Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus
Journal of Experimental Botany
Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus
Björn Scheler 0
Vera Schnepf 0
Carolina Galgenmüller 0
Stefanie Ranf 0
Ralph Hückelhoven 0
Katherine Denby, University of Warwick
0 Phytopathology, Technische Universität München , D-85354 Freising-Weihenstephan , Germany
RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh. RACB is required for positioning of the nucleus near the site of attack from Bgh. We therefore suggest that Bgh profits from RACB's function in cell polarity rather than from immunity-regulating functions of RACB.
Blumeria graminis; disease susceptibility; epidermis; MAP kinase; nucleus; oxidative burst; polarity; ROP GTPase
Plants possess an innate immunity, which constantly
monitors the cell surface and cytoplasm for the presence or
activity of pathogenic organisms. Plant immune receptors
can detect conserved molecular patterns that derive from
microbes (MAMPs, microbe-associated molecular patterns)
or from host cell damage (damage-associated molecular
patterns). Such receptors are called pattern recognition
receptors (PRRs). They are localized in the host plasma membrane
and function in basal resistance to non-adapted and virulent
pathogens (Macho an
d Zipfel, 2014
). Additionally, a second
class of plant immune receptors co-evolved with specific,
largely polymorphic virulence effectors
(Jones and Dangl,
. Most of these so-called resistance (R) proteins are
localized in the cytoplasm and nucleoplasm. For triggering
immunity, R proteins directly interact with effector proteins,
monitor functionality of effector targets, or mimic effector
(Dodds and Rathjen, 2010)
. Plant immunity is robust
in most environments. Nevertheless, microbes adapt to plant
hosts by evolution of virulence effectors that suppress or
circumvent host immunity.
Plant disease resistance can be observed as a result of
MAMP- or effector-triggered immunity but also as a
consequence of mutations of susceptibility genes. Susceptibility
genes encode host factors that are required for pathogenesis
in interactions of susceptible hosts with adapted virulent
pathogens. Mechanistically, loss of susceptibility can result
from de-regulated or primed immunity if susceptibility genes
code for negative regulators of plant defence. Alternatively,
loss of susceptibility can be explained by lack of or mutation
of effector targets that serve demands of the pathogen other
than suppressing immunity. Furthermore, susceptibility
factors might provide developmental or metabolic prerequisites
for attraction, accommodation, or feeding of the pat
(Hückelhoven et al., 2013
Lapin and Van den Ackerveken,
). Loss of susceptibility is usually recessively inherited
after loss of gene function and often accompanied by
pleiotropic effects that limit applicability in plant breeding (
et al., 2010
Hückelhoven et al., 2013
; van Schie an
). Thus a deeper understanding of susceptibility is
required to inform plant breeding.
Plant monomeric RHO GTPases (rat sarcoma
homologues, also called RAC for rat sarcoma-related C3
botulinum toxin substrate or ROP for RHO of plants) are
involved in immunity and susceptibility to plant diseases.
Type I RAC/ROPs possess a typical motif for
post-translational prenylation at their C-terminus, and can be
additionally palmitoylated after activation. In contrast, type II
RAC/ROPs are often constitutively S-acylated
. In rice, the type II RAC/ROP protein RAC1 is a
central regulator of immune response mediated by either PRRs
or R proteins
(Kawano et al., 2014)
. The rice chitin elicitor
PRR CERK1 can activate RAC1 via a plant-specific
guanine nucleotide exchange factor RACGEF1. RAC1
orchestrates the elicitor-activated production of reactive oxygen
species (ROS), mitogen-activated protein (MAP) kinase
(MAPK) activation, transcriptional responses, and changes
of the host proteome
(Kawano et al., 2014)
RAC1 is activated downstream of the R protein Pit, a
nucleotide-binding leucine-rich repeat protein, which
confers effector-triggered immunity to the rice blast fungus
(Kawano et al., 2010)
. However, in rice
and barley, there are also other type I and type II RAC/ROP
GTPases that limit basal resistance or support susceptibility
to fungal diseases
(Chen et al., 2010; Schultheiss et al., 2002,
. Barley RACB, a type I RAC/ROP, is required for
full susceptibility to barley powdery mildew caused by the
biotrophic ascomycete Blumeria graminis f.sp. hordei (Bgh).
Transient or stable gene silencing of RACB by RNAi limits
fungal entry and formation of fungal haustoria in barley
(Schultheiss et al., 2002; Hoefle et al., 2011)
Molecular cell biology suggests a role for RACB in
organization of the cytoskeleton
(Opalski et al., 2005; Hoefle
et al., 2011; Huesmann et al., 2012)
expression of constitutively activated (CA) RACB (RACBG15V)
enhances powdery mildew susceptibility, supports
establishment of haustoria in barley epidermal cells, but has little
effect on cellular defence reactions. When overexpressed in
single epidermal cells or transgenic plants, barley type II CA
RAC/ROPs (RAC1, RAC3, and ROP6) can support
susceptibility to powdery mildew too, whereas RACD, another
type I RAC/ROP, appears not to influence the outcome of
interaction with Bgh
(Schultheiss et al., 2003; Pathuri et al.,
. Little is known about the function of barley RAC/
ROPs in interaction with other microbes. However, when
ectopically expressed, barley CA RACB and CA RAC3 can
support susceptibility to Pseudomonas syringae pv. tabaci
in tobacco, and barley CA RAC1 can support penetration
resistance to hemibiotrophic M. oryzae in transgenic barley
(Pathuri et al., 2008, 2009)
RAC/ROPs function in plant cell polarity. This is well
established for root hair tip growth, pollen tube tip growth,
and epidermal pavement cell interdigitation
In particular, type I RAC/ROP GTPases of dicots function
in cell polarity. Little is known about RAC/ROP functions
in polar cell growth in monocots. It has been described that
barley CA RACB enhances epidermal cell size in leaves.
Root hair phenotypes of transgenic CA RACB barley were
reported as root hair swelling on solid medium, which is
typical in dicots expressing CA ROP genes
(Jones et al.,
2002; Pathuri et al., 2008, 2009)
. In contrast, silencing of
RACB by RNAi in transgenic barley led to a defect in the
ability to form root hairs
(Hoefle et al., 2011)
. In maize,
the development of stomatal complexes was reported to
depend partially on ROP2 and ROP9, two type I RAC/
ROP proteins very similar to barley RACB
et al., 2011)
It has not been studied whether RACB interferes with
pattern-triggered immunity or defence gene expression in
response to Bgh. Here, we show that RACB does not limit
early MAMP-triggered immune responses and supports
rather than limits expression of defence genes. However,
knock down of RACB strongly affects polar cell growth and
positioning of the nucleus in barley epidermal cells. Bgh may
hence profit from functions of RACB in cell polarity during
invasion of host cells.
