Vol. 19, No. 9, 2006 / 939
MPMI Vol. 19, No. 9, 2006, pp. 939–947. DOI: 10.1094/MPMI-19-0939.
Stage-Specific Suppression of Basal Defense
Discriminates Barley Plants Containing Fast- and
Delayed-Acting Mla Powdery Mildew Resistance Alleles
Rico A. Caldo,1 Dan Nettleton,2 Jiqing Peng,3 and Roger P. Wise1,4
1Department of Plant Pathology and Center for Plant Responses to Environmental Stresses, 2Department of Statistics,
3GeneChip Facility, Office of Biotechnology, 213L Molecular Biology Building, and 4Corn Insects and Crop Genetics
Research, USDA-ARS, Iowa State University, Ames, IA 50011, U.S.A.
Submitted 7 March 2006. Accepted 4 April 2006.
Nonspecific recognition of pathogen-derived general elicitors
triggers the first line of plant basal defense, which in turn,
preconditions the host towards resistance or susceptibility.
To elucidate how basal defense responses influence the onset
of Mla (mildew resistance locus a)–specified resistance, we
performed a meta-analysis of GeneChip mRNA expression
for 155 basal defense–related genes of barley (Hordeum
vulgare) challenged with Blumeria graminis f. sp. hordei,
the causal agent of powdery mildew disease. In plants con-
taining the fast-acting Mla1, Mla6, or Mla13 alleles, tran-
scripts hyper-accumulated from 0 to 16 h after inoculation
(hai) in both compatible and incompatible interactions.
Suppression of basal defense–related transcripts was ob-
served after 16 hai only in compatible interactions,
whereas these transcripts were sustained or increased in
incompatible interactions. By contrast, in plants containing
wild-type and mutants of the delayed-acting Mla12 allele,
an early hyper-induction of transcripts from 0 to 8 hai was
observed, but the expression of many of these genes is mark-
edly suppressed from 8 to 16 hai. These results suggest that
the inhibition of basal defense facilitates the development
of haustoria by the pathogen, consequently delaying the
onset of host resistance responses. Thus, we hypothesize
that the regulation of basal defense influences host-cell ac-
cessibility to the fungal pathogen and drives allelic diversi-
fication of gene-specific resistance phenotypes.
Additional keywords: innate immunity, pathogen-associated
molecular patterns, timing of resistance response.
Nonself recognition is an indispensable system for eukaryotes
in preventing microbial attack. In plants, perception of patho-
gen-derived general elicitors, which are similar to pathogen-
associated molecular patterns (PAMP), triggers nonspecific
basal defense (Kim et al. 2005; Navarro et al. 2004; Nürnberger
et al. 2004; Zeidler et al. 2004; Zipfel et al. 2004), while recog-
nition of pathogen avirulence effectors initiates gene-specific
resistance (Dangl and Jones 2001; Martin et al. 2003). Both
pathogen-derived general elicitors and avirulence gene effec-
tors are released during pathogen infection, and recent experi-
mental evidence indicates that basal and host-specific mecha-
nisms of innate immunity are linked (Caldo et al. 2004; Kim et
al. 2005; Navarro et al. 2004).
General elicitor or PAMP-induced defense is an important
component of the total immune response. Hyperactivation of
basal defense, in most cases, is one of the consequences of an R-
mediated pathogen recognition leading to resistance (Caldo et
al. 2004; Nimchuk et al. 2003). By contrast, suppression of basal
defense leads to pathogen establishment (Abramovitch and
Martin 2004; Caldo et al. 2004; Hückelhoven 2005; Panstruga
2003). In addition, it has been suggested that suppressor mole-
cules of general defense influence specificity in many plant-
pathogen interactions (Bushnell and Rowell 1981; Heath 1981;
Shiraishi et al. 1994). Taken together, these support the hypothe-
ses that basal defense is one of the initial targets of suppression
by the pathogen and that the host-specific resistance conceivably
evolved from R proteins guarding the plant against effector
molecules that trigger basal defense suppression (Alfano and
Collmer 2004; Caldo et al. 2004; Kim et al. 2005).
Powdery mildew of barley, caused by Blumeria graminis f.
sp. hordei, is an ideal system to explore the interactions of obli-
gate fungal biotrophs with their cereal hosts. Specific recogni-
tion in barley–B. graminis f. sp. hordei interactions is triggered
in a gene-for-gene manner by genes designated Ml (mildew-
resistance loci) (Jørgensen 1994; Schulze-Lefert and Vogel
2000; Wise 2000). Approximately 30 distinct resistance speci-
ficities have been identified at the Mla locus on chromosome 5
(1H) (Halterman and Wise 2004; Halterman et al. 2001, 2003;
Jørgensen 1994; Shen et al. 2003; Zhou et al. 2001), and well-
defined stages of powdery mildew disease development provide
multiple possibilities to interrogate the regulation of host genes
in response to Mla-specified incompatible and compatible
barley–B. graminis f. sp. hordei interactions (Clark et al. 1993;
Ellingboe 1972; Jørgensen 1988; Kunoh 1982). The Mla1,
Mla6 and Mla13 encoding coiled-coil-nucleotide binding-
leucine-rich repeat alleles trigger a rapid and absolute resistance,
while Mla7 and Mla12 trigger a delayed and intermediate re-
sponse. In rapid resistance, fungal growth is terminated on or
before haustorium formation, leading to a single cell death
(Boyd et al. 1995; Kruger et al. 2003; Wise and Ellingboe
Corresponding author: Roger P. Wise; USDA ARS Corn Insects and Crop
Genetics Research Unit, Department of Plant Pathology, Iowa State
University; Telephone: +1-515-294-9756; Fax: +1-515-294-9420; E-mail:
*The e-Xtra logo stands for “electronic extra” and indicates the HTML
abstract available on-line contains supplemental material describing basal
defense-related genes and showing timecourse expression patterns of
anthranilate N-benzoyltransferase in Mla1 and Mla6 alleles that is not
included in the print edition.
