analysis of RPW8.2 and identified three residues as critical sites
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for regulation of RPW8.2’s defense (cell death activation) function, two basic residue-enriched motifs as the core EHM targeting signals, and a minimum sequence of 60 amino acids
necessary and sufficient to enable EHM localization. In addition,
we observed localization of RPW8.2 variants to the nucleus and/or
a potentially novel membrane tightly associated with plastids
and plastid-derived stromules, provoking speculations on intrinsic properties and regulatory mechanisms of RPW8.2.
Allelic Polymorphisms Inform Mechanisms of RPW8.2’s
Regulation and Evolution
Figure 8. Minimal Sequence Requirement of RPW8.2 for EHM Targeting
and a Model for Intracellular Trafficking Pathways in Epidermal Cells
Invaded by Haustoria of Powdery Mildew.
YFP signal from the tagged RPW8.2 mutant protein is pseudo-colored
green, PI-stained structures are red, and autofluorescent chloroplasts
are blue. Images are representative Z-stack projections of 15 to 45 optical sections. h, haustorium; n, nucleus. Bars = 10 mm.
(A) to (C) R82Δ43-93+Δ113-174 is capable of reaching the EHM as mostly
diffuse signal (A) or both diffuse and small punctate signal ([B] and [C]).
Insets are single optical sections. Note weak YFP signal was also detected in ring structures surrounding the nucleus and plastids (arrows).
(D) R82Δ43-93+Δ113-174 was detected in the PSM 20 min after treatment
with 10 µM abscisic acid. Note the YFP-positive stromule-like membrane
filaments (arrows) extended from plastids into the nucleus.
(E) R82Δ43-93+Δ113-174 was detected in punctate rings surrounding plastids (asterisks) labeled by a stromule marker PT-mCherry (Nelson et al.,
2007) (pseudo-colored red).
(F) A cartoon illustrating vesicle trafficking in a leaf epidermal cell invaded
by a haustorium of powdery mildew. During the biogenesis of the EHM,
major trafficking at the trans-Golgi network is oriented toward the EHM
(1), while vesicle trafficking to PM (2) and other organelles such as the
nucleus remain active. Vesicles loaded with protein cargos containing
EHM targeting motifs such as RPW8.2 are specifically targeted to the
EHM, while vesicles containing proteins without a strong sorting signal
may passively enter the major EHM-oriented trafficking pathway (1). A
common feature(s) may be shared between the EHM and the PSM,
which may provide a trafficking cue for RPW8.2’s localization. The PSM
may further serve as a trafficking highway for RPW8.2 to reach the EHM
(3). In addition, RPW8.2 contains NLSs and export signals; consequently,
some RPW8.2 mutant proteins are nuclear localized. This raises a possibility that a small portion of RPW8.2 may be nuclear localized for defense activation (4) and suggests that balanced trafficking forces may be
required to ensure EHM targeting of RPW8.2. It has been proposed that
some PM-localized proteins may be translocalized to the EHM via the
endocytic vesicle trafficking pathway (Lu et al., 2012) (5); however, definitive evidence remains to be provided.
Natural allelic polymorphism at RPW8.2 should, to some extent,
reflect functional divergence of RPW8.2 (Orgil et al., 2007). Results from our site-directed mutagenesis guided by the natural
allelic polymorphism of RPW8.2 support this notion, albeit with
some unexpected surprise. For example, we previously reported
that the T64S polymorphism occurs in almost all the RPW8.2
alleles in mildew-susceptible accessions and Thr-64 may be
important for resistance (Orgil et al., 2007). We had thus predicted that T64S might compromise defense activation. Unexpectedly, transgenic lines expressing R82T64S showed strong
SHL, pronounced HR, and increased resistance to powdery
mildew, and this phenotype was essentially replicated in transgenic lines expressing R82T64A (Figure 2). Conversely, the T64E
mutation in RPW8.2 did not apparently affect RPW8.2 function.