Materials and methods
Plant material and growth conditions
For all experiments, the barley (Hordeum vulgare) cultivar Golden
Promise and transgenic RACB plants with the genetic background
of Golden Promise were used. The overexpressor line of CA RACB
17/1-11 and RACB RNAi 16/2-4B and 15/1-16 have been described
(Schultheiss et al., 2005; Hoefle et al., 2011)
were surface-sterilized in 20 ml of sterilization solution (4% NaOCl,
Tween-20) for 1.5 h with shaking. After washing with H2O for
30 min, husks were carefully removed without damaging the embryo
to guarantee equal germination of seeds. Seeds were pre-germinated
on wet filter paper for 2 d in the dark before being sown into soil (Typ
ED73, Einheitserde- und Humuswerke, Gebr. Patzer GmbH & Co
KG, Sinntal-Jossa, Germany). Plants were grown in a growth
chamber (Conviron, Winnipeg, Canada) at 18 °C with relative
humidity of 65% and a photoperiod of 16 h. Both transgenic genotypes
do not produce homozygous offspring. Offspring of transgenic T3
donor plants were genotyped according to previous studies to
separate transgenic offspring carrying the T-DNA from azygous
offspring that lost the T-DNA due to segregation. Azygous sister plants
are similar to the wild type
(WT; Schultheiss et al., 2005; Hoefle
et al., 2011)
and thus served as ideal controls. Arabidopsis thaliana
ecotype Columbia 0 (Col-0) seeds were purchased from Lehle Seeds
(Round Rock, USA) and stratified for 2^d at 4 °C before placing into
a growth chamber. Plants were grown at 22 °C with a photoperiod
of 10 h and a relative humidity of 65%.
The flagellin elicitor flg22
(Felix et al., 1999)
was synthesized as
(Ranf et al., 2011)
. Chitin from shrimp shells
(Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was ground
to a fine powder and suspended in H2O (20 mg ml–1). Insoluble chitin
fragments were removed by centrifugation (1900 g, 10 min) and the
supernatant was used for experiments.
For detection of activated MAPKs, we used 10 leaf discs of 5 mm
diameter from second leaves of 14-day-old barley plants or from
6-week-old Arabidopsis plants per time. Leaf discs were
incubated in 2 ml of H2O/well for 16 h in 24-well plates, transferred to
fresh H2O for 30 min, and subsequently elicited with 1 µM flg22 or
100 µg ml–1 chitin. Detection of activated MAPKs with anti-pTEpY
(α-phospho-p44/42-ERK, Cell Signaling Technology, Boston,
USA) was performed as previously described
(Saijo et al., 2009;
Ranf et al., 2011)
Detection of ROS production of barley leaves
ROS production was assayed by H2O2-mediated oxidation and
luminescence of the luminol derivative L-012 (Wako Chemicals GmbH,
Neuss, Germany). Leaf discs (5 mm diameter) of 7-day-old barley
plants were floated in 200 µl of H2O/well overnight in a 96-well plate.
After removal of H2O, leaf discs were incubated for 30 min in 2 µg
ml–1 horseradish peroxidase (HRP) and 10 µM L012. Subsequently,
leaf discs were elicited with 100 nM flg22 or 100 µg ml–1 chitin.
Luminescence was measured at 1 min intervals with a Tecan Reader
(infinite M200, Tecan, Männedorf, Switzerland) for 30min. We
calculated relative luminescence units (RLU) by subtraction of leaf
disc-specific background (recorded for 5min before elicitation) and
of mock treatment-associated blanks.
Quantitative reverse transcription PCR
Gene expression analysis was carried out by reverse
transcription quantitative real-time PCR (RT–qPCR) in a Mx3005P cycler
(Agilent Technologies, Santa Clara, CA, USA) using the Maxima
SYBR Green qPCR master mix (2×) (Thermo Fisher Scientific, St.
Leon-Rot, Germany). Reactions were performed in duplicate with
10 ng of cDNA and 330 nM forward and reverse primer each in a
final volume of 10 µl. Expression values of defence genes and RACB
were normalized to a barley housekeeping ubiquitin (HvUBI)
(Ovesna et al., 2012)
using primer efficiency correction as suggested
. The program consisted of an initial step at 95 °C
for 10 min and 95 °C for 30 s, followed by 40 cycles at 55 °C for 30 s
and at 72 °C for 1 min. The melting curve analysis was performed at
55–95 °C. All primers (Table 1) were designed using Primer3
(Untergasser et al., 2012)
and were checked for specificity using
the Basic Local Alignment Search Tool (BLAST) and therein with
nucleotide blast against the H. vulgare database (http://blast.ncbi.
nlm.nih.gov), and amplicon size assessment in agarose gels before
Scanning electron microscopy
For scanning electron microscopy (SEM), root and leaf material was
harvested and fixed in 4% paraformaldehyde (4% PFA) in 1×
phosphate-buffered saline buffer (1× PBS), pH 7.4 as described
and Friml, 2010)
. Fixed material was washed in 1× PBS, pH 7.4
three times for 10 min, followed by three washing steps in distilled
water for 10 min. Dehydration occurred in an increasing ethanol
series of 25% (v/v), 50% (v/v), and 75% (v/v) in distilled water and
pure ethanol three times each for at least 10 min. For critical point
drying, the EM CPD300 Automated Critical Point Dryer (Leica,
Vienna, Austria) was used and drying was done following the ‘Rice
Root’ protocol for root tissue and the ‘Tobacco Leaf ’ protocol for
barley leaf material as described in the manufacturer´s manual. The
imaging was done using the TM300 Tabletop Microscope (Hitachi,
Tokyo, Japan). The image editing program GIMP 2.8 was used to
merge single root pictures to generate root overviews and to colour
subsidiary cells in the leaf images.
Fluorescence microscopy of root tissue
Seedling roots were harvested, fixed, and washed as described
above. For staining, propidium iodide (PI; Applichem, Darmstadt,
Germany) was dissolved in distilled water to a final concentration of
100 µg ml–1 for RACB RNAi root material and 40 µg ml–1 for the
azygous control plants. The root material was incubated in the staining
solution for 1 h in the dark. Subsequently, stained roots were
transferred to a clearing solution, prepared by mixing chloral hydrate,
glycerol, and water in the ratio 4:1:2 (w/v/v) and kept there for 15 h.
After clearing, the roots were directly mounted in Hoyer’s solution
consisting of 1 g of glycerol, 10 g of chloral hydrate, and 1.5 g of
gum arabic dissolved in 2.5 ml of distilled water. Visualization
followed immediately using a Leica TCS SP5 Confocal Microscope
and the Leica LAS AF software (Leica Microsystems, Mannheim,
Germany). PI was excited by a 561 nm laser line and emission was
detected from 560 nm to 675 nm.
Measurement of the nucleus attraction index
At 8 h after inoculation, the leaf material was harvested and halved
along the longitudinal axis using a razor blade. One half of the leaf
blade was used for relative quantification of RACB expression.
Leaf pieces were fixed, de-waxed, and destained
(Sauer and Friml,
. For the last rehydration step, 1× PBS, pH 7.4 was used. To
remove RNA from the tissue, RNase A (DNase free, Applichem,
Darmstadt, Germany) was dissolved in 10 mM Tris–HCl, pH 7.5
to a final concentration of 10mg ml–1. The stock solution was
subsequently diluted in 1× PBS, pH 7.4 to 100 µg ml–1 to achieve the
RNase A solution in which leaf material was incubated for 1 h for
RNA digestion. Subsequently the leaves were placed in the staining
solution (100 µg ml–1 PI in distilled water) for at least 5 min. To
determine the nucleus attraction index (NAI), epidermal B cells
et al., 1990)
, which were attacked by a single fungal appressorium,
were imaged. A z-stack was recorded starting from the brightest
fluorescence of the fungal appressorium to the brightest fluorescence of
the plant nucleus. The picture number and increments were adjusted
for each cell and z-stack, depending on the vertical distance between
the appressorium and plant nucleus. The NAI was calculated as
follows: NAI = a2 + b2 / d, where a reflects the depth of the z-stack
and b the planar distance between the appressorium and the nucleus.