This article is in the public domain and not copyrightable. It may be freely
reprinted with customary crediting of the source. The American Phyto-
pathological Society, 2006.
940 / Molecular Plant-Microbe Interactions
1983). By contrast, in delayed resistance, termination of fungal
growth occurs after the formation of haustorium and secondary
hypha, leading to death of the infected as well as surrounding
cells (Boyd et al. 1995; Freialdenhoven et al. 1994; Kruger et
al. 2003). Although the physiology of the infection process is
well established (Green et al. 2002), the molecular mecha-
nisms that differentiate the onset of Mla-specified rapid and
delayed resistance remain unclear.
Parallel gene-expression profiling has become an integral
tool to interrogate the molecular mechanisms underlying bio-
logical phenomena. With the increasing availability of a large
number of microarray data sets in public repositories, it is bene-
ficial to use comparative meta-profiling strategies to draw con-
clusions that span multiple experiments (Ghosh et al. 2003;
Moreau et al. 2003; Pellegrino et al. 2004; Rhodes et al. 2002,
2004; Stevens and Doerge 2005; Wang et al. 2004). In addi-
tion, data integration from related expression profiling experi-
ments is critical to the success of the large investment made on
genomics studies (Cope et al. 2004). Here, we perform cross-
experiment analysis of transcript accumulation of genes in-
volved in basal defense and determine how the accumulation
of this early-induced defense differentiates responses in barley
plants infected with B. graminis f. sp. hordei. In genotypes
containing the fast-acting Mla1, Mla6, and Mla13 alleles,
highly parallel and significant hyper-accumulation of 207
basal defense-related transcripts was observed in both incom-
patible and compatible interactions up to 16 h after inoculation
(hai), coinciding with germination of B. graminis f. sp. hordei
conidiospores and formation of appressoria. After 16 hai, these
transcripts are sustained or increased in incompatible interac-
tions but are significantly down-regulated in compatible inter-
actions (Caldo et al. 2004). In plants containing wild-type and
mutant forms of the delayed-acting Mla12 allele, a similar
hyper-induction of transcripts is observed from 0 to 8 hai;
however, mRNA expression is markedly suppressed from 8 to
16 hai, before re-induction from 16 to 32 hai. We hypothesize
that suppression of basal defense may precondition epidermal
cells to become accessible to the pathogen and, thus, may influ-
ence the mechanisms of Mla-specified rapid and delayed resis-
tance, analogous to mammalian innate immunity, which influ-
ences the expression of the acquired immune response (Fearon
and Locksley 1996).
Experimental concept and design.
Basal defense is part of all plant-pathogen interactions; yet,
little is known about its influence on the onset of gene-specific
resistance. A previous experiment conducted on near-isogenic
lines containing fast-acting Mla1, Mla6 and Mla13 alleles
(Caldo et al. 2004) demonstrated that basal defense-associated
mRNA accumulation is dependent on the kinetics of powdery
mildew infection and that its modulation influences the outcome
of particular gene-for-gene interactions towards incompatibility
or compatibility. To further elucidate how basal defense re-
sponses modify the onset of Mla-specified resistance, we ex-
tended the analysis of general elicitor perception from the
Caldo et al. (2004) investigation to another large expression-
profiling experiment involving variants of cultivar Sultan 5
harboring the delayed-acting allele, Mla12. In addition to Sultan
5, we examined its EMS (ethyl methane sulfonate)–derived
mla12-M66 loss-of-function Mla12 allele (Shen et al. 2003;
Torp and Jørgensen 1986), the NaN3-derived rar1-1 (M82) and
rar1-2 (M100) mutants (Freialdenhoven et al. 1994; Jørgensen
1996; Shirasu et al. 1999; Torp and Jørgensen 1986), and
rom1, a restorer of Rar1-independent, Mla12-specified resis-
tance (Freialdenhoven et al. 2005). The experiment conducted
on Sultan 5 and its derived mutants was based on a split-split
plot design described by Caldo et al. (2004), except for the
parallel inclusion of control noninoculated plants, with barley
first leaves harvested at 0, 8, 16, 20, 24, and 32 h after inocula-
tion with B. graminis f. sp. hordei isolate 5874 (AvrMla1,
AvrMla6, AvrMla12) (discussed below). The Sultan 5 experi-
ment consisted of 180 Barley1 GeneChip hybridizations (5
genotypes × 6 timepoints × 2 inoculation treatments × 3 bio-
logical replications), and the Caldo et al. (2004) study consisted
of 108 hybridizations (3 genotypes × 6 timepoints × 2 isolates
× 3 biological replications) resulting in a total of 96 treatment
combinations for the two experiments. To alleviate cross-
experiment variability, both studies were conducted under iden-
tical conditions. Because of the differences in genetic back-
ground of Manchuria-type isolines containing Mla1, Mla6, and
Mla13 and variants of Sultan 5 harboring Mla12, each experi-
ment was treated independently, although analyzed side-by-side,
and interpretation of results was based on gene-expression data
within each experiment (Stevens and Doerge 2005).