These results suggest that in uninfected cells Thr-64 but not
Ser-64 is subject to phosphorylation, and this phosphorylation
may keep RPW8.2 in an off state. Because EDR1, a mitogenactivated protein kinase kinase kinase (Frye et al., 2001), is
a negative regulator of RPW8-mediated HR and resistance (Xiao
et al., 2005), it is possible that EDR1 may exert its negative
regulation of RPW8.2 via phosphorylating Thr-64. Further
molecular and biochemical studies are required to test this
speculation.
The second functionally important site is Asp-116. This residue appears to be critical for RPW8.2’s defense function as the
single D116G substitution in RPW8.2 not only largely abolished
defense function of the R82Ms-0 wild-type protein (Figure 2E) but
also suppressed SHL triggered by R82T64S (Figure 2K). Based
on the functional consequences of these two mutations and
their strict co-occurrence in alleles from most susceptible accessions (Orgil et al., 2007), we speculate that T64S might have
occurred first to confer constitutive and stronger resistance
against very aggressive powdery mildew pathogens and D116G
subsequently occurred to counter the effect of T64S when the
disease pressure was lessened.
The third interesting residue that contributes to cell death
function of RPW8.2 is Val-68. The V68F substitution in RPW8.2
only occurred in Bg-1 and Bg-1-like Arabidopsis accessions.
Given that Val-68 is conserved among all RPW8 family members
(Xiao et al., 2004), and Phe is a bulky amino acid, it seems likely
that the V68F mutation may cause structural perturbation of
RPW8.2, thereby triggering cell death. Unlike T64A or T64S,
V68F-mediated cell death did not seem to tightly couple with
defense activation and could not be suppressed by D116G
(Figure 2O), providing an explanation for the massive mildewinduced cell death with only partial resistance in Bg-1 plants
Mutational Analysis of RPW8.2
(Wang et al., 2009). The selective advantage that leads to the
maintenance of Bg-1–like accessions in the natural population is
currently unknown.
There are other sites in RPW8.2 that also appear to be important for keeping RPW8.2 from triggering inappropriate cell
death. For example, the massive to lethal cell death phenotypes
caused by three NAAIRS mutants, R82N83-88, R82N137-142, and
R82N143-148, suggest that amino acid 83-VEENAE-88, amino
acid 137-ISTKLD-142, and amino acid 143-KIMPQP-148 contain residues that are important for negative regulation of cell
death of RPW8.2. Further site-directed mutagenesis is required
to identify the exact functionally relevant residues in these
regions.
EHM-Oriented Trafficking: How Specific Is It?
Specific localization of RPW8.2 at the EHM (Wang et al., 2009)
and the exclusion of eight PM-resident proteins from the EHM
(Koh et al., 2005) together imply the existence of an EHMspecific secretory pathway. However, more specific evidence
has yet to be provided to allow us to make a conclusion.
Pumplin et al. (2012) recently proposed a mechanism (i.e.,
precise temporal expression coupled with a transient reorientation of secretion) to explain specific localization of a
Medicago phosphate transporter (PT4) to the periarbuscular
membrane (PAM) surrounding the arbuscules of mycorrhizal
fungi in root cells. The critical supporting evidence for this
model is that when expressed from the PT4 promoter, PT1 (a
PM-resident protein homologous to Medicago PT4) is specifically localized to the PAM (Pumplin et al., 2012). Considering
that the PAM is >83 of the PM in infected cells in terms of
membrane surface area (Bonfante and Genre, 2010; Pumplin
et al., 2012), it is possible that the PAM-oriented trafficking
becomes a predominant (perhaps default) pathway during
PAM biogenesis.
Because RPW8.2 expression is induced upon powdery mildew invasion (Wang, et al., 2009), its EHM-specific localization
could also be a consequence of precise temporal expression
coupled with a transient EHM-oriented default secretion during
EHM biogenesis. However, the membrane surface area of EHM
is <1/53 of the PM (our estimation based on 20 haustoriuminvaded epidermal cells), a default EHM-oriented secretion is
less likely. In fact, our observations suggest that multiple protein
trafficking pathways are active in leaf epidermal cells invaded by
haustoria (Figure 8F). For example, R82Δ5-12 and R82Δ5-14 were
found in the EHM and the fireworks-like domain, which presumably represents the fungus-perturbed PM (Figure 3);
R82Δ138-174 was localized to both the EHM and the nucleus
(Figure 4); TMDACBP2-R82Δ2-18 (see Supplemental Figure 3B
online) and YFP-RPW8.2 (Wang et al., 2010) appeared to be
localized to both the EHM and the PM. Therefore, as a corollary,
we reasoned that exclusive EHM localization of RPW8.2(wt)YFP must be a consequence of the activation of a selective
EHM-oriented trafficking pathway in mildew-infected epidermal
cells (Figure 8F) and the identification of two EHM targeting
signals in RPW8.2 critical for EHM localization further supports
this conclusion.