Both represent the legs of a right-angled triangle. The diagonal of
the B cell is represented by d. Cell size measurement was performed
using the software ImageJ
(Schneider et al., 2012)
Rhodamine 123 staining and trichoblast quantification
Rhodamine 123 (R123) selectively stains mitochondria in living
. A stock solution was prepared by dissolving R123
(Sigma-Aldrich, St Louis, MO, USA) in DMSO to a final
concentration of 10 mg ml–1 For the staining solution, the stock solution was
diluted in 0.5× Murashige and Skoog medium with modified
vitamins (0.5× MS; Duchefa Biochemie, Harleem, The Netherlands)
mixed with sucrose to 1% (w/v) and
2-(N-morpholino)ethanesulphonic acid (MES; Carl Roth, Karlsruhe, Germany) to 0.05% (w/v)
final concentration, pH 5.6 to 1 µg ml–1. Intact seedlings were
incubated in the staining solution for 10 min in the dark. After staining,
seedlings were briefly rinsed in an excess of 0.5× MS, pH 5.6 and
immediately visualized by confocal microscopy. R123 was excited
by a 488 nm laser line and the emission was detected from 515 nm to
575 nm. In an early developmental state, trichoblasts were counted
before root hair initiation each in an area of 0.024 mm2.
RACB does not control early MAMP responses in barley
Specific RAC/ROP GTPases modulate immune responses
and NADPH oxidase-dependent ROS production in plants.
Therefore, we tested barley WT Golden Promise and
corresponding transgenic barley plants silenced for RACB
by RNAi or overexpressing CA RACB for their ability to
respond to MAMPs by production of ROS. Transgenic lines
used have been validated before as being silenced or
overexpressors, respectively, and are representatives of several (CA
RACB) or two (RACB RNAi) independent lines with
consistent transgene-associated phenotypes
(Schultheiss et al., 2005;
Hoefle et al., 2011)
. The barley type I RAC/ROP gene RACD
is co-silenced in the RACB RNAi line, whereas other RAC/
ROPs show WT-like expression
(Hoefle et al., 2011)
WT barley plants showed a typical MAMP-triggered
oxidative burst when challenged with a chitin elicitor preparation.
No ROS burst was recordable in mock-treated plants. After
elicitor treatment, barley leaf discs rapidly produced ROS.
The kinetics and amount of ROS produced appeared similar
in WT, CA RACB, and RACB RNAi barley (Fig. 1A). To
test whether RACB might influence the oxidative burst
elicited by a fungus-unrelated MAMP, we included the bacterial
flagellin-derived elicitor flg22 in our experiments
(Felix et al.,
. The flg22 peptide elicited an oxidative burst that was,
when taking variance of biological repetitions into account,
indistinguishable between WT, CA RACB, and RACB-RNAi
barley (Fig. 1B).
Fig. 1. MAMP-triggered ROS burst is unaffected by RACB transgenes.
(A) Chitin- (100 µg ml−1) triggered ROS in barley is unaffected by
overexpression of CA RACB (RACB OE) or by silencing RACB (RACB
RNAi). (B) Flagellin- (100 nM flg22) triggered ROS in barley is unaffected by
CA RACB (RACB OE) or by silencing RACB (RACB RNAi). Elicitors were
added to leaf discs at 0 min and ROS-dependent luminol luminescence
recorded over 30 min. Data show relative luminescence units (RLU) that
have been corrected by subtraction of leaf disc-specific background
(recorded for 5 min before elicitation) and mock treatment-associated
blanks (average of eight leaf discs). Error bars show the SE over the mean
of four (A) or three (B) experiments each with eight elicited leaf discs per
MAPK activation is another typical early MAMP response
and potentially modified by plant RAC/ROPs. Activation of
MAPKs can be detected by immunodetection of a
phosphorylated MAPK-typical TEY motif (pTEpY), such as that
present in Arabidopsis MPK3 and MKP6
(Ranf et al., 2011)
detected phosphorylated MAPKs (MAPK-P) in Arabidopsis
and barley leaf discs treated in parallel either with chitin or
with flg22 (Fig. 2A). In Arabidopsis, typical
phosphorylation of MAPKs was detected after elicitation with chitin or
flg22. Both elicitors induced a similar pattern of activated
MAPK-P in protein extracts from barley leaf discs. However,
one or two bands appeared predominant in most
experiments, whereas in Arabidopsis extracts two to three bands
were detected. This suggested that barley reacts to MAMPs
with activation of MAPKs. We then compared patterns of
MAPK-P in WT barley, CA RACB barley, and RACB RNAi
barley (Fig. 2B). This revealed, at the given level of detection,
similar MAPK activation in all three genotypes, with a slight
increase visible after 5 min and a decline between 20 min and
40 min after elicitation. This suggests that activity and
abundance of RACB do not strongly influence barley competence
to react to MAMPs with typical early MAMP responses.
We further studied pathogenesis-related (PR) PR1, PR3,
PR5, and PR10 gene expression after high density
inoculation with Bgh, because the strength of PR gene expression has
been linked to penetration resistance in barley
et al., 1997; Molitor et al., 2011)
. We further wanted to test
whether RACB possibly acts as a negative regulator of PR
or jasmonate-associated gene expression (jasmonate-induced
genes JIP23 and JIP60)
(Kogel et al., 1995)
. We compared
gene expression after mock inoculation and at 12 h and 32 h
after inoculation (HAI), because these times represent stages
of fungal penetration attempts and haustorium expansion,
and both processes are influenced by RACB
(Hoefle et al.,
. PR genes showed an enhanced expression level in
supersusceptible CA RACB barley 12 h after mock
inoculation. Conversely, PR genes were underexpressed 12 h after
mock inoculation in less susceptible RACB RNAi barley
when compared with the WT (Fig. 3A). At 32 h, similar and
partially stronger de-regulation of PR genes was observed in
RACB-transgenic barley. However, this was not statistically
significant for each individual PR gene (Fig. 3A). All four
PR genes were up-regulated after inoculation with Bgh in
the WT. At 12 and 32 HAI, CA RACB barley and RACB
RNAi barley reacted similarly to the WT when inoculated
with Bgh (Fig. 3B). Quantitative differences in the strength
of the PR gene expression post-inoculation are explained by
differences in constitutive gene expression. CA RACB barley
reacted to Bgh with a less strong PR gene expression response
because genes were already expressed at a higher level
without inoculation. JIP gene expression was not strongly
deregulated in RACB-transgenic barley. However, there was slightly
enhanced expression of JIP23 and JIP60 in CA RACB
barley (Fig. 3A, C). Other effects of genotype or inoculation
on JIP gene expression were less consistent between the two
sampling times. At 12 and 32 HAI with Bgh, the differences
in gene expression between WT and RACB-transgenic
genotypes were less pronounced when compared with the
situation without inoculation (compare Fig. 3A and C). However,
inoculated CA RACB barley still expressed single PR genes
and JIP23 on an up to a 2.7-fold higher level than the WT,
whereas RACB RNAi barley expressed the same genes at the
level of the WT or below. Together, the RACB transgenes
influenced defence gene expression but this did not reflect
altered susceptibility of RACB-transgenic barley.