Initial analysis strategy.
In our previous analysis (Caldo et al. 2004), we focused on
22 highly significant genes identified from contrasting incom-
patible (Mla/AvrMla) and compatible (Mla/avrMla) interactions.
These selected genes had a cutoff P value that was <0.0001, a
false discovery rate (FDR) that was <7%, and displayed highly
synchronized patterns of transcript upregulation among all
incompatible and compatible interactions up to approximately
16 hai, coinciding with germination of B. graminis f. sp. hordei
conidiospores and formation of appressoria. By contrast, sig-
nificant divergent expression was observed from 16 to 32 hai,
during membrane-to-membrane contact between fungal haus-
toria and host epidermal cells, with notable suppression of most
transcripts identified as differentially expressed in compatible
interactions. These observations were consistent with the hy-
pothesis that these 22 genes encode proteins that are involved
in nonspecific basal defense pathways.
The multiple-test situation described in the split-split plot
design of the Caldo et al. (2004) study also allows the total
number of differentially expressed genes to be estimated by ana-
lyzing the distribution of P values. To begin our cross-experi-
ment, we used the histogram-based method described by Mosig
et al. (2001) and identified 1,432 differentially expressed genes
among incompatible vs. compatible interactions over the period
of 0 to 32 hai (Fig. 1). Then, to ultimately extract the most
biologically meaningful results, we used timecourse expres-
sion patterns that correlated to the kinetics of fungal infection
as our criteria for gene selection.
Keeping in mind our model described above, we first con-
sidered the threshold P value of <0.001 and identified 81 genes
in the comparison of incompatible and compatible interactions.
Although the FDR was higher (20%), by evaluating the indi-
vidual timecourse expression graphs, we found that 28 of these
81 genes also showed the same pattern of expression as the
first 22 highly significant genes identified by Caldo et al.
(2004). To then extract the majority of coexpressed genes, we
collected the most significant 500 with P values that were
<0.01 and performed cluster analysis of mean signal intensities
(Fig. 2). As shown in Figure 2A, this analysis grouped the
genes into three major clusters based on their expression pro-
files. Of the three main clusters, cluster 3 contained 207 genes,
including 21 out of the original 22 identified by threshold P
values of <0.0001 (Caldo et al. 2004) and the subsequent 28
genes identified above (P < 0.001), which had identical time-
course patterns of expression. Notably, 20 predicted genes
from cluster 3 had annotations associated to the shikimate path-
way leading to the biosynthesis of secondary metabolites (Fig.
Vol. 19, No. 9, 2006 / 941
2B, right side). Because of the observed coexpression of these
207 genes in regards to the kinetics of fungal infection, they
were selected as a point of reference to analyze basal defense-
related expression in plants with fast-acting (Mla1, Mla6, and
Mla13; Caldo et al. 2004) and delayed-acting (Sultan 5 loss-
of-function mutants and Mla12) resistance alleles.
Transcript accumulation shows evidence
of nonspecific pathogen recognition by the plant.
To determine if the 207 coexpressed transcripts identified
above accumulated in response to pathogen inoculation in the
Sultan 5 experiment, we performed a test for differential expres-
sion patterns over time between noninoculated and inoculated
plants, using a contrast statement in SAS (Statistical Analysis
Software). A total of 155 of the 207 genes in Sultan 5 were
identified as differentially expressed with P values below 0.05
and an estimated FDR of 1.5% (Storey and Tibshirani 2003).
Of these 155 differentially expressed genes, 46 (29.7%) had a
predicted function in cellular metabolism, 22 (14.2%) had a
predicted function in the shikimate pathway or secondary me-
tabolism, and 37 (24%) are of unknown function (Table 1).
Transcript accumulation of these genes was induced at the
very early stages of infection, specifically at 8 hai. A test of
fixed effects showed nonsignificant genotype, genotype × treat-
ment, genotype × time, and genotype × treatment × time interac-
tions for most of the genes (data not shown), indicating a high
degree of similarity in the mRNA expression among wild type
and mutants derived from Sultan 5. These results indicate that
the expression profiles of these selected genes are similar among
all genotypes and most likely are due to the perception of gen-
Basal defense expression in delayed-acting Mla12 plants
and derived mutants is similar at 0 and 16 hai.
