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The Plant immunity inducer pipecolic acid accumulates in the xylem
sap and leaves of soybean seedlings following Fusarium virguliforme
infection.
Nilwala S. Abeysekara1&2*, Sivakumar Swaminathan2*, Nalini Desai3, Lining Guo3, and Madan
K. Bhattacharyya2
1
Department of Plant Pathology and Microbiology, and 2Department of Agronomy, Iowa State
University, Ames, IA, USA. 3Metabolon Inc, Durham, NC, USA.
*Contributed equally
Corresponding author: mbhattac@iastate.edu; Phone: 515-294-2505; Fax: 515-294-1363
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Abstract
The causal agent of the soybean sudden death syndrome (SDS), Fusarium virguliforme, remains
in infected roots and secretes toxins to cause foliar SDS. In this study we investigated the xylem
sap, roots, and leaves of F. virguliforme-infected and -uninfected soybean seedlings for any
changes in a set of over 3,000 metabolites following pathogen infection by conducting GC/MS
and LC/MS/MS, and detected 273 biochemicals. Levels of many intermediates of the TCA cycle
were reduced suggesting suppression of this metabolic pathway by the pathogen. There was an
increased accumulation of peroxidated lipids in leaves of F. virguliforme-infected plants
suggesting possible involvement of free radicals and lipoxygenases in foliar SDS development.
Levels of both isoflavone conjugates and isoflavonoid phytoalexins were decreased in infected
roots suggesting degradation of these metabolites by the pathogen to promote root necrosis. The
levels of the plant immunity inducer pipecolic acid (Pip) and the plant hormone salicylic acid
(SA) were significantly increased in xylem sap (in case of Pip) and leaves (in case of both Pip
and SA) of F. virguliforme-infected soybean plants compared to the control plants. This suggests
a major signaling role of Pip in inducing host defense responses in above ground parts of the F.
virguliforme-infected soybean. Increased accumulation of pipecolic acid in foliar tissues was
associated with the induction of GmALD1, the soybean homolog of Arabidopsis ALD1. This
metabolomics study generated several novel hypotheses for studying the mechanisms of SDS
development in soybean.
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Keywords: Fusarium virguliforme; xylem sap; metabolites; soybean; sudden death syndrome;
pipecolic acid
Abbreviations: BLOB, binary large object; BSTFA, bistrimethyl-silyl-triflouroacetamide;
ESI, electrospray ionization; FT-ICR, Fourier transform ion cyclotron resonance; 13-HODE, 13(S)-hydroxyoctadecadienoic acid; 9-HODE, 9-Hydroxy-10,12-octadecadienoic acid; LAN, local
area network; LIMS, laboratory information management system; LIT, linear ion-trap; Pip,
pipecolic acid; QC, quality control; SDS, sudden death syndrome
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1. Introduction
Interactions of pathogens with plants lead to an array of changes in transcriptomes,
proteomes, and metabolomes in both hosts and pathogens. Metabolic changes are well known
for their roles in defending plants from invading pathogens. For example, phytoalexins and
lignins are two long studied secondary metabolite classes that are considered to play major roles
in plant defense. Depending on the host cultivar, there can be large qualitative differences in the
accumulation of plant metabolites in response to different pathogen infections [1]. For instance,
Fusarium oxysporum-infected Brassica rapa plants have been shown to accumulate more
phenylpropanoids, flavonoids, and fumaric acid than those infected with other pathogenic fungi
like Aspergillus niger and Leptosphaeria maculans [1]. Degradation of isoflavanoid phytoalexins
by pathogens has been shown to be necessary for disease development [2].
Investigation of the global changes of metabolites can help answer plant biological
questions. The utility of metabolomics in dissecting plant-fungal interactions has been reviewed
recently [3]. Metabolic profiling was used to study the basal metabolism in Fusarium spp. [4].
The roles of small metabolites, such as methyl salicylate, azelaic acid, and pipecolic acid, in the
expression of systemic acquired resistance have recently been considered [5]. Xylem sap is
known to be involved in long-distance signaling in response to pathogen infection [6]. Changes
are observed in xylem sap proteomes and metabolomes in response to abiotic and biotic stresses
[6,7,8]. Pathogen infections can result in increases in phenolic compounds [9] and/or salicylic
acid and its derivatives in the xylem sap [10]. Changes in the contents of phenolic and indolic
compounds, amino acids, nitrogen compounds, disaccharides, glucosinolates and molecules that
lead to an increase in reactive oxygen species have been observed in Arabidopsis thaliana
infected with Pseudomonas syringae pv. tomato [11].
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Sudden death syndrome (SDS) is a soybean fungal disease that can cause drastic annual
yield losses of nearly 300 million dollars [12]. In North America, this disease is caused solely by
Fusarium virguliforme, formally known as F. solani (Mart.) Sacc. f. sp. glycines; whereas, in
South America five different Fusarium species including F. virguliforme are known to cause the
disease [13,14,15]. F. virguliforme is a hemi-biotrophic, soil borne, asexually propagated fungus
possessing only one mating gene idiomorph, MAT1-1; in contrast, F. tucumaniae, one of the SDS
pathogens in South America, comprises two idiomorphs each having either the Mat1-1 or Mat1-2
gene and is therefore able to sexually propagate [13,16]. F. virguliforme causes root necrosis and
has never been detected in diseased foliar tissues. Host-selective toxins produced by the fungus
have been hypothesized to cause foliar SDS symptoms as the pathogen restricted to infected
soybean roots [17,18,19]. Recently, a major F. virguliforme toxin FvTox1 has been shown to
induce foliar SDS in soybean [20,21,22]. In addition, five minor candidate toxins have been
detected in xylem sap proteomes [7]. Thus, it is becoming apparent that multiple host-selective
toxins produced by F. virguliforme could be transported to the leaves via the vascular system to
cause foliar SDS in soybean [7,20,21,22].