RACB operates in root trichoblast polarity of barley
Because RACB did not regulate basal immune responses
in a way that would explain its function as a susceptibility
factor, we hypothesized that RACB’s role in plant cell
development could support pathogenesis. We therefore studied
RACB RNAi-mediated developmental failure in more detail.
We first confirmed by SEM that knock down of RACB
mediates inability to form root hairs (Fig. 4A, B). The roots
showed a dramatic reduction of root hair outgrowth and,
even if occasionally trichoblast protrusions were formed,
they remained short. This has been similarly reported before
for two independent transgenic RACB RNAi events
et al., 2011)
. Since barley root hairs can only develop from
short epidermal trichoblasts, we compared the epidermal
Fig. 4. Root hair phenotype of RACB RNAi plants. (A) SEM of barley
roots. Root hairs develop on azygous controls (non-transgenic segregants
from RNAi plants). (B) RACB RNAi plants do not show root hair outgrowth.
(C) Detailed view of the azygous control barley root stained with propidium
iodide. Propidium iodide intensely stains root hairs. (D) Detailed view on
a root segment of RACB RNAi plants, which corresponds to that of the
control in Fig. 3C. (E) Rhodamin-123 staining of a root segment close
to the tip of an azygous control root in which plasma-rich trichoblasts
differentiate from more vacuolized atrichoblasts. (F) Rhodamin-123 staining
of a root segment of RACB RNAi plants, which corresponds to that of
the control in Fig. 3E. (G) Counting of trichoblasts reveals no differences
in relative frequencies of cell types in azygous versus RACB RNAi barley
roots. Columns show the mean of 30 root samples with a total of 1844
(control) or 1846 cells (RACB RNAi) counted. Error bars show the SD of
the mean. Scale bars: A, B, 1 mm; C, D, 100 µm; E, F, 25 µm. (This figure
is available in colour at JXB online.
cell size pattern and number of trichoblasts in RACB RNAi
plants and non-transgenic sister plants that lost the silencing
cassette due to segregation (azygous control). Trichoblasts
are shorter than atrichoblasts at late stages of root
outgrowth (Marzec et al., 2013
). PI staining of comparable root
sections of root hair initiation showed the occurrence of a
typical pattern of shorter and longer cells in both RACB
RNAi roots and the control (Fig. 4C, D). However,
identification of short cells as trichoblasts was only possible after
root hair outgrowth. Thus we used the fact that trichoblasts
differ from atrichoblasts in size of vacuoles, density of
cytoplasm, and number of cell organelles
(Marzec et al., 2013)
and stained barley roots with the mitochondrial dye R123.
Trichoblasts showed a more intense staining by R123 due to
their higher number of mitochondria and denser cytoplasm
compared with atrichoblasts. This allowed for the
identification and quantification of trichoblasts before any obvious
differences in cell expansion occurred. Qualitative and
quantitative evaluation revealed that RACB RNAi plants were
able to form trichoblasts in the same amount and pattern as
azygous controls (Fig. 4E–G). Hence, RACB RNAi plants
are able to specify root epidermal cells as trichoblasts but
fail at bulging or tip growth at subsequent stages of root hair
RACB is involved in leaf stomatal subsidiary cell formation
Type I ROPs are required for the asymmetric cell division of
the subsidiary mother cell (SMC) resulting in the formation
of a stomatal subsidiary cell and a pavement cell in Zea mays
(Humphries et al., 2011)
. We hence visualized patterns of
epidermal cells in barley leaves using SEM. The subsidiary
cells of RACB RNAi barley showed deformations of
different degrees of severity or were often completely lacking. The
SMC-derived pavement cells also exhibited serious defects in
shape (Fig. 5B, D; Supplementary Fig. S1 at JXB online). The
guard cells and other cell types of the leaf epidermis, however,
were normally developed. We detected defective subsidiary
cell formation on the leaf blade as well as on the leaf sheath,
which develop from different meristems. Quantification of
cell shape defects revealed that RACB RNAi barley failed to
form normal stomatal subsidiary cells in ~20% of stomatal
complexes whereas in azygous sister plants, this failure was
observed in only 3.5% of stomata and was restricted to
moderately distorted subsidiary and pavement cells in most cases
RACB is involved in positioning of the nucleus in cells attacked by Bgh
Positioning of the polarized nucleus is a common element
of root hair outgrowth, subsidiary cell formation, and
powdery mildew infection
(Kita et al., 1981; Opalski et al., 2005;
Humphries et al., 2011; Griffis et al., 2014)
. To analyse the
influence of RACB on the position of nuclei in spatial
association with fungal attack, we measured distances between nuclei
and fungal appressoria in controls and RACB RNAi barley
(Fig. 6A, B). To avoid mistakes due to cell shape effects, we
focused on epidermal B-cell files between stomata and
stomata-associated A-cell files
(Koga et al., 1990)
we normalized distances to the cell sizes to obtain an index
(NAI, see material and methods) for each attacked cell that
displays the distances in a cell size-independent manner. We
chose 8 HAI as pre-penetration stage when appressoria are
fully developed but haustoria are not yet established. At this
point in time, nuclei were more distant from appressoria in
RACB RNAi plants when compared with azygous control
sister plants. This is evident by grouping the attacked cells by their
individual NAIs (Fig. 6C). However, even without
normalization to the cell sizes, the reduced attraction of the nucleus to
the site of attempted penetration is apparent (Fig. 6D). To
confirm that RACB RNAi plants are indeed less susceptible
already at early stages of cellular interaction with Bgh, we
scored frequencies of immature haustoria at 16 HAI, when
haustoria reach a size that can be readily detected after
staining with fluorescent wheat germ agglutinin (WGA). Similar
to that observed previously for 48 HAI
(Hoefle et al., 2011)
RACB RNAi plants allowed 35% less frequent establishment
of haustoria when compared with azygous control sister plants
(28.5% instead of 44.3% in the control) (Fig. 6F). We also
confirmed that the RACB transcript amount was indeed reduced
in the RACB RNAi plants when compared with azygous
sister plants at 8 and 16 HAI. Therefore, we had cut the leaves
at the mid rib and fixed one half of the leaf for determining
the nucleus to appressorium distance or the haustorium
frequency, respectively. The other halves of the leaves were used
to measure RACB transcript abundance by RT–qPCR. RACB
transcript abundance in RACB RNAi plants corresponded to
28% of the control level at 8 HAI and to 31% of the control
level at 16 HAI (Fig. 6E, G). Together, the susceptibility factor
RACB is involved in positioning of the nucleus in cells, which
Bgh attempts to penetrate. In plants in which RACB
expression is reduced by RNAi, the nucleus is more distant from the
appressorium before the fungus penetrates and the fungus is
subsequently less successful in penetration.