To analyze time-specific responses to pathogen inoculation,
we performed condition clustering of the different treatment-
factor combinations in the Sultan 5 and Caldo et al. (2004) ex-
periments, using the Spearman correlation based on the 155
differentially expressed genes identified in the analysis above.
The Spearman correlation was used to measure the correlation
of ranks of data values rather than correlation between the actual
data values themselves, which is required in this clustering, as
the conditions being compared were derived from two separate
experiments. Since our objective here was to compare responses
to pathogen inoculation, conditions involving noninoculated
plants (Sultan 5 experiment) were excluded.
As shown in Figure 3, three major clusters resulted from this
analysis, one cluster consisting of 0-h timepoints and the other
two clusters distinguishing between delayed and rapid responses
after pathogen inoculation. In cluster 1, all responses at the 0-h
timepoint immediately after inoculation clustered together re-
gardless of genotype × isolate combination, indicating similar-
ity in transcription profiles for all genotypes prior to infection.
Also, in cluster 1, expression profiles at 16 hai in the delayed-
acting Mla12 and Sultan 5 loss-of-function mutants were
equivalent to the responses at 0 hai for all genotypes used in
the two experiments. This is worth noting, because 16 hai is
the timeframe when membrane-to-membrane contact is made
between fungal haustorium and the host cell. In cluster 2, ex-
pression profiles at each timepoint for delayed-acting Mla12
and Sultan 5 loss-of-function mutants clustered together regard-
less of the interaction types. This result is also consistent with
the above statistical analysis that shows the fixed effects in-
volving genotypes are nonsignificantly different for most
genes. In cluster 3, plant responses in incompatible and com-
patible interactions at the early stages clustered together, while
grouping of responses at the later stages is influenced by
genotypes and isolates, consistent with the findings of Caldo et
al. (2004). Taken together, these results show that important
differences in the expression patterns of the basal defense-
related genes occurred at the early stages of infection in barley
plants containing fast- and delayed-acting Mla alleles.
Suppression of basal defense coincides
with haustorial formation.
Following the cluster analysis above, we examined the ex-
pression profiles for the first 32 h after pathogen inoculation for
each of the 155 selected genes. Figure 4 illustrates the mRNA
expression of a gene encoding a putative B12D protein, one of
the 155 basal defense-related genes that differentiate responses
of plants containing rapid- (Mla1, Mla6, and Mla13; Caldo et al.
2004) and delayed-acting (Mla12 and Sultan 5 loss-of-function
mutant) alleles. In barley lines containing Mla1, Mla6, and
Mla13, mRNA expression of most selected genes displayed sig-
nificant upregulation from 0 to 16 hai, coincident with termina-
tion of fungal growth. These genes also displayed steady up-
regulation at the later stages of infection in incompatible interac-
tions, whereas suppression of transcript levels occurs after 16
hai in compatible interactions (Caldo et al. 2004). By contrast,
in plants containing wild-type and mutant alleles of Mla12, an
early upregulation was observed but with significant suppression
of transcript levels from 8 to 16 hai, coincident with attempted
fungal penetration. Using a threshold P value of <0.05 for up-
regulation from 0 to 16 hai in the Caldo et al. (2004) experiment
and downregulation from 8 to 16 hai in the Sultan 5 experiment,
27 genes were identified in the analysis of C.I. 16151 (Mla6)
and Sultan 5 (Mla12), 26 genes in the analysis of C.I. 16137
(Mla1) and Sultan 5, and 21 genes in the analysis of C.I. 16155
Fig. 1. Estimate of the number of differentially expressed genes based on
histogram of P values as described by Mosig and associates (2001). Histo-
gram of 22,840 P values for the comparison of incompatible and compatible
interactions in the Caldo et al. (2004) study were distributed into 20 bins.
The nonshaded area below the horizontal line designates the uniform distri-
bution associated to non–differentially expressed genes. The shaded region
of the histogram corresponds to an excess of 1,432 genes that had time-
specific differences in the expression between compatible and incompatible
942 / Molecular Plant-Microbe Interactions
(Mla13) and Sultan 5. Of these, 21 genes were consistently up-
regulated from 0 to 16 hai in all plants containing fast-acting
Mla alleles and also were significantly down-regulated from 8 to
16 hai in plants containing the slow-acting Mla12 allele and
mutant derivatives (Table 2; Fig. 4).
Sequence identities revealed that seven of the 21 differentially
expressed genes at 16 hai are putatively involved in the shiki-
mate pathway. Notably, three of these seven genes, cinnamoyl-
CoA reductase, anthranilate N-benzoyl transferase, and agma-
tine coumaroyltransferase, are implicated in phenylalanine me-
tabolism leading to the synthesis of lignins and phenylpropa-
noid phytoalexins. The other four genes (anthranilate synthase
alpha 2 subunit, phosphoribosylanthranilate isomerase, and two
corresponding to tryptophan synthase beta-subunit) are involved
in tryptophan biosynthesis in the anthranilate synthase branch
of the shikimate pathway (Table 2). Only four of the total 37
basal defense-related genes that were annotated as “unknown”
had significant differential expression at 16 hai in plants under-
going rapid and delayed resistance.