SDS is an emerging disease with no known genes conferring complete resistance against
the SDS pathogen, F. virgulifome. Toxin-induced foliar SDS is the highly destructive component
of the disease and can lead to total yield losses. To date, there are no fungicides available for
controlling this disease. Therefore it is essential to understand the mechanisms of disease
development in order to design appropriate SDS management practices. In this study we
monitored the accumulation patterns of 273 biochemicals in roots, xylem sap and leaves of F.
virgulifome-infected and uninfected SDS-susceptible soybean cultivar "Spencer" to determine if
there are any biochemicals that are accumulated during SDS development. Elevated levels of the
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plant immunity inducer pipecolic acid were observed in the xylem sap and leaves of F.
virguliforme-infected soybean plants. In addition, the levels of peroxidated lipids were increased
in leaves of F. virguliforme-infected plants, compared to those of uninfected soybean plants. This
suggests a possible involvement of free radicals and lipoxygenases in developing foliar SDS
symptoms following root infection of soybean with F. virguliforme.
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2. Materials and Methods
2.1. Inoculum preparation, plant material, and xylem sap collection
The cultivar “Spencer”, a highly F. virguliforme-susceptible soybean cultivar, was used
in this study. Mixed inoculum from two F. virguliforme isolates, Scott and Clinton, was prepared
in sorghum meal as described by Hartman et al. [23]. Mixed inoculum was used to increase the
disease pressure. The isolates were grown in 1/3 strength potato dextrose agar (PDA) prior to
inoculation of sorghum meal. Detailed descriptions of growth conditions of the plants, inoculum
preparation, and xylem sap collection are reported in Abeysekara and Bhattacharyya [7]. In brief,
120 seedlings were grown in soil mixed with either the F. virguliforme inocula grown in
sorghum as the treatment or with just the sorghum meal as the control [23,24]. Approximately
between 14-21 days after emergence, xylem sap was collected from the plants showing uniform
levels of foliar disease severity. The experiment was repeated four more times. Roots and leaves
from each of the infected and uninfected seedlings were collected separately, ground in liquid
nitrogen, and stored at -80°C along with the xylem sap samples until analysis. The experiment
was repeated three more times for collecting leaf and root samples to conduct RT-PCR analysis
for a few selected genes.
2.2. Sample Preparation
Metabolomic analysis of the xylem sap samples was carried out at Metabolon Inc.,
Durham, NC, USA. The sample preparation process was carried out using the automated
MicroLab STAR® system from Hamilton Company. Recovery standards were added prior to
extraction for quality control (QC) purposes. Sample preparation was conducted using a
proprietary series of organic and aqueous extractions to remove the protein fraction while
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allowing maximum recovery of small molecules. The resulting extract was divided into two
fractions; one for analysis by LC and the other one for analysis by GC. Samples were placed
briefly on a TurboVap® (Zymark) to remove the organic solvent. Each sample was then frozen
and dried under vacuum prior to reconstitution in another solvent depending on the following
analysis (see 2.3 and 2.4).
2.3. Liquid chromatography/mass spectrometry (LC/MS, LC/MS/MS) and accurate mass
measurement
The LC/MS was conducted on a Waters ACQUITY UPLC and a Thermo-Finnigan LTQ
mass spectrometer, which consisted of an electrospray ionization (ESI) source and a linear iontrap (LIT) mass analyzer. The sample extract was split into two aliquots, dried, then reconstituted
in acidic or basic LC-compatible solvents, each of which contained at least 11 injection standards
at fixed concentrations. One aliquot was analyzed using acidic conditions optimized for positive
ion detection and the other one using basic conditions optimized for negative ion detection. After
injection, each sample aliquot was separated on separate acid/base dedicated 2.1 mm × 100 mm
Waters BEH C18 1.7 µm particle columns (Waters, Milford, MA, USA). Extracts reconstituted
in acidic conditions were eluted using a gradient of water and methanol containing 0.1% formic
acid, while for elution of the basic extracts, the gradient of water and methanol contained 6.5
mM ammonium bicarbonate. For ions with counts greater than 2 million, an accurate mass
measurement was performed. Accurate mass measurements could be made on the parent ion as
well as on the fragments. The typical mass error was less than 5 ppm. Ions with less than two
million counts require a greater amount of effort to characterize. Fragmentation spectra (MS/MS)
were typically generated in data dependent manner, but when necessary, targeted MS/MS was
also employed, such as in the case of metabolites with lower level signals.
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2.4. Gas chromatography/mass spectrometry (GC/MS)
The samples destined for GC/MS analysis were dried by vacuum desiccation for a
minimum of 24 h prior to derivatization under nitrogen using bistrimethyl-silyltriflouroacetamide (BSTFA). A 20 m x 0.18 mm
(0.18 mm film phase consisting of 5%
phenyldimethyl silicone) GC column (Thermo Finnegan, San Jose, CA, USA) was used and the
temperature ramp used was from 40° to 300°C in a 16 min period. Samples were analyzed on a
Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole mass spectrometer using electron
impact ionization.
2.5. Bioinformatics
The informatics system consisted of four major components: (i) the laboratory
information management system (LIMS); (ii) the data extraction and peak-identification
software, a modified version of “Array Studio” software (OmicSoft Corporation, Cary, NC,
USA); (iii) data processing tools for QC and compound identification [25,26]; and (iv) a
collection of information interpretation and visualization tools for use by data analysts [25,26].
The hardware and software foundations for these informatics components were the local area
network (LAN) backbone, and a database server running Oracle 10.2.0.1 Enterprise Edition.
2.6. Data Extraction and Quality Assurance
Information from the data extraction of the raw mass spectrometry data files was loaded
into a relational database and manipulated without resorting to binary large object (BLOB)
manipulation. Once in the database, the information was examined and appropriate QC limits
were imposed. Peaks were identified using Metabolon’s proprietary peak integration software,
and component parts were stored in a separate and specifically designed complex data structure
[25,26].
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2.7. Compound identification
Compounds were identified by comparison to >3,000 library entries of purified standards.
Identification of chemical entities was based on comparison to metabolomic library entries of
purified standards. The combination of chromatographic properties and mass spectra gave an
indication of a match to the specific compound or an isobaric entity [25,26].
2.8. Data imputation and statistical analysis
The missing value for a given metabolite was imputed with the observed minimum
detected value, based on the assumption that they were below the limits of detection sensitivity
of the instrument.
Statistical analysis of the data was performed using JMP (SAS,
http://www.jmp.com) and “R” (http://cran.r-project.org/). Following log transformation and
imputation with minimum observed values for each compound, Welch’s two sample t-tests was
used to identify biochemicals that differed significantly between experimental groups [27]. An
estimate of the false discovery rate (q-value) [28] was also calculated to account for the multiple
comparisons that normally occur in metabolomic-based studies.