15 of 20
Notably, Lu et al. (2012) observed differential localization of
a number of membrane proteins (that are normally localized to
the PM or function in secretory transport or endocytic trafficking) to the EHM induced by two oomycete pathogens.
Therefore, it seems possible that both the secretory and endosomal pathways may play a role in the delivery of membrane
materials to the EHM during its biogenesis and that endocytosis might occur at the EHM to exclude other PM-resident
proteins (Lu et al., 2012). These observations hint at the existence of multiple trafficking routes to establish the EHM while
certain selectivity of protein targeting or exclusion has to be
established (Figure 8F). Since we did not observe PM localization of RPW8.2-YFP even after treatment with the endocytosis inhibitor wortmannin (data not shown), EHM-specific
localization of RPW8.2 is unlikely because of selective exclusion from the PM via endocytosis.
R/K-R/K-x-R/K: A ZIP Code for the EHM?
The most significant result from this mutational analysis is that
we defined a minimum sequence of 60 amino acids consisting
of the putative TMD and two basic residue-enriched regions
from RPW8.2 to be sufficient for rendering EHM localization of
the YFP fusion protein (Figures 8A to 8E).
Considering that the single putative TMD in RPW8.2 is
probably required for membrane anchorage, it is not surprising
that removing the entire TMD (1 to 22) resulted in mislocalization
of RPW8.2 as puncta in an unknown compartment (Wang et al.,
2010). However, unexpectedly, removal of amino acids 5 to 12,
or 5 to 14 within the TMD, each of which should completely
disrupt the presumable TMD function as predicted by TMpred
(www.ch.embnet.org/software/TMPRED_form.html) or TMHMM,
(www.cbs.dtu.dk/services/TMHMM/) the two RPW8.2 mutant
proteins are still capable of EHM localization (Figure 3), albeit at
a lower efficiency. This result implies that RPW8.2 may be
subject to lipidation for membrane association and that amino
acids 15 to 22 may be required for this lipidation. Given that
RPW8.2 with a TMD from SYP122 or ACBP2 showed only limited EHM localization and lower level accumulation (see
Supplemental Figure 3 online), and failed to activate defense, it
is likely that the native TMD may have an additional role besides
membrane insertion: It may promote EHM targeting of RPW8.2
by suppressing nuclear localization of RPW8.2 since there is
a predicted nuclear export signal (NES; LGLALSVL) within the
TMD (amino acids 11 to 18) and removal of this NES resulted in
nuclear localization (Figure 3F). Indeed, when fused with 2xYFP,
this NES can target nuclear-localized 2xYFP to the cytoplasm
(Y. Huang, S. Xiao, and W. Wang unpublished data). Because
nucleus-targeted RPW8.2 variants seemed to be rapidly degraded (data not shown), suppressing nuclear localization of
RPW8.2 should consequently promote adequate accumulation
of RPW8.2 at the EHM for activation of effective resistance
against haustorial invasion.