Biological questions addressed via
Expression profiling data sets deposited in public repositories
are good substrates for continued analyses, as they contain
thousands of datapoints that can and should be re-interrogated
(Pellegrino et al. 2004). In this regard, gene-expression patterns
from published results can be the focus of more detailed analy-
sis as well as being utilized as a point of reference for meta-
analysis involving several experiments. Experimental differ-
ences should be acknowledged up front to avoid misleading
conclusions (Grütsmann et al. 2005); however, careful selec-
tion of studies driven by a particular biological question makes
possible access to previously undescribed phenomena.
In this study, we focused on the analysis of basal defense re-
sponses induced by nonspecific pathogen recognition in two
independent but related parallel expression experiments. Al-
though the results of the two experiments were analyzed side-
by-side (Stevens and Doerge 2005), they were treated independ-
ently, because of the differences in genotypic background and
other possible variations in experimental conditions. The focal
point of this analysis was based on data that indicated that basal
defense expression in the host was dependent on the kinetics of
pathogen infection. Due to its nonspecific nature, the perception
of general elicitors from the same pathogen isolate regardless of
plant genotypic background leads to the induction of host gen-
eral defense. These basal defense responses are, in turn, poten-
tial targets for suppression by the pathogen effector molecules to
establish successful infection (Alfano and Collmer 2004; Caldo
et al. 2004; Espinosa and Alfano 2004). Thus, differences in the
patterns of expression of such general elicitor-triggered re-
sponses should provide insights into how pathogens respond or
counterattack early plant-defense mechanisms.
Fig. 2. Basal defense-related genes differentially expressed among in-
compatible and compatible barley–powdery mildew interactions involv-
ing fast-acting Mla (mildew resistance locus a) alleles. A, Cluster analy-
sis of the top 500 differentially expressed genes (Caldo et al. 2004).
Genes with P values below 0.01 were collected, and a data matrix of
mean signal intensities in incompatible (–) and compatible (+) interac-
tions of the identified 500 genes was uploaded in GeneSpring 6.2. Low
(green), normal, and high (magenta) expressions in the heat map were
based on the GeneSpring 6.2 color scheme. Hierarchical clustering was
performed using the Pearson correlation. Individual timepoints, within a
genotype-isolate interaction from 0 to 32 h after inoculation, are sepa-
rated by white lines. B, A total of 20 genes represented by 24 Barley1
probe sets had annotations related to the shikimate pathway. Genes des-
ignated in red were identified via a P value < 0.0001 (Caldo et al. 2004),
whereas additional genes identified through cluster analysis are desig-
nated in blue.
Vol. 19, No. 9, 2006 / 943
In plants containing fast-acting Mla alleles, an upregulation
of mRNA expression of most basal defense-related genes was
observed in compatible and incompatible interactions at the
early stages of powdery mildew infection. The peak of transcript
accumulation was attained at 16 hai and continuously up-regu-
lated in incompatible interactions at the later stages of infec-
tion, which coincides with the timing of the termination of
fungal growth (Figs. 2 and 4; Caldo et al. 2004). By contrast,
suppression of basal defense in compatible interactions was
observed after 16 hai, which may be necessary for the forma-
tion of haustoria (Caldo et al. 2004). However, in plants con-
taining wild type and mutants impacting the delayed-acting
Mla12 allele, a biphasic transcript accumulation was observed
in many of these basal defense-related genes, which includes
those involved in the later steps of the shikimate pathway (Fig.
4; Table 2). The first phase of mRNA induction was observed
from 0 to 8 hai, followed by marked suppression at 16 hai, cor-
responding to attempted fungal penetration.
Further confirmation of the timecourse expression profiles
from plants with fast-acting Mla alleles was also possible by in-
vestigating additional barley–powdery mildew data sets. Indeed,
similar expression for plants with fast-acting alleles was ob-
served in another independent timecourse expression experiment
consisting of 144 Barley1 GeneChip hybridizations (R. A. Caldo
and R. P. Wise, unpublished data). B. graminis f. sp. hordei iso-
late 5874–inoculated C.I. 16151 (Mla6) and C.I. 16137 (Mla1)
plants displayed consistent upregulation of all 21 basal defense-
related genes that were the final focus of the analysis described
here. In addition, RNA blot analysis of barley cv. Pallas isolines
demonstrated stage-specific suppression of phenylalanine am-
monia lyase, one of the basal defense genes identified in this
study, in plants harboring the delayed-acting Mla12 allele but
not the fast-acting Mla1 allele (Kruger et al. 2003).
Powdery mildew haustoria develop from 14 to 18 hai. Coin-
cident with haustorial formation, suppression of basal defense
occurred at 16 hai in all plants containing the delayed-acting
Fig. 3. Time-specific expression of 155 differentially expressed genes in plants containing fast- and delayed acting Mla (mildew resistance locus a) alleles.
Clustering of responses based on a combination of 66 conditions from the study by Caldo et al. (2004) and the Sultan 5 experiment reported here. Mean ex-
pression was computed using Microsoft Excel 2002. A data matrix was constructed based on 155 genes that were found to be differentially-expressed be-
tween inoculated and noninoculated Sultan 5 plants. Data were uploaded in GeneSpring 6.2, and clustering was performed using the Spearman correlation.