2.9. Visualization of the study data
For the convenience of data visualization, the raw area counts for each biochemical
compound were re-scaled by dividing the value for a specific biochemical in each sample by the
median value observed for that specific biochemical (median scaled). Box plots were used to
convey the spread of the data with 50% of the data points closest to the median represented by
shaded boxes and whiskers reporting the overall range of the data. The solid horizontal bar
within a shaded box represents the median while the + indicates the mean values. Data were
scaled such that the median value measured across all samples was set to 1.0. Any outliers were
shown as dots outside the whiskers of the plot [25,26].
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2.10. RT-PCR analysis
Total RNA was extracted from the 14-day old leaves and roots of uninfected and infected
soybean plants using the SV Total RNA Isolation System (Promega, Madison, WI, USA). RNAs
were eluted from the column in nuclease-free water, and aliquots were stored at - 80◦C prior to
analysis. From RNA, first strand cDNA amplification was carried out with Superscript III
reverse transcriptase enzyme kit (Invitrogen, Life Technologies, Grand Island, NY, USA) by
using an oligo(dT)20 primer.
The PCR was carried out with the Taq DNA polymerase
(Invitrogen, Life Technologies, Grand Island, NY, USA) by using the cDNAs as template. The
PCR amplification conditions were as follows: 30 PCR cycles of denaturation temperature at
94°C for 30 s, re-annealing at 52°C for 30 s, and extension at 72°C for 40 s followed by a final
extension cycle of 10 min at 72°C. The PCR products were ran on 1% agarose gel and
photographed with a gel doc imaging system (FOTODYNE Incorporated, Hartland, WI, USA).
2.11. Molecular phylogenetic analysis of ALD1 proteins
The evolutionary relationship of ALD1 proteins of Arabidopsis with other species was
inferred by using the Maximum Likelihood method with bootstrap (1,050 replicates) by
analyzing the MUSCLE alignment of the reported ALD1 sequences. Evolutionary analyses were
conducted in MEGA7 software program (Center for Evolutionary Medicine and Informatics,
Tempe, AZ, USA). The tree with the highest log likelihood (-5296.8872) is shown. Initial tree(s)
for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ
algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the
topology with superior log likelihood value. The analysis involved 5 amino acid sequences. All
positions with less than 95% site coverage were eliminated with fewer than 5% alignment gaps,
missing data, and ambiguous bases at any position. There were 449 positions in the final dataset.
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3. Results and Discussion
In this study we investigated the metabolomes of root, xylem sap, and leaves of F.
virgulifome-infected and uninfected plants of an SDS susceptible soybean cultivar, “Spencer.” A
total of 273 biochemicals were detected in this study. The number of biochemicals with
statistical significance at p ≤ 0.05 and 0.05 < p <0.1 are summarized in Table 1. An overview of
the major biochemicals is presented as a heat map in Fig. 1. A more detailed heat map can be
found in Fig. S1. The data of all three matrices (viz., leaves, xylem sap and roots) were treated as
stemming from as a single experiment (Fig. S1). Differential accumulation of many metabolites
between F. virguliforme-infected and -uninfected soybean plants was observed for all three
matrices.
3.1. Altered accumulation of isoflavonoids and their conjugates following F. virguliforme
infection
In this study, we observed a general decline in isoflavone biosynthetic precursors,
isoliquiritigenin and liquiritigenin, and the isoflavone conjugate daidzin (daidzein 7-O-glucoside)
in F. virguliforme-infected root tissues as compared to control root tissues (Fig. 2). The pathogen
has been shown to induce the accumulation of the isoflavonoid glyceollin in infected hairy
soybean roots developed from an SDS resistant cultivar [29]. The isoflavone, formononetin but
not coumestrol, was substantially elevated in both xylem sap and leaf samples of infected plants
as compared to that of the control plants. Daidzein, the precursor of formononetin, was also
significantly more abundant in infected leaf tissues than in the control leaf tissues (Fig. 2).
Increased levels of both daidzein and formononetin were also recorded in xylem sap of F.
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virguliforme-infected plants compared to controls. We observed significantly lower levels of
coumestrol and daidzin in infected roots as compared to the uninfected roots.
Phytoalexin biosynthesis is a general defense response, and soybean leaves recognize F.
virguliforme toxin-induced disease symptoms as pathogen invasion or recognize toxins as
effectors to activate the isopropanoid biosynthetic pathway for phytoalexins accumulation. A
recent transcriptomic study demonstrated that genes involved in phytoalexin biosynthesis are
expressed or induced in leaves following treatment of soybean seedlings with F. virguliforme
culture filtrates containing toxins [30].
3.2. Increased levels of free and peroxidated fatty acids accumulate in soybean leaves following
F. virguliforme infection
An increase in free fatty acids [2-hydroxypalmitate, azelate (nonanedioate), caproate
(6:0), oleate (18:1n9), linoleate (18:2n9), palmitate (16:0), sebacate (decanedioate)] including
some peroxidated fatty acids [13-(S)-hydroxyoctadecadienoic acid (13-HODE) + 9-Hydroxy10,12-octadecadienoic
acid
hydroxyoctadec-12(Z)-enoic
(9-HODE),
acid,
9,
10-hydroxyoctadec-12(Z)-enoic
10-hydroxyoctadec-12(Z)-enoic
acid,
acid
or
[99,10-
dihydroxyoctadec-12(Z)-enoic acid], 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoate, 12,13epoxy-9-keto-10 (trans-octadecenoate)] were observed in leaf samples of infected plants as
compared to that in uninfected controls (Fig. 3). The increased levels of free fatty acids suggest
an increase in membrane degradation triggered by F. virguliforme toxins through unknown
mechanisms, which could include fatty acid peroxidation via reactive oxidative species (ROS) or
by lipoxygenases (LOX) [31]. Peroxidated fatty acids are oxidized metabolites of linoleic acid
and are markers of membrane lipid damage caused in the presence of excess ROS or LOX
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activity. Additionally, LOX may also be involved in degradation of linoleic acid to the oxoepoxyoctadecenoic acid [32]. Lipid peroxidation leads to generation oxylipins that have been
shown to have signaling function across the kingdoms, both animal and plants [33]. Recently,
Aliferis et al. [34] reported in their metabolomics study on the soybean-Rhizoctonia solani
interaction various oxylipin species enzymatically produced and possessing a plant signaling
function which were differentially regulated by pathogen infection.