Susceptibility factors are plant components that serve the
demands of a pathogen during disease development. However,
the mode of action of susceptibility factors is often not well
understood. They might be classified as negative regulators
of host immunity or as host factors that support metabolic
or developmental processes required for successful
hogenesis (Hückelhoven, 2005
Hückelhoven et al., 2013
Van den Ackerveken, 2013
; van Schie an
d Takken, 2014
latter, however, is challenging to provide evidence for, since
it is difficult to distinguish whether failure of a pathogen to
infect is because the host mutant does not properly support
pathogenesis or, is due to an enhanced basal defence of that
mutant. Our data support that RACB acts as a
susceptibility factor through its function in cell polarity rather than
by suppressing early MAMP-triggered immune responses
or defence gene expression. Indeed, CA RACB enhanced
PR gene expression in non-infected plants, and silencing of
RACB lowered the level of PR gene expression. This is,
however, not reflected in the resistance status of RACB-transgenic
(Schultheiss et al., 2005; Hoefle et al., 2011)
and therefore counterintuitive. Interestingly, expression of a
dominant negative form of the Arabidopsis type I RAC/ROP
protein DN ROP6 also causes enhanced defence gene
expression. DN ROP6 expression further led to reduced
penetration success and reduced reproductive success of the powdery
mildew fungus Golovinomyces orontii in Arabidopsis. Genetic
experiments with salicylic acid biosynthesis and signalling
mutants suggested that defence gene expression can be
uncoupled from powdery mildew resistance in t
(PoratyGavra et al., 2013
). Arabidopsis ROP6 might be involved in
susceptibility to adapted powdery mildew independent of its
function in salicylic acid signalling and defence gene
expression. Together, perturbation of RAC/ROP signalling appears
to alter plant defence gene expression but this cannot explain
enhanced or reduced susceptibility of RAC/ROP mutants to
Developmental host reprogramming is observed for
mutualistic symbiosis such as root nodule development. However,
there are few examples of pathogenic interaction with plant
(Evangelisti et al., 2014)
barley susceptibility factor RACB is involved in plant
development and cytoskeleton organization
(Opalski et al., 2005;
Pathuri et al., 2008; Hoefle et al., 2011)
. However, other
RAC/ROP proteins are involved in regulating typical immune
(Kawano et al., 2014)
. We therefore studied
typical early MAMP responses in RACB-misexpressing barley.
This showed that barley reacts to MAMPs like other plants
by early ROS production and MAPK activation. However,
neither expression of CA RACB nor suppression of RACB
expression greatly influences the ability of barley to respond
quickly to fungal MAMP chitin or to the bacterial MAMP
flg22. This suggests that RACB and the co-silenced RACD
do not regulate canonical MAMP-triggered immunity in
RACB RNAi effects on fungal success to develop
haustoria can be observed in a cell-autonomous manner after
transient induced gene silencing or after transient overexpression
of CA RACB in single barley epidermal cells
et al., 2002, 2003)
. This shows that RACB is required for
fungal entry in a WT background, and reduced susceptibility of
RACB RNAi barley is not a secondary consequence of
developmental alterations. However, we considered developmental
effects of RACB RNAi as instrumental to better understand
the physiological role of RACB from which Bgh might profit.
The involvement of RACB-like RAC/ROPs of dicots in
development of root hairs and pollen tubes provoked the
‘inverted tip growth’ hypothesis
(Schultheiss et al., 2003)
According to this, Bgh profits from RACB’s function in polar
cell growth for inward growth of the fungal haustorium into
an intact epidermal cell that surrounds the haustorium with
a host-derived extrahaustorial membrane and matrix. Stable
transgenic RNAi-mediated silencing of RACB provided first
evidence for this hypothesis because a reduction of the RACB
transcript level in RACB RNAi plants caused a dramatic
reduction of frequency and size of hairs on the root epidermis
and a strong reduction of frequency and size of Bgh
haustoria in the leaf epidermis at 48 HAI (Hoefle et al., 2011). In
contrast, stable or transiently overexpressed CA RACB
supports establishment of haustoria but induces isotropic instead
of polar root hair growth
(Schultheiss et al., 2003; Pathuri
et al., 2008, 2009)
. In Arabidopsis, root hair development is
dependent on ROP signalling. ROP proteins accumulate at
the site of root hair initiation, and constitutively activated
ROPs abolish root hair polarity whereas dominant negative
ROPs restrict root hair development
(Molendijk et al., 2001;
Jones et al., 2002)
. The receptor-like kinase Feronia activates
ROP2 for root hair development
(Duan et al., 2010)
positioning of root hairs is spatially controlled by the
ROPGDP dissociation inhibitor SCN1, and different ROP-GEFs
influence the number, localization, and length of root hairs
(Carol et al., 2005; Huang et al., 2013)
. The NADPH oxidase
RBOHC is a potential ROP effector protein and required for
root hair formation
(Foreman et al., 2003; Jones et al., 2007)
Also in barley a SCN1 homologue and ROS might function
in root hair development
(Kwasniewski et al., 2010, 2013)
Actin microfilaments, microtubules, Ca2+ gradients, and ROS
together appear to orchestrate polar root hair initiation and
growth. Interestingly, all these components are influenced by
ROP signalling in root hairs
(Molendijk et al., 2001; Jones
et al., 2002; Carol and Dolan, 2006; Yang et al., 2007; Takeda
et al., 2008)
and play a role in interactions of plants with
powdery mildew fungi (Kobayashi et al., 1997;
Kim et al.,
Hückelhoven and Kogel, 2003
; Felle et al., 2004; Hoefle
et al., 2011;
Dörmann et al., 2014
). In the interaction of
barley with Bgh, microfilament and microtubule organization
are strongly influenced by RACB or by RACB-associated
(Opalski et al., 2005; Hoefle et al.,
2011; Huesmann et al., 2012)
. In barley, only short
plasmarich epidermal cells, which gained identity as trichoblasts,
are capable of initiating root hairs, whereas long expanding
and highly vacuolized atrichoblasts remain
et al., 2013
). We analysed cell identity of epidermis cells in the
root differentiation zone and root transition zone of RACB
(Verbelen et al., 2006)
. This suggests that the
knock down of RACB does not change trichoblast identity
(Fig. 4E, G) but limits the ability of trichoblasts to undergo
root hair initiation, bulging, and tip growth. Concerning the
role of ROPs in root hair development
(Molendijk et al.,
2001; Jones et al., 2002; Singh et al., 2008)
, this is probably
caused by the inability to establish and maintain cell polarity
in the trichoblasts, which is required for root hair
development. Together, this supports that RACB acts in cell
polarization during root hair initiation and tip growth. Bgh might
profit from a similar function for RACB during initiation of
and progressive ingrowth of the fungal haustorium into the
leaf epidermis. Root hair formation always goes along with
specific nucleus positioning in the trichoblasts throughout
all phases of root hair development
(Ketelaar et al., 2002;
Čiamporová et al., 2003)
(Fig. 7). Similar to this, the precise
positioning of the nucleus of the SMC next to the guard
mother cell (GMC) is the first visible indication for SMC
polarization during stoma development in barley and maize.