Our mutational study also identified two basic residueenriched short regions (amino acids 20 to 30 and amino acids
95 to 100) in RPW8.2 critical for EHM localization. We found a
common R/K-R/K-x-R/K signature in these two regions and
we thus tentatively proposed that the duplex R/K-R/K-x-R/K
16 of 20
The Plant Cell
motifs are the core EHM targeting signals. This inference is not
only supported by results from deletion and NAAIRS scanning
results (Figures 4 to 6), but also by results from site-directed
mutagenesis (Figure 7). For example, the two single mutations
K26A and R95A together completely abolished RPW8.2’s EHM
targeting (Figure 7E). However, these two motifs probably
differ from each other despite having a common R/K signature
because the Phe-98 in the second motif (i.e., KKFR) also appears to be critical for EHM targeting as evidenced by the
compromised EHM localization of R82 F98A (Figure 7B),
whereas there is an Ala at the corresponding position (i.e.,
KRAK) in the first motif. In addition, the neighboring residues of
these two motifs are also quite different (Figure 6A). Thus,
more detailed site-directed mutagenesis is needed to define
the boundaries of these two functional elements. Future work
is also required to determine whether and how these two
motifs may act synergistically to facilitate protein sorting to the
EHM.
EHM Targeting versus Nuclear Localization: Which Way
to Go?
We found that six C-terminally truncated RPW8.2 mutants (from
R82Δ138-174 to R82Δ112-174) showed both EHM and nuclear
localization and four C-terminally truncated mutants (from
R82Δ111-174 to R82Δ88-174) exhibited exclusive nuclear localization (Figure 4). Sequence analysis of RPW8.2 with cNLS Mapper
(Kosugi et al., 2009) (nls-mapper.iab.keio.ac.jp) also identified
two NLSs: amino acids 66 to 77 (RKVNKRLKLLLE) with a medium score (4.5) and amino acids 91 to 101 (RRNVRKKFRYM)
with a high score (9.5). The latter NLS coincides with the second
ETS (Figure 6A). Given that the two R/K-R/K-x-R/K motifs play
a critical role in guiding RPW8.2 to the EHM, it is interesting to
note that an RRxR motif has been reported to be critical for
nucleolus localization of several proteins (Scott et al., 2001;
Meder et al., 2005; Müller et al., 2010). Nuclear-localized
RPW8.2 mutant proteins were often detected as variable-sized
speckles in the nucleus (Figure 4), resembling nucleolar localization patterns. Therefore, the wild-type RPW8.2 protein may be
partitioned between the EHM and the nucleus and that the
second R/K-R/K-x-R/K motif may be engaged for targeting
RPW8.2 to these two distinct destinations. Several immunity
proteins, including MLa10, RPS4, EDS1, and NPR1, partition
between the cytoplasm and the nucleus (Kinkema et al., 2000;
Feys et al., 2005; Shen et al., 2007; Bhattacharjee et al., 2011;
Heidrich et al., 2011) and NPR1 gets degraded in the nucleus
(Spoel et al., 2009; Fu et al., 2012). However, since we have not
observed YFP-tagged RPW8.2 wild-type protein in the nucleus, it is possible that either wild-type RPW8.2 is exclusively
targeted to the EHM or only a very small portion of wild-type
RPW8.2 is partitioned to the nucleus (possibly for activation of
defense gene expression) and then rapidly degraded. Consistent
with the above speculation, we found that the C-terminal 37
amino acids plays a role in suppressing nuclear localization
(Figure 4), and there is a predicted NES in this region (i.e., 127IKELKAKMSEI-137).
Taken together, our results suggest that RPW8.2 might be subject to regulation by multiple (and potentially opposing) trafficking
forces and that its EHM-specific localization probably requires
accurate spatiotemporal expression and engagement of proper
trafficking machinery (Figure 8F).
Formation of a Fireworks-Like Domain: The Water
Ripple Effect?
A small portion of both R82Δ5-12 and R82Δ5-14 was detected in
the fungal penetration site as fireworks-like puncta radially
distributed around the penetration site (Figure 3) where R82Ms-0
has not been observed. Because this domain seemed to align
well with the cell wall around the penetration site when viewed
horizontally (Figure 3C), we speculate that the fireworks-like
domain represents the portion of the PM affected by fungal
penetration. If so, the fungal penetration of the host cell wall
may produce a physical impact on the portion of the PM surrounding (10 to 30 µm) the penetration site, resembling the
water rippling effect when a stone is tossed into a still pond.