The key F. virguliforme toxin, FvTox1, initiates foliar SDS in presence of light [20]. It
has been previously shown that rapid degradation of the RubisCo large subunit and the
accumulation of free radicals occur in soybean leaves exposed to F. virguliforme toxins and light
implicating that photosynthesis could be negatively affected by the pathogen toxins [35]. The
accumulated free fatty acids in leaves of F. virguliforme-infected compared to control plants
might get converted to the corresponding oxidized products by ROS produced via the direct or
indirect action of fungal toxins such as FvTox1. It is plausible that F. virguliforme toxins utilizes
jasmonic acid to cause senescence and disease development, a mechanisms reported to be used
by F. oxysporum to cause disease in Arabidopsis [36]. Further studies are warranted to test this
hypothesis and to disentangle the possible roles of the free fatty acids in foliar SDS development.
3.3. Decreased levels of intermediates of the tricarboxylic acid cycle in xylem sap and leaves
following F. virguliforme infection
Decreased accumulation of intermediates of the tricarboxylic acid (TCA) cycle, viz., αketoglutarate, cis-aconitate, citrate, malate, and succinate as well as pyruvate in xylem sap, and
α-ketoglutarate, cis-aconitate, isocitrate, and malate in leaves of the F. virguliforme-infected as
compared to the uninfected plants were observed (Fig. 4). This overall decrease in intermediates
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of the TCA cycle was associated with an increase in lactate in xylem sap of F. virguliformeinfected as compared to the uninfected plants. Depletion of pyruvate levels most likely led to
reduced accumulation of intermediates of the TCA cycle in xylem sap of F. virguliformeinfected plants.
3.4. Increased levels of allantoin and allantoic acid in xylem sap and leaves following F.
virguliforme infection
The levels of allantoin and allantoic acid were significantly higher in leaf samples of
infected plants than in the controls (Fig. 5). The level of allantoic acid was also elevated in the
xylem sap. Ureides, allantoin, and allantoic acid are well known nitrogen carriers in plants.
Legumes have a sophisticated symbiotic relationship with nitrogen fixing microorganisms.
Rhizobia inhabit roots and provide nitrogen to the plants, and plant provides C4 carbon
metabolites to the microbial TCA cycle for generating energy. The products of nitrogen fixation,
mainly asparagine or allantoin and allantoic acid, are transported to the rest of the plant, for use
in metabolic processes or for storage. These molecules also play a role in the co-ordinate
regulation of nitrogen assimilation and energy expenditure in the plant. It has been suggested that
the signals in plant response to pathogen infection may be similar to those produced in its
response to symbiotic microbes [37]. For example, the isoflavones daidzein, formononetin and
coumestrol not only accumulate after F. virguliforme infection, but also modulate the expression
of Nod genes required for the establishment of nodules in legumes [38, 39].
As in our soybean-F. virguliforme interaction study, a recent metabolomics study of the
soybean-F. tucumaniae interaction also revealed increases in the levels of nitrogen-containing
compounds and decreased in levels of the intermediates of the TCA cycle [40]. The observation
of increased nitrogen carrier accumulation in sap and leaves of F. virguliforme infected plants is
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interesting and raises several questions for further study. This host response following F.
virguliforme infection is comparable to the one observed following colonization by plant
symbiotic microorganisms to enhance nitrogen assimilation. It’s very unlikely that there was any
enhanced nitrogen fixation by symbiotic microorganisms because soil was steam-sterilized prior
to inoculation. This observation could imply that increased nitrogen assimilation may be a
consequence of shared mechanism(s) used by soybean in response to invasion by pathogenic and
symbiotic microorganisms. It appears also plausible that plants initiate resource re-allocation
processes after both pathogen and symbiont colonization for the biosynthesis of plant protectant
molecules during pathogen invasion.
3.5. Elevation in the levels of the plant immunity inducers pipecolic and salicylic acid
High levels of pipecolic acid (Pip), a catabolite of lysine, which has been reported to act
as an osmo-protectant, was elevated in both xylem sap and leaves of infected plants (Fig. 6).
Increased accumulation of Pip was associated with a decrease of its precursor lysine in the xylem
sap. Pip has recently been shown to be a critical regulator of inducible plant immunity [41].
Upon pathogen recognition, Pip accumulates in the petiole exudates of infected leaves and
induces systemic acquired resistance (SAR) in Arabidopsis [41]. Accumulation of Pip in leaves
in response to pathogen recognition has been previously reported in several plant species
including soybean [42,43,44]. It positively regulates salicylic acid (SA) biosynthesis [42].
SA levels were also elevated in F. virguliforme-infected leaf tissues compared to that in
leaves of uninfected plants (Fig. 7). However, no significant difference was observed between
salicylic acid levels of the xylem sap of infected and the uninfected soybean plants (Fig. 7). SA
is a signal for the activation of disease resistance responses and also generation of long lasting
and broad-spectrum systemic acquired resistance in soybean and other plant species [45]. Our
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data suggest that SA is induced in response to toxin-induced foliar SDS symptoms in soybean.
Presumably SA accumulates due to recognition of general defense-related signals from the toxininduced foliar SDS symptoms characterized by rapid cell death. Alternatively, it may be possible
that Pip uploaded from xylem sap may signal for SA synthesis in leaves of F. virguliformeinfected soybean plants [41]. Further study is warranted to determine whether these important
metabolites might serve as a plant signal to defend in foliar tissues as a result of the: (i)
recognition of toxins as effectors by elusive soybean toxin receptors in leaves, or (ii) outcomes of
the complex interaction of F. virguliforme with soybean roots, or (iii) both processes (i) and (ii)
mentioned above.
Increased accumulation of Pip in xylem sap of infected soybean plants suggests that the
molecule may be transported to apical parts of the soybean plants through xylem to serve as a
signal to initiate defense responses [41]. In leaves of F. virguliforme infected plants, the
increased Pip accumulation results from Pip transport via the xylem sap because the lysine
content in leaves of diseased plants was increased while that for xylem sap went down. However,
we cannot rule out the possibility of the lysine biosynthesis pathway being induced by its
catabolism to Pip and its increased demand in leaves during defense responses.