This SMC polarization is required for asymmetric cell
division, resulting in a small-volume subsidiary cell and a
largevolume epidermal pavement cell (Fig. 7) (Facette and Smith,
2012). The site-directed nucleus migration and polarization
of the SMC is highly ROP regulated. It is thought that maize
ROP2 and ROP9 stimulate the formation of a polar actin
patch after their own accumulation at the anticlinal interface
of the GMC. Maize rop2/rop2;rop9/+ mutants show a similar
subsidiary cell formation defect to that which we examined
on our barley RACB RNAi plants (Fig. 5)
(Humphries et al.,
2011; Facette and Smith, 2012)
. Considering that ROP2 and
ROP9 are extremely similar homologues of barley RACB
(98% and 99% amino acid sequence identity, respectively), we
suggest that RACB is involved in similar processes of SMC
polarization and nuclear direction during subsidiary cell
formation. The observed defects in subsidiary cell formation of
RACB RNAi lines are best explained by a failure of
asymmetric cell division. As a result of this, in most cases one cell,
which originates from the undivided SMC (Fig. 5D), instead
of two developed. In some other cases, the cell wall between
the subsidiary cell and the SMC did not show the usual
longitudinal orientation but was twisted, such that the cell wall
of the subsidiary cell did not have a typically convex shape.
There is increasing awareness of an effector-triggered
influence of microbes on plant development processes
et al., 2014)
. Positioning of the nucleus is dynamic in both
parasitic and mutualistic plant–microbe interactions. The
formation of the pre-penetration apparatus in the response of
legumes to hyphopodia formation by arbuscular mycorrhiza
fungi involves attraction of the nucleus and its movement in
front of the penetration hyphae (Genre and Bonfante, 2007).
Therefore, we examined RACB RNAi plants for their
capability for single cell polarization after fungal attack. We used
positioning of the nucleus as a marker because it is common
to root hair and subsidiary cell formation and to cell
polarization in plant–microbe interactions (Fig. 7). The data
support that nuclei closely associate with fungal appressoria in
non-transgenic plants, as observed earlier
(Kita et al., 1981;
Schmelzer, 2002; Opalski et al., 2005)
. However, when we
observed nuclei at 8 HAI before Bgh actually penetrated,
nuclei appeared less attracted by fungal appressoria in RACB
RNAi plants because NAIs were shifted to higher values and
the absolute nucleus to appressorium distance was higher
when compared with azygous controls (Fig. 6). This may
indicate reduced single cell polarization of the attacked RACB
RNAi cells at an early stage of plant–pathogen interaction
and that RACB may be involved in positioning the nucleus in
response to a fungal penetration attempt. The nucleus is
confined and connected to the site of attack by both microtubules
(Opalski et al., 2005; Hoefle et al., 2011)
Since RAC/ROP proteins are key regulators of the plant
cytoskeleton, and the cytoskeleton is an important target of
(Cheong et al., 2014; Porter and Day, 2015)
it is logical that RACB mutants show nucleus positioning
phenotypes in combination with an altered susceptibility. The
role, however, of nucleus positioning during plant–pathogen
interaction is hardly understood. Movement of the nucleus
correlates with cytoplasmic aggregation at the sites of fungal
attack and with subsequent secretion events for formation of
cell wall appositions. In interaction with filamentous
pathogens, cell polarization, and nuclear attraction is often less
frequent and more transient in compatible interactions when
compared with resistance
. Invasive hyphae
of the Cowpea rust fungus are connected via host actin
microfilaments and microtubules to the host plant nucleus in both
compatible and incompatible interactions. However, nuclei are
more often close to fungal hyphae in compatible interactions.
The actin inhibitor cytochalasin E inhibits positioning of the
nucleus close to fungal hyphae and hypersensitive cell death in
(Skalamera and Heath, 1998)
E also inhibits penetration resistance of barley to Bgh and
(Kobayashi et al., 1997; Miklis et al., 2007)
Barley actin microfilaments and microtubules are strongly
re-organized in cells that defend against fungal penetration
(Kobayashi et al., 1997; Opalski et al., 2005; Hoefle et al.,
. Quantification of actin cytoskeleton patterns at 14–36
HAI suggested an association of cell polarity with penetration
resistance to Bgh (Opalski et al., 2005). In interaction with
Bgh, stability of microtubules and polarization of both
microfilaments and microtubules is influenced by RACB or by the
RACB-interacting proteins MAGAP1
(MICROTUBULEASSOCIATED ROP GTPASE ACTIVATING PROTEIN
1) and RBK1 (ROP BINDING KINASE 1)
(Opalski et al.,
2005; Hoefle et al., 2011; Huesmann et al., 2012)
this strongly suggests a function of polarity in penetration
resistance. On the other hand, polar secretory events are also
required for fungal accommodation in intact cells. Similar
components of membrane transport act in penetration
resistance and in formation of perimicrobial compartments in
compatible plant–microbe interactions (
Dörmann et al.,
). Additionally, the nucleus is a target of virulence
effectors of diverse plant pathogens including pow
(Wessling et al., 2014
). Cell polarization and movement of the
nucleus may thus be important for basal penetration
resistance. However, Bgh might also co-opt this during host cell
reprogramming for fungal accommodation. Additionally, Bgh
might profit from a host cell developmental programme for
polar growth including local cell wall remodelling and supply
with sufficient building blocks for formation of the haustorial
complex. As an obligate biotroph that has lost some essential
gene functions during co-evolution with its host
(Spanu et al.,
, Bgh might partially depend on support from its host.
Our data support that RACB is a susceptibility factor that
supports accommodation of fungal infection structures by its
function in polar cell development.
Supplementary data are available at JXB online.
Figure S1. Polar cell development and the nucleus
positioning phenotype of RACB RNAi event 15/1-16.
We are grateful to undergraduate student Christin Gebhardt for
contributing to the ROS assays, and to Caroline Hoefle, Christopher McCollum, and
Mathias Nottensteiner for technical advice. This study is supported by a
research grant to RH in the framework of the German Research Foundation
Collaborative Research Centre SFB924 (TP B08).
Carol RJ , Dolan L. 2006 . The role of reactive oxygen species in cell growth: lessons from root hairs . Journal of Experimental Botany 57 , 1829 - 1834 .
Carol RJ , Takeda S , Linstead P , Durrant MC , Kakesova H , Derbyshire P , Drea S , Zarsky V , Dolan L. 2005 . A RhoGDP dissociation inhibitor spatially regulates growth in root hair cells . Nature 438 , 1013 - 1016 .
Chen L , Shiotani K , Togashi T , Miki D , Aoyama M , Wong HL , Kawasaki T , Shimamoto K. 2010 . Analysis of the Rac/Rop small GTPase family in rice: expression, subcellular localization and role in disease resistance . Plant and Cell Physiology 51 , 585 - 595 .
Cheong MS , Kirik A , Kim J-G , Frame K , Kirik V , Mudgett MB . 2014 .
AvrBsT acetylates Arabidopsis ACIP1 , a protein that associates with microtubules and is required for immunity . PLoS Pathogens 10 , e1003952 .