Purple lines in the cluster tree are conditions from the experiment involving fast-acting Mla alleles, while red lines in the cluster tree are conditions from the
experiment involving wild type (Mla12) and mutants of Sultan 5. The GeneSpring 6.2 heat-map color scheme was used, with green designating low expres-
sion and magenta designating high expression for the 155 genes.
Table 1. Predicted functional classification of 155 differentially expressed
genesa among inoculated and noninoculated Sultan 5 (Mla12) plants
Predicted functional classificationb
Number of genes
Nucleic acid binding
a Based on p value < 0.05 in the comparison between inoculated and
noninoculated Sultan 5 (Mla12) plants from 0 to 32 h after inoculation.
b Functional classification based on Uniprot BlastX results.
944 / Molecular Plant-Microbe Interactions
Mla12 allele but only after 16 hai in susceptible plants contain-
ing fast-acting Mla alleles. Likewise, chemical inhibition of
genes related to phenylpropanoid metabolism (phenylalanine
ammonia lyase and cinnamoyl CoA dehydrogense), which are
downstream of the shikimate pathway, increased B. graminis f.
sp. hordei haustorial formation and led to the suppression of
Mla1-mediated powdery mildew resistance (Kruger et al.
2002; Zeyen et al. 1995). Results of these previous inhibition
studies, therefore, support the involvement of basal defense
suppression in conditioning host cells to allow formation of
fungal haustoria, conceivably contributing to the delay in the
timing of resistance responses.
Haustorial development is critical in pathogenesis because
only this specialized structure has direct contact with the host
Fig. 4. Representative differential expression of 155 basal defense-related genes in plants undergoing Mla (mildew resistance locus a)–specified rapid and
delayed resistance. A, mRNA expression detected by Contig8605_s_at, a gene encoding a predicted B12D protein in plants containing fast-acting Mla
alleles. Normalized mean signal intensities were plotted from 0 to 32 h after inoculation (hai) in incompatible (–) and compatible interactions (+) from the
experiment by Caldo et al. (2004), using Microsoft Excel 2002. The peak of mRNA expression is coincident with the termination of fungal growth leading to
single-cell death. B, Transcription profiles of the same gene in panel A for plants containing delayed-acting Mla12 allele. Normalized average signal intensi-
ties were calculated from three independent replications. Gene expression was plotted from 0 to 32 hai in inoculated and noninoculated wild type and mu-
tants of Sultan 5, using Microsoft Excel 2002. A total of 21 basal defense-related genes were consistently up-regulated from 0 to 16 hai in all plants contain-
ing fast acting Mla alleles and also were significantly down-regulated from 8 to 16 hai in plants containing the slow-acting Mla12 allele (P value <0.05).
Table 2. Basal defense-related genes (total of 21) differentially-accumulateda in plants containing fast- and delayed-acting Mla alleles at 16 h after powdery
probe set ID
Putative anthranilate N-benzoyltransferase
Tryptophan synthase beta-subunit
Tryptophan synthase beta-subunit
Anthranilate synthase alpha 2 subunit
Putative pectin methylesterase
Protein kinase family
Ras-related GTP-binding protein
Blue copper binding protein
Putative serine palmitoyltransferase
Ubiquitin family protein
Putative Na+/K+/Cl cotransport protein
a Based on P value < 0.05 upregulation from 0 to 16 h after inoculation (hai) in plants containg fast-acting Mla alleles and downregulation from 8 to 16 hai
in plant containing delayed Mla allele.
b BarleyBase/PLEXdb annotations were based on the consensus of multiple searches. NCBI/TIGR/ATH1 searches were performed using Harvest:Barley
assembly 25, and best BLASTX nonredundant was performed using HarvEST:Barley assembly 31.
c No organism designated for genes with nonsignificant E value.
Vol. 19, No. 9, 2006 / 945
cell to acquire plant nutrients for further fungal growth (Freial-
denhoven et al. 1994; Panstruga 2003; Schulze-Lefert and Pan-
struga 2003). Thus, it is reasonable to hypothesize that inhibition
of early-induced innate immunity preconditions the cell to allow
the pathogen to develop a haustorium, resulting in delayed
mechanisms of barley powdery mildew resistance. Previous
studies have shown that suppressor molecules not only inhibit
host defense responses but also condition host cell accessibility
to pathogens (Hayami et al. 1982; Kohmoto et al. 1987; Kunoh
2002; Shiraishi et al. 1994). In addition, it has been suggested
that suppressors might act as determinants of pathogen specific-
ity by establishing basic compatibility through suppression of
host general resistance (Shiraishi et al. 1994). Because of the
differences in fungal development, which appear to be influ-
enced by suppression of early basal defense, specific avirulence
effectors may be released at different stages, triggering variation
in the timing of the hypersensitive response for fast and delayed
Mla-specified resistance (Kruger et al. 2003).