To determine if Pip is synthesized in leaf tissues, we investigated the regulatory enzyme
for Pip biosynthesis, LL-diaminopimelate aminotransferase encoded by the ALD1 gene. In
soybean, we identified four genes that showed high sequence identity to Arabidopsis ALD1. One
of these genes, GmALD4 (Glyma.14G167600) (Supplementary Table 1) is most likely a
pseudogene because it encodes a truncated protein and we did not detect any transcripts for this
gene. We studied the other three genes for their steady state transcript levels (Table 2; Fig. 8;
Supplementary Table 1). GmALD1 with the highest similarity to Arabidopsis ALD1 was induced
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in the leaves of F. virguliforme-infected soybean plants. Phylogenetic analysis of Arabidopsis
ALD1 and its homologs revealed that GmALD1 grouped with Arabidopsis ALD1, and one
Arabidopsis ALD1 homolog each from Medicago truncatula, Oryza sativa and Zea mays in a
clade (Fig. 9). Our results suggest that most likely GmALD1 is orthologous to Arabidopsis
ALD1 and the Pip biosynthetic pathway is induced in leaves of the F. virguliforme-infected
soybean plants. We conclude that the increased Pip levels in leaves of F. virguliforme-infected
soybean plants could be contributed from both xylem sap and local synthesis in leaf tissues. Our
results also suggest that F. virguliforme toxins are recognized by host factors to initiate host
defense mechanisms, one of which is most likely regulated by Pip.
We investigated if ALD1 homologs of other plant species are also induced following
pathogen infection. Study of the available transcriptomic data revealed that all ALD1 homologs
included in generating the phylogenetic tree are transcriptionally activated following pathogen
infection (Supplementary Table 2). This observation strongly indicates that Pip may be an
immunity inducer across plant species including both eudicots and monocots, and further study
of this signaling molecule is warranted.
Acknowledgements
The authors wish to thank United Soybean Board and Ms. Katelynn Davis, Iowa State
University, for technical assistance. The authors declare to have no conflict of interests. We
thank Jordan Baumbach and David Grant for reviewing the manuscript.
Appendix A. Supplementary data
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Figure Legends
Fig. 1. The heat map showing the fold changes in major biochemicals in roots, xylem sap, and
leaves of soybean seedlings infected with F. virguliforme. The fold changes were formatted in a
graduated three-color system; green = 0.33 (three fold decrease), white = 1 (no change between
the groups), and red = 3 (three-fold increase). Each line represents a metabolite.
Fig. 2. Altered accumulation of conjugates and isoflavanoid metabolites in leaves and roots of F.
virguliforme-infected as compared to the control plants. Formononetin levels were increased in
both the xylem sap and leaves of the F. virguliforme-infected plants *p ≤ 0.1; **p ≤ 0.05. C,
Control; I, Infected
Fig. 3. Elevated free fatty acids in leaves of F. virguliforme-infected as compared to the F.
virguliforme-uninfected soybean plants. HODE levels were decreased in the roots of the F.
virguliforme-infected plants *p ≤ 0.1; **p ≤ 0.05. C, Control; I, Infected
Fig. 4. General reduction in the levels of intermediate metabolites of the TCA cycle in xylem sap
and leaves of soybean plants following F. virguliforme infection. * p ≤ 0.1; **p ≤ 0.05. C,
Control; I, Infected.
Fig. 5. Increased levels of nitrogen-rich metabolites in xylem sap and leaves of soybean plants
following F. virguliforme infection. *p ≤ 0.1; ** p ≤ 0.05. C, Control; I, Infected.
Fig. 6. Increased accumulation of pipecolate levels in xylem sap and leaves of F. virguliformeinfected as compared to the control soybean plants. Lysine levels were decreased in the xylem
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sap while increased in the leaves of F. virguliforme-infected plants . **p ≤ 0.05. C, Control; I,
Infected.
Fig. 7. Increased accumulation of salicylate levels in leaves of the F. virguliforme-infected as
compared to the control soybean plants. *p ≤ 0.1. C, Control; I, Infected.
Fig 8. Soybean GmALD1 is transcriptionally activated in soybean leaves in response to F.
virguliforme toxins.
RT-PCR was carried out for the following four samples. Un-L, leaves of uninfected plant; In-L,
leaves of infected plants; Un-R, uninfected roots; In-R, infected roots. GmALD1, GmALD2, and
GmALD3 are three soybean homologs of Arabidopsis ALD1 gene. Two soybean defense genes,
GmIFR1 and GmPR1 encoding isoflavone reductase and pathogenesis-related protein,
respectively, and a F. virguliforme gene, FvMEP1 encoding a metalloproteinase were used as
controls (Supplementary Table 1). The constitutively expressed soybean gene GmEF1B
encoding elongation factor EF-1 beta subunit was used to determine the levels of transcripts
among the samples. Results of three biological replications are presented.
Fig. 9. Soybean GmALD1 protein clustered with Arabidopsis ALD1 in a clad of an un-rooted
phylogenetic tree.
The tree was generated for the following Arabidopsis ALD1-like proteins identified from the
Phytozome v10.3 website. The accession numbers of the ALD1-like proteins are: Arabidopsis
thaliana AT2G13810; A. thaliana AT4G33680; Glycine max Glyma.08G180600; G. max
Glyma.08G063500; G. max Glyma.07G185700; Oryza sativa LOC_Os03g18810; O. sativa
26
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
LOC_Os03g09910; Zea mays GRMZM2G415117; Z. mays GRMZM2G119150; Medicago
truncatula Medtr2g008430; M. truncatula Medtr4g092620.
Supporting Information
Fig. S1. Detailed heat map of all detected biochemicals under each major category. The fold
changes are shown with colors: green = 0.3 (three-fold decrease), white = 1 (no change between
the groups), and red = 3 (three-fold increase).
27
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Table 1. Summary of the number of compounds which significantly differ between experimental
groups either at p ≤ 0.05 or 0.05 < p < 0.10 (Welch´s two-sample t-test) within a pool of 273
plant metabolites termed ´biochemicals´ in this study.
Welch's Two Sample tTests
Total number of
biochemicals with
p ≤ 0.05
Biochemicals (↑↓)
Total number of
biochemicals with
0.05 < p < 0.10
Biochemicals (↑↓)
I - LEAF
C - LEAF
I - SAP
C - SAP
I - ROOT
C - ROOT
29
21
1
23|6
3|18
1|0
25
19
3
21|4
4|15
0|3
C, Control; I, Infected.