This disturbance may rapidly change the chemical nature of
the affected PM, possibly resulting in formation of microdomains (lipid rafts) where proteins such as R82Δ5-12 and
R82Δ5-14 can be selectively incorporated. Such fireworks-like
domains are reminiscent of concentric rings forming a bull’s
eye at the penetration hole as visualized by green fluorescent
protein–tagged PM resident proteins such as the syntaxin
SYP121 (Assaad et al., 2004). Our observations also imply that
R82Δ5-12 and R82Δ5-14 may be altered in such a manner that
these mutant proteins have a reduced affinity to the EHM and/or
gain a higher affinity to the fungus-perturbed PM. How the
fireworks-like domain differs from the PM and the EHM and
whether the EHM is formed via invagination and differentiation
of the perturbed PM remain interesting questions for future
studies.
The PSM: An Ancient Host–Microbe Interface?
Several RPW8.2 mutants were targeted to the stromule-like
membrane. Based on the tight periplastid localization of
RPW8.2 mutant proteins such as R82Δ65-93+Δ138-174 and
R82Δ120-174 (Figures 5A and 5J), we initially thought that these
mutant proteins were targeted to the stromule. Subsequent
colocalization analysis with a stromule marker indicated that
R82Δ65-93+Δ138-174 was actually in a membrane that wraps
around the stromule (Figures 5K and 5L). We thus propose that
there exists a novel peristromule (and plastid) membrane (PSM)
that is distinct from the stromule. Considering that plastids
evolved from cyanobacteria through primary endosymbiosis
with a primitive eukaryote more than one billion years ago
(Yoon et al., 2009; Price et al., 2012), it is possible that the PSM
may represent an ancient host-microbe interface. Because
several RPW8.2 mutant proteins were found at both the EHM
and the PSM in the same invaded cells (Figures 8B to 8E), it
is tempting to speculate that the EHM (a newly formed hostmicrobe interface) and the PSM may share some common features that allow localization of RPW8.2 variants. In addition, as
long tubular structures labeled by R82Δ65-93+Δ138-174 connect
the PSM with the EHM in some epidermal cells (Figure 5I), it
Mutational Analysis of RPW8.2
seems possible that the dynamic PSM and the extended tubules may serve as trafficking routes for these mutant or even
wild-type RPW8.2 proteins to reach the EHM (Figure 8F).
However, we have never observed wild-type RPW8.2 in the
PSM tubules or in the punctate ring structures surrounding
chloroplasts in the mesophyll cells, despite repeated close
examination. One possibility is that vesicles carrying wild-type
RPW8.2 travel too fast to allow detectable protein accumulation in the trafficking pathway, whereas vesicles loaded with
RPW8.2 mutant proteins move more slowly and thus accumulate along their trafficking pathway toward the EHM, making
them visible. Alternatively, RPW8.2 may be targeted to the
EHM via a different, yet fast, route (Figure 8F).
The endoplasmic reticulum (ER) and Golgi network may be
associated with the formation of stromules (Isakoff et al., 1998),
and stromule branching coincides with contiguous ER tubules
(Schattat et al., 2011). Therefore, one might think it possible that
RPW8.2 variants are retained at the ER network tightly associated with stromules, forming ER-membrane tubules. However,
the thin-thread tubules labeled by RPW8.2 variants (Figure 5) are
clearly different from the ER tubular network associated with
stromules (Figure 2 in Schattat et al., 2011). Hence, a more likely
explanation is that the intimate association of the ER network
with dynamic stromules provides short conduits for instantaneous targeting of lipids and membrane proteins to form the
PSM under inductive conditions. How the PSM differs from
other endomembranes, how its biogenesis is coordinated with
the plastid status and stromule formation, and how it might be
involved in plastid-cytoplasm and even plastid-nucleus (Figure
8D) intercommunication during stress response are interesting
questions for future studies. In this regard, it is worth noting that
NRIP1, a chloroplast-localized protein, was found in the stromule upon interaction with the coat protein of an RNA virus, the
Tobacco mosaic virus (Caplan et al., 2008), and that a plant
DNA-virus has also been shown to induce stromule formation in
epidermal leaf tissues (Krenz et al., 2012).
What May be the Trafficking Cue for EHM Localization
of RPW8.2?