The level of R protein accumulation offers an alternative hy-
pothesis to explain the differences in the timing of host-specific
resistance. Shen et al. (2003) showed that overexpression of
the Mla12 allele can change the kinetics of infection from an
intermediate to a rapid response, similar to those conferred by
Mla1 or Mla6, suggesting that the level of R protein is rate
limiting for the onset of resistance (Bieri et al. 2004; Holt et al.
2005). MLA proteins accumulate to a higher level in Rar1-
independent as compared with Rar1-dependent genotypes. In
rar1 mutants, MLA protein accumulation is impaired, compro-
mising resistance function in Rar1-dependent types (Bieri et
al. 2004). Because of the differences in R protein accumula-
tion, impairment of MLA protein levels in rar1 backgrounds
may still be above the threshold level of activation for Rar1-
independent but not in Rar1-dependent types, leading to the
idea of the “threshold model” (Bieri et al. 2004; Holt et al.
2005). In relation to this model, we speculate that the modula-
tion of general elicitor-induced defense responses affects the
threshold of activation of MLA12 proteins or key components
of R signaling, leading to the evolution of delayed resistance.
This hypothesis supports the finding of Holt et al. (2005) on a
potential linkage of Rar1 (and most likely R protein accumula-
tion) and basal defense. Consistently, in mammalian immunity,
innate mechanisms have an influence on the expression of ac-
quired immune response by modulating the threshold level of
adaptive antigen-recognition receptors and by inducing key co-
stimulatory molecules and cytokines (Fearon and Locksley
1996; Girardin et al. 2002; Medzhitov and Janeway 1998).
Taken together, the effect of suppressors that target early induc-
tion of basal defense likely influences intracellular mechanisms
of pathogen recognition, triggering the timing of effective resis-
Cross-experiment analysis of basal defense responses has
advanced our understanding of the mechanisms that influence
the onset of resistance responses. Cellular conditioning through
basal defense accumulation appears to be important in modu-
lating host accessibility and inaccessibility to the pathogen.
Thus, the interplay of nonspecific and specific host recognition
of pathogen-derived molecules, depending on the nature of
microbial infection, conceivably triggers the diversification of
R gene–mediated resistance phenotypes.
MATERIALS AND METHODS
Blumeria graminis f. sp. hordei isolate 5874 (Torp et al. 1978;
Wei et al. 1999; AvrMla1, AvrMla6, AvrMla12) was propagated
on Hordeum vulgare cv. Manchuria (C.I. 2330) in controlled
growth chambers at 18°C (16 h light and 8 h dark).
C.I. 16151 (Mla6), C.I. 16155 (Mla13), and C.I. 16137
(Mla1) plants exhibit rapid and absolute resistance responses
when challenged by B. graminis f. sp. hordei isolates that carry
cognate AvrMla6, AvrMla13, and AvrMla1 genes, respectively
(Boyd et al. 1995; Wise and Ellingboe 1983). Sultan 5 loss-of-
function mutants mla12-M66, rar1-1 (M82), and rar1-2 (M100)
were generated by EMS and NaN3 mutagenesis (Torp and
Jørgensen 1986). rom1 was generated by NaN3 mutagenesis of
rar1-2 (M100) (Freialdenhoven et al. 2005). Sultan 5 (Mla12)
and rom1 exhibit delayed and intermediate resistance to B.
graminis f. sp. hordei 5874 while mla12-M66, rar1-1 (M82)
and rar1-2 (M100) are susceptible.
Planting, stage of seedlings, harvesting, and experimental
design were as described by Caldo et al. (2004), except for the
inclusion of noninoculated seedlings. Briefly, two 20 × 30-cm
flats per genotype of Sultan 5 (Mla12) and derived mutants
mla12-M66, rar1-1 (M82), rar1-2 (M100), and rom1 were
planted in sterilized potting soil. Each experimental flat con-
sisted of six rows of 15 seedlings, with rows randomly assigned
to one of the six harvest times. Seedlings were grown in a
20°C controlled glasshouse to 10 cm (first leaf unfolded,
GRO:0007060) prior to the application of treatment. Noninocu-
lated plants were processed before the inoculated plants to avoid
accidental inoculation. First leaves of noninoculated seedlings
in a row assigned for the 0-h timepoint were harvested into liq-
uid nitrogen. Immediately after harvesting, the flats were trans-
ferred to the growth chamber at 18°C (16 h light and 8 h dark).
For the treated plants, inoculation was performed, starting at 4
PM Central Standard Time (CST), by dusting the plants with a
high density of fresh conidiospores (84 ± 19 spores/mm2). Im-
mediately after inoculation, the row of seedlings designated
for the 0-h timepoint was harvested into liquid nitrogen. Simi-
larly, the flats of inoculated plants were also transferred to the
growth chamber immediately after harvesting. Noninoculated
and inoculated plants were stored side-by-side in randomly
assigned positions in the growth chamber. For the 8- to 32-h
timepoints, flats were removed one at a time from the growth
chamber, and leaves were harvested into liquid nitrogen. Fol-
lowing harvest, flats were immediately returned (approximately
30 s) to randomized positions within the chamber. Lights in
the growth chamber were “off” from 4 p.m. to 12 a.m. and
were “on” from 12:01 a.m. to 3:59 p.m. CST. The entire experi-
ment was repeated three times in a split-split plot design, with
genotype, inoculation type, and harvest time as whole-plot,
split-plot, and split-split plot factors, respectively (Kuehl 2000).