Numbers in bold font are the number of biochemicals that were up-regulated and the ones
without bold font are the number of biochemicals that were down-regulated.
28
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Table 2. ALD1 homologs1 of different plant species.
Species
Arabidopsis thaliana
A. thaliana
Glycine max
G. max
G. max
G. max
Oryza sativa
O. sativa
Zea mays
Z. mays
Medicago truncatula
M. truncatula
1
Gene ID *
% similarity to
AT2G13810
67.3
72.8
66.9
66.7
21.92
66.4
63.6
64.0
64.7
66.9
67.3
AT2G13810 (ALD1)
AT4G33680
Glyma.08G180600
Glyma.08G063500
Glyma.07G185700
Glyma.14G167600
LOC_Os03g18810
LOC_Os03g09910
GRMZM2G119150
GRMZM2G415117
Medtr2g008430
Medtr4g092620
The ALD1-like genes were identified by blasting (TBlastN) the Arabidopsis ALD1 protein
sequence in the Phytozome v10.3 website.
2
The low level of similarity with Arabidopsis ALD1 was due to truncated GmALD4 enzyme
caused by premature stop codon.
29
Figure(s)
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
ALD1
Fig. 7
Fig. 8
In-R
Un-R
In-L
Replication 3
Un-L
In-R
Un-R
In-L
Replication 2
Un-L
In-R
Un-R
In-L
Un-L
Replication 1
GmALD1
GmALD2
GmALD3
GmIFR1
GmPR1
FvMEP1
GmEF1B
Fig. 9
Supplementary Figure 1
Figure S1. Detailed heat map with all the biochemicals in
change is shown as: Green =0.33 (Three fold decrease), w
the groups), and Red =3 (Three fold increase ). Values in b
significant at p<0.05; values in blue font were statistically
values in black font were not significantly different.
SUPER
PATHWAY
SUB PATHWAY
BIOCHEMICAL
4-guanidinobutanoate
beta-hydroxypyruvate
Serine family
glycine
(phosphoglycerate
N-acetylserine
derived)
O-acetylserine
Amino acid
serine
4-hydroxycinnamate
Aromatic amino kynurenate
acid metabolism N-acetyltryptophan
(PEP derived)
phenylacetate
phenylalanine
shikimate
tryptophan
tyrosine
2-aminoadipate
alanine
asparagine
aspartate
beta-alanine
cyano-alanine
glutarate (pentanedioat
homoserine
Aspartate family
lysine
(OAA derived)
methionine
methionine sulfoxide
N-acetylasparagine
N-acetylmethionine
pipecolate
3-oxoadipate
threonine
homocitrate
2-aminobutyrate
2-pyrrolidinone
4-acetamidobutanoate
4-hydroxybutyrate (GHB
5-aminovalerate
arginine
carboxyethyl-GABA
gamma-aminobutyrate (
Glutamate family
glutamate
(alphaketoglutarate
glutamine
derived)
histidine
N-acetylglutamate
N-acetylornithine
N-acetylproline
proline
trans-4-hydroxyproline
N-methyl-4-aminobutyr
4-hydroxy-2-oxoglutaric
Branched Chain
Amino Acids (OAA isoleucine
derived)
2,3-dihydroxyisovalerate
3-methyl-2-oxobutyrate
Branched Chain 3-methyl-2-oxovalerate
Amino Acids
4-methyl-2-oxopentano
(pyruvate derived) leucine
valine
2-isopropylmalate
Amines and
polyamines
Glutathione
metabolism
Carbohydrate Glycolysis
5-methylthioadenosine
5-oxoproline
glucose
glucose-6-phosphate (G6
glycerate
lactate
pyruvate
TCA cycle
alpha-ketoglutarate
cis-aconitate
citrate
fumarate
isocitrate
malate
succinate
Photorespiration glycolate (hydroxyaceta
Amino sugar and
arabinose
nucleotide sugar
arabitol
arabonate
erythritol
erythronate*
fucose
gluconate
maltol
N-acetylglucosamine
ribitol
ribose
threitol
xylitol
xylonate
xylose
Inositol
metabolism
chiro-inositol
myo-inositol
pinitol
Sucrose, glucose,
fructose
3-deoxyoctulosonate
metabolism
erythrose
fructose
galactinol
galactose
glucono-1,5-lactone
isosorbide
maltose
mannitol
raffinose
rhamnose
sucrose
Isobar: 1-kestose, levan
trehalose
C5 branched
dibasic acid
metabolism
citraconate
citramalate
2-hydroxyglutarate
2-hydroxypalmitate
2-hydroxystearate
4-octenedioate
8-hydroxyoctanoate
adipate
arachidate (20:0)
azelate (nonanedioate)
Free fatty acid
caproate (6:0)
ethylmalonate
Lipids
linoleate (18:2n6)
linolenate [alpha or gam
6)]
malonate (propanedioat
n-Butyl Oleate
oleate (18:1n9)
palmitate (16:0)
sebacate (decanedioate
Oxylipins
13-HODE + 9-HODE
9,10-hydroxyoctadec-12
Glycerolipids
Phospholipids
13S-hydroperoxy-9Z,11E
octadecatrienoate
12,13-epoxy-9-keto-10(t
octadecenoate
1-palmitoylglycerol (1-m
glycerol
1-palmitoylglycerophosp
1palmitoylglycerophosph
1-palmitoylglycerophosp
glycerol 3-phosphate (G
Choline
metabolism
choline phosphate
ethanolamine
3-hydroxy-3-methylgluta
Sterols
beta-sitosterol
campesterol
squalene
Cofactors,
Prosthetic
Groups,
Electron
Carriers
CoA metabolism pantothenate
Nicotinate and
nicotinamide
metabolism
nicotinamide
nicotinamide riboside*
nicotinate
nicotinate ribonucleosid
trigonelline (N'-methyln
acetylphosphate
Oxidative
methylphosphate
phosphorylation
phosphate
riboflavin (Vitamin B2)
Riboflavin and
FAD metabolism flavin mononucleotide (
Quinone
phytonadione (Vitamin
metabolism