Čiamporová M , Dekánková K , Hanáčková Z , Peters P , Ovečka M , Baluška F. 2003 . Structural aspects of bulge formation during root hair initiation . Plants and Soil 255 , 1 - 7 .
Dodds PN , Rathjen JP . 2010 . Plant immunity: towards an integrated view of plant-pathogen interactions . Nature Reviews Genetics 11 , 539 - 548 .
Dörmann P , Kim H , Ott T , Schulze-Lefert P , Trujillo M , Wewer V , Hückelhoven R. 2014 . Cell-autonomous defense, re-organization and trafficking of membranes in plant-microbe interactions . New Phytologist 204 , 815 - 822 .
Duan Q , Kita D , Li C , Cheung AY , Wu H-M. 2010 . FERONIA receptorlike kinase regulates RHO GTPase signaling of root hair development .
Proceedings of the National Academy of Sciences, USA 107 , 17821 - 17826 .
Evangelisti E , Rey T , Schornack S. 2014 . Cross-interference of plant development and plant-microbe interactions . Current Opinion in Plant Biology 20 , 118 - 126 .
Facette MR , Smith LG . 2012 . Division polarity in developing stomata .
Current Opinion in Plant Biology 15 , 585 - 592 .
Felix G , Duran JD , Volko S , Boller T. 1999 . Plants have a sensitive perception system for the most conserved domain of bacterial flagellin .
The Plant Journal 18 , 265 - 276 .
2004. Apoplastic pH signaling in barley leaves attacked by the powdery mildew fungus Blumeria graminis f. sp hordei . Molecular Plant-Microbe Interactions 17 , 118 - 123 .
Foreman J , Demidchik V , Bothwell JH , et al. 2003 . Reactive oxygen species produced by NADPH oxidase regulate plant cell growth . Nature 422 , 442 - 446 .
Genre A , Bonfante P. 2007 . Check-in procedures for plant cell entry by biotrophic microbes . Molecular Plant-Microbe Interactions 20 , 1023 - 1030 .
Griffis AH , Groves NR , Zhou X , Meier I. 2014 . Nuclei in motion: movement and positioning of plant nuclei in development, signaling, symbiosis, and disease . Frontiers in Plant Science 5 , 129 .
Hoefle C , Huesmann C , Schultheiss H , Boernke F , Hensel G , Kumlehn J , Hückelhoven R. 2011 . A barley ROP GTPase ACTIVATING PROTEIN associates with microtubules and regulates entry of the barley powdery mildew fungus into leaf epidermal cells . The Plant Cell 23 , 2422 - 2439 .
Huang G-Q , Li E , Ge F-R , Li S , Wang Q , Zhang C-Q , Zhang Y. 2013 .
Arabidopsis RopGEF4 and RopGEF10 are important for FERONIAmediated developmental but not environmental regulation of root hair growth . New Phytologist 200 , 1089 - 1101 .
Hückelhoven R. 2005 . Powdery mildew susceptibility and biotrophic infection strategies . FEMS Microbiology Letters 245 , 9 - 17 .
Hückelhoven R , Eichmann R , Weis C , Hoefle C , Proels RK . 2013 .
Genetic loss of susceptibility: a costly route to disease resistance? Plant Pathology 62 , 56 - 62 .
Hückelhoven R , Kogel K-H. 2003 . Reactive oxygen intermediates in plant-microbe interactions: who is who in powdery mildew resistance? Planta 216 , 891 - 902 .
Huesmann C , Reiner T , Hoefle C , Preuss J , Jurca ME , Domoki M , Feher A , Hückelhoven R. 2012 . Barley ROP binding kinase1 is involved in microtubule organization and in basal penetration resistance to the barley powdery mildew fungus . Plant Physiology 159 , 311 - 320 .
Humphries JA , Vejlupkova Z , Luo A , Meeley RB , Sylvester AW , Fowler JE , Smith LG . 2011 . ROP GTPases act with the receptor-like protein PAN1 to polarize asymmetric cell division in maize . The Plant Cell 23 , 2273 - 2284 .
Jones JD , Dangl JL . 2006 . The plant immune system . Nature 444 , 323 - 329 .
Jones MA , Raymond MJ , Yang Z , Smirnoff N. 2007 . NADPH oxidasedependent reactive oxygen species formation required for root hair growth depends on ROP GTPase . Journal of Experimental Botany 58 , 1261 - 1270 .
Jones MA , Shen JJ , Fu Y , Li H , Yang ZB , Grierson CS . 2002 . The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth . The Plant Cell 14 , 763 - 776 .
Kawano Y , Akamatsu A , Hayashi K , et al. 2010 . Activation of a Rac GTPase by the NLR family disease resistance protein pit plays a critical role in rice innate immunity . Cell Host and Microbe 7 , 362 - 375 .
Kawano Y , Kaneko-Kawano T , Shimamoto K. 2014 . Rho family GTPase-dependent immunity in plants and animals . Frontiers in Plant Science 5 , 522 .
Ketelaar T , Faivre-Moskalenko C , Esseling JJ , de Ruijter NC , Grierson CS , Dogterom M , Emons AM . 2002 . Positioning of nuclei in Arabidopsis root hairs: an actin-regulated process of tip growth . The Plant Cell 14 , 2941 - 2955 .
Kim MC , Panstruga R , Elliott C , Muller J , Devoto A , Yoon HW , Park HC , Cho MJ , Schulze-Lefert P. 2002 . Calmodulin interacts with MLO protein to regulate defence against mildew in barley . Nature 416 , 447 - 451 .
Kita N , Toyoda H , Shishiyama J. 1981 . Chronological analysis of cytological responses in powdery-mildewed barley leaves . Canadian Journal of Botany 59 , 1761 - 1768 .
Kobayashi Y , Kobayashi I , Funaki Y , Fujimoto S , Takemoto T , Kunoh H. 1997 . Dynamic reorganization of microfilaments and microtubules is necessary for the expression of non-host resistance in barley coleoptile cells . The Plant Journal 11 , 525 - 537 .
Koga H , Bushnell WR , Zeyen RJ . 1990 . Specificity of cell type and timing of events associated with papilla formation and the hypersensitive reaction in leaves of Hordeum vulgare attacked by Erysiphe graminis f. sp.
hordei. Canadian Journal of Botany 68 , 2344 - 2352 .
Kogel KH , Ortel B , Jarosch B , Atzorn R , Schiffer R , Wasternack C. 1995 . Resistance in barley against the powdery mildew fungus (Erysiphe graminis f. sp. hordei) is not associated with enhanced levels of endogenous jasmonates . European Journal of Plant Pathology 101 , 319 - 332 .
Lapin D , Van den Ackerveken G. 2013 . Susceptibility to plant disease: more than a failure of host immunity . Trends in Plant Science 18 , 546 - 554 .
Macho AP , Zipfel C. 2014 . Plant PRRs and the activation of innate immune signaling . Molecular Cell 54 , 263 - 272 .
Marzec M , Melzer M , Szarejko I. 2013 . Asymmetric growth of root epidermal cells is related to the differentiation of root hair cells in Hordeum vulgare (L.) . Journal of Experimental Botany 64 , 5145 - 5155 .