Having identified the putative EHM targeting signals, a challenging question then is why RPW8.2 is specifically targeted to
the EHM? What is special about the EHM? It is reasonable
to speculate that during its biogenesis, the EHM may attain
a special lipid composition as a result of the intimate host–
haustorium interaction; consequently, this unique membrane
feature may determine its protein constitution by selective recruitment. More specifically, we hypothesize that (1) a particular
lipid species may be (transiently) enriched in the EHM during its
biogenesis, and (2) this lipid molecule interacts with RPW8.2,
providing a trafficking cue for EHM-specific targeting. In this
regard, it is interesting to note that several characterized lipid
binding protein domains, such as the PH domain [KXn(K/R)XR]
(Isakoff et al., 1998; Lemmon, 2008) and the FYVE domain (RR/
KHHCR) (Misra et al., 2001; Kutateladze, 2006), are also enriched in basic residues similarly to the two ETSs in RPW8.2.
Future work will be focused on characterizing the lipid characteristics of the EHM (and the PSM) in comparison with other
17 of 20
endomembranes and investigating whether RPW8.2 binds any
lipid molecule(s) enriched in the EHM for realization of its EHMspecific localization.
METHODS
Plant Lines, Growth Conditions, and Transformation
Arabidopsis thaliana accession Col-0 (or Col-gl) was used for generation
of all transgenic lines expressing each of the >100 constructs from the
native RPW8.2 promoter. Accession Ms-0 containing RPW8.1 and
RPW8.2 (together referred to as RPW8 unless otherwise indicated) and/or
a homozygous Col-0 transgenic line R2Y4 expressing RPW8.2-YFP under
control of the native promoter (Wang et al., 2009) were used as control for
resistance phenotypes and a homozygous Col-0 transgenic line C15
expressing YFP under control of the 35S promoter (Wang et al., 2007) as
control for susceptibility phenotypes.
Unless otherwise indicated, seeds were sown in Sunshine Mix #1
(Maryland Plant and Suppliers) and cold treated (4°C for 2 d), and
seedlings were kept under 22°C, 75% relative humidity, short-day (8 h
light at ;125 µmol$m22$s21, 16 h dark) conditions for 5 to 6 weeks before
pathogen inoculation or other treatments.
DNA Constructs
All deletion, point mutation, and NAAIRS replacement RPW8.2 mutant
constructs were generated by extension-overlap PCR (Vallejo et al., 2003)
using the high-fidelity thermostable Pyrococcus furiosus (Pfu) DNA
polymerase according to the manufacturer’s instructions (Fermentas). To
simplify cloning methods, we first made the core binary vector pP2Y39 via
two cloning steps. First, the RPW8.2 native promoter was amplified with
primers EcoR82PF (59-CAGAATTCACCGAAATTGTTAGTATTCA-39) and
BamR82PR (59-ATGGATCCGAAATTAGTTTGTTAGCTCTCGAG-39), digested with EcoRI and BamHI, and cloned into the EcoRI-BamHI site of
pPZP211, generating an intermediate vector pPR8R5. Then, the cassette
containing eYFP and the 39-untranslated region of RPW8.2 was amplified
with primers BamYFPF1 (59-TCGGATCCATGGTGAGCAAGGGCGAG-39)
and BglR823’R (59-TGAGATCTTTTGTTGTTTTTTACTCT-39) from
pPR82EYFP (Wang et al., 2007), digested with BamHI and BglII, cloned
into the BamHI site of pPR8R5, generating the core binary vector pP2Y39.
All deletion and site-specific substitution RPW8.2 mutants were cloned
into the BamHI site of pP2Y39. The NAAIRS replacement RPW8.2 mutants
were made following two-round PCR (Rairdan et al., 2008) in which two
PCR products from RPW8.2 containing 59-AATGCTGCTATACGATCG-39
to replace 18 nucleotides encoding the targeted six amino acids were
amplified (with one being 59 and one being 39 of the mutation site) and
used for further amplification of the RPW8.2 mutant using primers
BamR82F (59-CACCGGATCCATGATTGCTGAGGTTGCCGCA-39) and
BamR82R (59-CGCGGATCCAGAATCATCACTGCAGAACGTAAA-39). All
site-specific mutants had the same start and termination sites as the wildtype constructs. All constructs generated by PCR were verified by sequencing and introduced into Arabidopsis accession Col-gl.