ascorbate (Vitamin C)
Ascorbate
metabolism
dehydroascorbate
glucarate (saccharate)
gulono-1,4-lactone
threonate
Tocopherol
alpha-tocopherol
metabolism
Vitamin B
pyridoxal
metabolism (B6 or
pyridoxate
B12)
Chlorophyll and pheophorbide A
heme metabolism phytol
Nucleotide
Purine
2'-deoxyadenosine
metabolism
2'-deoxyguanosine
2'-deoxyinosine
adenine
adenosine
allantoic acid
allantoin
guanine
guanosine
hypoxanthine
inosine
xanthine
2'-deoxycytidine
cytidine
Pyrimidine
metabolism
ectoine
pseudouridine
thymidine
uracil
uridine
Glucose derivative methyl-beta-glucopyran
Peptide
gamma-glutamyl gamma-glutamylleucine
gamma-glutamylphenyl
gamma-glutamyltryptop
gamma-glutamylvaline
Dipeptide
alanylisoleucine
alanylleucine
alanylphenylalanine
alanylvaline
alpha-glutamyltyrosine
aspartylphenylalanine
glycylisoleucine
glycylleucine
glycylphenylalanine
glycylvaline
isoleucylisoleucine
isoleucylphenylalanine
isoleucylleucine
leucylisoleucine
valylphenylalanine
leucylleucine
leucylglycine
leucylhistidine
histidylisoleucine*
pyroglutamylvaline
arginylleucine
isoleucylarginine
isoleucylglutamate
isoleucylglycine
isoleucylserine
isoleucyltyrosine
leucylarginine
threonylphenylalanine
aspartylleucine
histidylleucine
isoleucylalanine
isoleucylvaline
leucylalanine
leucylasparagine
leucylglutamate
leucylphenylalanine
leucylserine
valylglutamate
lysylleucine
serylisoleucine*
serylleucine
serylphenyalanine
threonylleucine
valylisoleucine
valylleucine
isoleucyltryptophan
valyltryptophan
valylvaline
arginylisoleucine
Hormone
Abscisic acid
metabolism metabolism
Secondary
Alkaloids
metabolism
Amine derived
abscisate
caffeate
phenethylamine (isobar
phenylethanamine)
tryptamine
tyramine
Benzenoids
2,4,6-trihydroxybenzoat
2,4-dihydroxybenzoic ac
4-hydroxybenzoate
gentisate
p-hydroxybenzaldehyde
vanillate
Fatty acid and
galactarate (mucic acid)
sugar derivatives
Flavonoids
afromosin
apigenin
daidzein
daidzin
formononetin
genistein
genistin
glycitin
kaempferol 3-O-beta-glu
luteolin
luteolin-7-O-glucoside
naringenin
naringenin-7-O-glucosid
rutin
syringic acid
coumestrol
chrysoeriol
Phenylpropanoids cinnamate
ferulate
salicylate
vanillin
liquiritigenin
isoliquiritigenin
Terpenoids
beta-amyrin
stigmasterol
2-(hydroxymethyl)pheno
3-(2-piperidinyl)pyridine
Supplementary Table 1
Supplementary Table1. Primers used in the RT-PCR analyses.
Gene *
GmALD1
GmALD2
GmALD3
GmALD4
GmELF1B
GmIFR1
Gene ID
Glyma.08G180600
Glyma.08G063500
Glyma.07G185700
Glyma.14G167600
Glyma.14G039100
Glyma04g16270
Forward Primer
CGGACACTGCACAAAGGTGCCCA
GCCAGACTGGCCTCTTCCAGAAGAATG
GGATTCTACAAAGAAAATACCGACATT
CCAAGATAAGCAAGTGAAAG
CACACCGAAGAGGGCATCAAATC
GAGATTCCTTCCATCAGATTTTGGG
Reverse Primer
TGTTGAAAGGCCATGCACAAAATC
GTTCCCTTGTTGCCACAGCACCAGTAGG
CAGGTCCAAAACCGCTGCCAGGA
ATCTGGTCGAGAAATTGAAGACA
CTCAACTGTCAAGCGTTCCTCAAC
CGTAAGGACGAAGCAAATAATTGAC
Amplicon (bp)
208
168
187
192
592
185
GmPR1
Glyma13g32530
GCCAACTGATCCACTCTGGTGGTG
CTACAGTTTGTAGGGTCTTTCACC
287
FvMEP1
g14032
GGAGTCCATGCTATTGGAACCTACTG
CGACAACAAGCTTCATGGTCAAGTAC
157
*AGD2- Like Defense response protein (ALD); Isoflavone reductase (IFR); Pathogenesis-related protein (PRP); Metalloproteinase
(MEP); Elongation factor EF-1 beta subunit (EF1B)
Supplementary Table 2
Supplementary Table 2. Expression of Arabidopsis ALD1 and its homologs in response to
pathogen infection. PLEXdb (Plant Expression Database) was used to obtain the information.
a) Transcriptional activation of Arabidopsis ALD1 and ALD1-like gene
Treatment
Erysiphae
cichoracearum
Pseudomonas
syringae
Pseudomonas
syringae
Blumeria graminis
f. sp. hordei
Botrytis cinerea
AT2G13810 AT4G33680 Reference #
13
1
GEO Accession: GSE431
Erysiphe orontii
Golovinomyces
orontii
Xanthomonas
strain, Xcc147
Botrytis cinerea
and powdery
mildew
Pseudomonas
syringae ES4326
Hyaloperonospora
parasitica
Hyaloperonospora
arabidopsidis
Pseudomonas
syringae pv.
tomato (Pto)
9
4
GEO Accession: GSE6176
10
1
24
3
TAIR Accession
ExpressionSet:1008031517
1
14
3
GEO Accession: GSE5684
9
20
2
1
*
2
31
2
3
22
5
4
22
7
GEO Accession: GSE18978
12
2
5
18
3
6
20
7
GEO Accession: GSE21920
The values represent CV in %
Database
NCBI GEO
via PLEXdb
NCBI GEO
via PLEXdb
NCBI GEO
via PLEXdb
NCBI GEO
via PLEXdb
(CV, coefficient of variation of the treatment means)
*Not specified at the plexdb website
References:
1.
Transcriptional regulation by a NAC (NAM-ATAF1,2-CUC2) transcription factor
attenuates ABA signalling for efficient basal defence towards Blumeria graminis f.sp.
hordei in Arabidopsis, Jensen MK, Hagedorn PH, de Torres-Zabala M, Grant MR, Rung
JH, Collinge DB, Lyngkjaer MF. Plant J. 2008 Dec;56(6):867-80.
1
2.