Miklis M , Consonni C , Bhat RA , Lipka V , Schulze-Lefert P , Panstruga R. 2007 . Barley MLO modulates actin-dependent and actinindependent antifungal defense pathways at the cell periphery . Plant Physiology 144 , 1132 - 1143 .
Molendijk AJ , Bischoff F , Rajendrakumar CSV , Friml J , Braun M , Gilroy S , Palme K. 2001 . Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth . EMBO Journal 20 , 2779 - 2788 .
Molitor A , Zajic D , Voll LM , Pons KHJ , Samans B , Kogel KH , Waller F. 2011 . Barley leaf transcriptome and metabolite analysis reveals new aspects of compatibility and Piriformospora indica-mediated systemic induced resistance to powdery mildew . Molecular Plant-Microbe Interactions 24 , 1427 - 1439 .
Opalski KS , Schultheiss H , Kogel KH , Hückelhoven R. 2005 . The receptor-like MLO protein and the RAC/ROP family G-protein RACB modulate actin reorganization in barley attacked by the biotrophic powdery mildew fungus Blumeria graminis f.sp hordei . The Plant Journal 41 , 291 - 303 .
Ovesna J , Kucera L , Vaculova K , Strymplova K , Svobodova I , Milella L. 2012 . Validation of the beta-amy1 transcription profiling assay and selection of reference genes suited for a RT-qPCR assay in developing barley caryopsis . PLoS One 7 , e41886 .
Pathuri IP , Imani J , Babaeizad V , Kogel K-H , Eichmann R , Hückelhoven R. 2009 . Ectopic expression of barley constitutively activated ROPs supports susceptibility to powdery mildew and bacterial wildfire in tobacco . European Journal of Plant Pathology 125 , 317 - 327 .
Pathuri IP , Zellerhoff N , Schaffrath U , Hensel G , Kumlehn J , Kogel K-H , Eichmann R , Hückelhoven R. 2008 . Constitutively activated barley ROPs modulate epidermal cell size, defense reactions and interactions with fungal leaf pathogens . Plant Cell Reports 27 , 1877 - 1887 .
Pavan S , Jacobsen E , Visser RF , Bai Y. 2010 . Loss of susceptibility as a novel breeding strategy for durable and broad-spectrum resistance .
Molecular Breeding 25 , 1 - 12 .
Peterhänsel C , Freialdenhoven A , Kurth J , Kolsch R , Schulze-Lefert P. 1997 . Interaction analyses of genes required for resistance responses to powdery mildew in barley reveal distinct pathways leading to leaf cell death . The Plant Cell 9 , 1397 - 1409 .
Pfaffl MW . 2001 . A new mathematical model for relative quantification in real-time RT-PCR . Nucleic Acids Research 29 , e45 .
2013 . The Arabidopsis Rho of Plants GTPase AtROP6 functions in developmental and pathogen response pathways . Plant Physiology 161 , 1172 - 1188 .
Porter K , Day B. 2015 . From filaments to function: the role of the plant actin cytoskeleton in pathogen perception, signaling, and immunity .
Ranf S , Eschen-Lippold L , Pecher P , Lee J , Scheel D. 2011 . Interplay between calcium signalling and early signalling elements during defence responses to microbe- or damage-associated molecular patterns . The Plant Journal 68 , 100 - 113 .
Saijo Y , Tintor N , Lu X , Rauf P , Pajerowska-Mukhtar K , Haweker H , Dong X , Robatzek S , Schulze-Lefert P. 2009 . Receptor quality control in the endoplasmic reticulum for plant innate immunity . EMBO Journal 28 , 3439 - 3449 .
Sauer M , Friml J. 2010 . Immunolocalization of proteins in plants.
Methods in Molecular Biology 655 , 253 - 263 .
Schiefelbein JW . 2000 . Constructing a plant cell. The genetic control of root hair development . Plant Physiology 124 , 1525 - 1531 .
Schmelzer E. 2002 . Cell polarization, a crucial process in fungal defence .
Trends in Plant Science 7 , 411 - 415 .
Schneider CA , Rasband WS , Eliceiri KW . 2012 . NIH Image to ImageJ: 25 years of image analysis . Nature Methods 9 , 671 - 675 .
Schultheiss H , Dechert C , Kogel KH , Hückelhoven R. 2002 . A small GTP-binding host protein is required for entry of powdery mildew fungus into epidermal cells of barley . Plant Physiology 128 , 1447 - 1454 .
Schultheiss H , Dechert C , Kogel KH , Hückelhoven R. 2003 .
Functional analysis of barley RAC/ROP G-protein family members in susceptibility to the powdery mildew fungus . The Plant Journal 36 , 589 - 601 .
Schultheiss H , Hensel G , Imani J , Broeders S , Sonnewald U , Kogel KH , Kumlehn J , Hückelhoven R. 2005 . Ectopic expression of constitutively activated RACB in barley enhances susceptibility to powdery mildew and abiotic stress . Plant Physiology 139 , 353 - 362 .
Singh SK , Fischer U , Singh M , Grebe M , Marchant A. 2008 . Insight into the early steps of root hair formation revealed by the procuste1 cellulose synthase mutant of Arabidopsis thaliana . BMC Plant Biology 8 , 57 .
Skalamera D , Heath MC . 1998 . Changes in the cytoskeleton accompanying infection-induced nuclear movements and the hypersensitive response in plant cells invaded by rust fungi . The Plant Journal 16 , 191 - 200 .
Spanu PD , Abbott JC , Amselem J , et al. 2010 . Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism .
Science 330 , 1543 - 1546 .
Takeda S , Gapper C , Kaya H , Bell E , Kuchitsu K , Dolan L. 2008 .
Science 319 , 1241 - 1244 .
Untergasser A , Cutcutache I , Koressaar T , Ye J , Faircloth BC , Remm M , Rozen SG . 2012 . Primer3-new capabilities and interfaces .
Nucleic Acids Research 40 , e115 .
van Schie CC , Takken FL . 2014 . Susceptibility genes 101: how to be a good host . Annual Review of Phytopathology 52 , 551 - 581 .
Verbelen JP , De Cnodder T , Le J , Vissenberg K , Baluska F. 2006 .
The root apex of Arabidopsis thaliana consists of four distinct zones of growth activities: meristematic zone, transition zone, fast elongation zone and growth terminating zone . Plant Signaling and Behavior 1 , 296 - 304 .
Wessling R , Epple P , Altmann S , et al. 2014 . Convergent targeting of a common host protein-network by pathogen effectors from three kingdoms of life . Cell Host Microbe 16 , 364 - 375 .
Wu FS . 1987 . Localization of mitochondria in plant cells by vital staining with rhodamine 123 . Planta 171 , 346 - 357 .
Yalovsky S. 2015 . Protein lipid modifications and the regulation of ROP GTPase function . Journal of Experimental Botany 66 , 1617 - 1624 .
Yang G , Gao P , Zhang H , Huang S , Zheng Z-L. 2007 . A mutation in MRH2 kinesin enhances the root hair tip growth defect caused by constitutively activated ROP2 small GTPase in Arabidopsis . PLoS One 2 , e1074 .
Yang Z. 2008 . Cell polarity signaling in Arabidopsis . Annual Review of Cell and Developmental Biology 24 , 551 - 575 .