Data were collected from 180 GeneChips, one for each row of
Barley1 GeneChip probe array.
The Barley1 GeneChip probe array (part number 900515) is
distributed by Affymetrix (Santa Clara, CA, U.S.A.). The array
includes 22,792 probe sets derived from worldwide contribution
of 350,000 high-quality expressed sequence tags clustered
from 84 cDNA libraries in addition to 1,145 barley gene se-
quences from the National Center for Biotechnology Informa-
tion nonredundant database (Close et al. 2004). Array annotation
information is hosted on the Harvest:Barley and BarleyBase/
PLEXdb (Shen et al. 2005) databases.
Target synthesis and GeneChip hybridization.
Total RNA was isolated using a hot (60°C) phenol/guanidine
thiocyanate method as described by Caldo et al. (2004). Probe
synthesis and labeling followed, using One Cycle and GeneChip
IVT labeling protocols based on the Affymetrix manual, and
946 / Molecular Plant-Microbe Interactions
were performed at the Iowa State University GeneChip Core
facility. A total of 15 μg of fragmented cRNA was used to
make each hybridization cocktail containing 10% dimethyl
sulfoxide, and an equivalent of 10 μg was hybridized to
Barley1 GeneChip probe array (Affymetrix #900515; Close et
al. 2004). All detailed protocols can be accessed online within
the BarleyBase/PLEXdb parallel expression database (Shen et
Normalization and data analysis.
The top 500 differentially expressed genes reported by
Caldo et al. (2004) were identified by a P value of <0.01 and
were analyzed using hierarchical clustering based on Pearson
correlation in GeneSpring 6.2 (Silicon Genetics, Redwood
City, CA, U.S.A.) software. The average scaled signal intensities
(mean of signal intensities that were scaled using marker-
assisted selection 5.0 algorithm) were calculated from three
replications, using Microsoft Excel 2002. Normalization, data
transformation, and mixed linear model analysis (Wolfinger et
al. 2001) for the Sultan 5–derived microarray data were pat-
terned after the methods used by Caldo et al. (2004) for 207
selected Barley1 probe sets. The mixed linear model analysis
was performed using the SAS mixed procedure. Contrast state-
ments in SAS were made to compare mRNA expression over
time in noninoculated and inoculated plants for the individual
genotypes. Subsequently, a data matrix was constructed of 155
differentially expressed genes from the Sultan 5 and Caldo et
al. (2004) experiments but without the conditions involving
noninoculated plants. This data matrix was loaded in
GeneSpring 6.2 (Silicon Genetics) for hierarchical clustering
using the Spearman correlation.
of differentially expressed genes.
The histogram-based method described by Mosig et al.
(2001) was followed to estimate the number of differentially
expressed genes (false null hypotheses) by Caldo et al. (2004).
The P values of 22,840 probesets with interval of (0,1) were
partitioned into 20 bins. An iterative algorithm was used to
estimate the number of P values in excess of the uniform dis-
tribution. The excess sum was the estimate of genes with time-
specific differences that were not constant across the 32 hai in
the comparison of compatible and incompatible interactions.
All detailed data and protocols from these experiments have
been deposited in BarleyBase/PLEXdb, a MIAME (minimum
information about a microarray experiment)–compliant expres-
sion database for plant GeneChips (Shen et al. 2005). Files are
categorized under accession number BB4 for the 108 Gene-
Chips from the Caldo et al. (2004) study and BB2 for the 180
GeneChip Sultan 5 experiment. Data files have also been de-
posited in ArrayExpress as accession number E-MEXP-142
for the Caldo et al. (2004) study and E-TABM-82 for the
Sultan 5 experiment.
The authors thank P. Schulze-Lefert and A. Freialdenhoven for the gift
of rom1 seeds and S. Whitham for the critical review of the manuscript.
This research was supported by the United States Department of Agricul-
ture (USDA) Initiative for Future Agriculture and Food Systems grant
2001-52100-11346, USDA National Research Initiative grant 02-35300-
12619, and National Science Foundation Plant Genome grant 05-00461.
This article is a joint contribution of the Corn Insects and Crop Genetics
Research Unit, USDA-Agricultural Research Service, and The Iowa Agri-
culture and Home Economics Experiment Station. Mention of trade names
or commercial products in this publication is solely for the purpose of pro-
viding specific information and does not imply recommendation or en-
dorsement by the U.S. Department of Agriculture.
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AUTHOR-RECOMMENDED INTERNET RESOURCES
ArrayExpress repository for microarray data: www.ebi.ac.uk/arrayexpress
BarleyBase database for plant microarrays: barleybase.org
HarvEST EST database-viewing software and Harvest:Barley website:
PLEXdb database for plant and plant pathogen microarrays: plexdb.org
Uniprot BlastX website: www.pir.uniprot.org