C-terminal, N-terminal, and internal deletion mutant constructs were
made by PCR with appropriate primers based on the sequence of the
target regions. For replacing the RPW8.2 transmembrane domain with
that from SYP122 or ACBP2, the TMD of SYP122 (amino acids 285 to 304)
or ACBP2 (amino acids 7 to 29) was amplified using TMs’82F and
TMs’82R, or TMa’82F and TMa’82R, respectively, and then translationally
fused with RPW8.2 (amino acids 1 to 19) using 19R82F and BamR82R.
Sequences of all the primers used in this study are provided in
Supplemental Table 2 online.
18 of 20
The Plant Cell
Pathogen Infection and Microscopy
The powdery mildew isolate Golovinomyces cichoracearum UCSC1 was
maintained on live eds1-2 or Col-0-nahG plants for generation of fresh
fungal spores. Inoculation and visual scoring of disease reaction phenotypes were done as previously described (Xiao et al., 2003, 2005).
Confocal laser scanning microscopy images were acquired as previously
described (Wang et al., 2007, 2009) using the Zeiss LSM 710 microscope.
All pictures presented in the figures are projections from Z-stacks of 15 to
65 images unless otherwise indicated. The image data were processed
using Zeiss laser scanning microscopy Image Browser or the ZEN microscope software (2012 edition) and Adobe Photoshop CS4.
Phenotypic evaluation and microscopy examination were done with all
T1 lines for each DNA construct, and the results were confirmed with T2 or
T3 generations for some selected DNA constructs (indicated by an asterisk in Table 1 and Supplemental Table 1 online).
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under accession number
AF273059 (RPW8).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Representative Confocal Images Showing
Normal EHM Localization of Eight Mutant RPW8.2 Proteins.
Supplemental Figure 2. Unusual Subcellular Localization of R82Δ5-12
and R82Δ5-14 in Haustorium-Invaded Cells.
Supplemental Figure 3. The Transmembrane Domain of RPW8.2 Is
Important for Efficient EHM Localization.
Supplemental Figure 4. Cell Death and/or Resistance Phenotypes of
Representative Transgenic Lines Expressing RPW8.2 Mutant Proteins.
Supplemental Figure 5. Subcellular Localization of C-Terminally
Truncated RPW8.2 Mutant Proteins.
Supplemental Figure 6. Subcellular Localization of Various RPW8.2
Mutant Proteins.
Supplemental Table 1. A Summary of RPW8.2 Mutant Constructs for
Mapping the EHM Targeting Signal.
Supplemental Table 2. Primers Used in This Study.
Supplemental Movie 1. Z-Stack Confocal Images Showing the
Localization of R82D5-12-YFP in a Haustorium-Invaded Epidermal Cell.
ACKNOWLEDGMENTS
We thank Ryan Cooper for maintaining plant growth facility and Amy
Beaven for technical help with confocal imaging. This project was
supported by National Science Foundation grants (IOS-0842877 and
IOS-1146589) to S.X. and a grant from the National Natural Science
Foundation of China (31071670) to W.W.
AUTHOR CONTRIBUTIONS
W.W. and Y.Z. performed most of the experiments with support from
S.X., Y.W., R.B., X.M., Z.P., D.B., H.K., and Q.Z. W.W. and S.X. designed
the experiments and wrote the article.
Received August 7, 2013; revised September 15, 2013; accepted
September 24, 2013; published October 22, 2013.
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A Comprehensive Mutational Analysis of the Arabidopsis Resistance Protein RPW8.2 Reveals Key
Amino Acids for Defense Activation and Protein Targeting
Wenming Wang, Yi Zhang, Yingqiang Wen, Robert Berkey, Xianfeng Ma, Zhiyong Pan, Dipti
Bendigeri, Harlan King, Qiong Zhang and Shunyuan Xiao
Plant Cell; originally published online October 22, 2013;
DOI 10.1105/tpc.113.117226
This information is current as of June 17, 2017
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