Temporal global expression data reveal known and novel salicylate-impacted processes and
regulators mediating powdery mildew growth and reproduction on Arabidopsis, Chandran
D, Tai YC, Hather G, Dewdney J et al. Plant Physiol. 2009 Mar;149(3):1435-51
3.
A MYB transcription factor regulates very-long-chain fatty acid biosynthesis for activation
of the hypersensitive cell death response in Arabidopsis, Raffaele S, Vailleau F, Léger A,
Joubès J, Miersch O, Huard C, Blée E, Mongrand S, Domergue F, Roby D. Plant Cell 2008
Mar;20(3):752-67
4.
Tryptophan-derived metabolites are required for antifungal defense in the Arabidopsis
mlo2 mutant, Consonni C, Bednarek P, Humphry M, Francocci F, Ferrari S, Harzen A, Ver
Loren van Themaat E, Panstruga R. Plant Physiol. 2010 Mar;152(3):1544-61.
5.
WRKY72-type transcription factors contribute to basal immunity in tomato and
Arabidopsis as well as gene-for-gene resistance mediated by the tomato R gene Mi-1,
Bhattarai KK, Atamian HS, Kaloshian I, Eulgem T. Plant J. 2010 Jul;63(2):229-40
6.
Timing of plant immune responses by a central circadian regulator, Wang W, Barnaby JY,
Tada Y, Li H, Tör M, Caldelari D, Lee DU, Fu XD, Dong X. Nature 2011
Feb;470(7332):110-4
2
b) Transcriptional activation of soybean homologs of the Arabidopsis ALD1 gene
Treatment
Glyma.08
G180600
21
Glyma.08G063500/
Glyma.07G185700
6
Reference # Database
Phytophthora
sojae
PT2004C2.S1
isolate
Phakopsora
pachyrhizi
15
5
GEO
Accession:
GSE9687
12
1
2
Phytophthora
sojae
22
2
NCBI GEO
via PLEXdb
Phakopsora
pachyrhizi
31
8
GEO
Accession:
GSE7124
3
Virus induced
gene silencing
of GmMPK4
Phytophthora
sojae
Phakopsora
pachyrhizi
Phakopsora
pachyrhizi
26
6
NCBI GEO
via PLEXdb
13
31
GEO
Accession:
GSE29653
4
41
2
5
33
5
NCBI GEO
via PLEXdb
Bradyrhizobium
japonicum
Phakopsora
pachyrhizi
36
5
GEO
Accession:
GSE29741
6
12
0
GEO
Accession:
GSE41724
NCBI GEO
via PLEXdb
Phakopsora
pachyrhizi
The values represent CV in %
1
NCBI GEO
via PLEXdb
(CV, coefficient of variation of the treatment means)
References:
1. Distinct biphasic mRNA changes in response to Asian soybean rust infection, van de
Mortel M, Recknor JC, Graham MA, Nettleton D, Dittman JD, Nelson RT, Godoy CV,
Abdelnoor RV, Almeida AMR, Baum TJ, Whitham SA, Mol. Plant-Microbe Interact.
2007 Aug;20(8):887-99
2. Gene expression analysis in soybean in response to the causal agent of Asian soybean rust
(Phakopsora pachyrhizi Sydow) in an early growth stage, Panthee DR, Yuan JS, Wright
3
DL, Marois JJ et al. Funct. Integr. Genomics 2007 Oct;7(4):291-301
3. A microarray analysis for differential gene expression in the soybean genome using
Bioconductor and R, Gregory Alvord W, Roayaei JA, Quiñones OA, Schneider KT
Brief Bioinform. 2007 Nov;8(6):415-31
4. The Phytophthora sojae avirulence locus Avr3c encodes a multi-copy RXLR effector
with sequence polymorphisms among pathogen strains, Dong S, Qutob D, Tedman-Jones
J, Kuflu K, Wang Y, Tyler BM, Gijzen M. PLoS One 2009;4(5):e5556
5. Biphasic gene expression changes elicited by Phakopsora pachyrhizi in soybean correlate
with fungal penetration and haustoria formation, Schneider KT, van de Mortel M,
Bancroft TJ, Braun E, Nettleton D, Nelson RT, Frederick RD, Baum TJ, Graham MA,
Whitham SA. Plant Physiol. 2011 Sep;157(1):355-71
6. Complete transcriptome of the soybean root hair cell, a single-cell model, and its alteration
in response to Bradyrhizobium japonicum infection, Libault M, Farmer A,
Brechenmacher L, Drnevich J, Langley RJ, Bilgin DD, Radwan O, Neece DJ, Clough SJ,
May GD, Stacey G. Plant Physiol. 2010 Feb;152(2):541-52
c) Transcriptional activation of Medicago truncatula homologs of the Arabidopsis ALD1 gene
Treatment
Medtr2g008430
Sinorhizobium 20
meliloti and
Meloidogyne
incognita
Ralstonia
3
solanacearum
The values represent CV in %
Medtr4g092620
4
Reference #
GEO
Accession:
GSE19872
Database
NCBI GEO via
PLEXdb
2
GEO
Accession:
GSE18473
NCBI GEO via
PLEXdb
(CV, coefficient of variation of the treatment means)
4
d) Transcriptional activation of rice homologs of the Arabidopsis ALD1 gene
Treatment
Magnaporthe
grisea
Magnaporthe
grisea
Xanthomonas
oryzae pv.
oryzicola
Xanthomonas
oryzae pv.
oryzicola
Magnaporthe
grisea
LOC_Os03g18810 LOC_Os03g09910 Reference #
7
3
1
2
12
2
2
3
2
6
1
7
Xanthomonas
oryzae pv.
oryzicola
Magnaporthe
grisea
43
5
1
1
Xanthomonas
oryzae pv.
oryzicola
Blumeria
graminis f. sp.
hordei
Xanthomonas
oryzae pv.
oryzicola
1
2
1
3
2
5
GEO
Accession:
GSE16793
GEO
Accession:
GSE33411
GEO
Accession:
GSE18361
GEO
Accession:
GSE36272
GEO
Accession:
GSE30941
GEO
Accession:
GSE36093
GEO
Accession:
GSE30942
3
The values represent CV in %
Database
NCBI GEO via
PLEXdb
NCBI GEO via
PLEXdb
NCBI GEO via
PLEXdb
NCBI GEO via
PLEXdb
NCBI GEO via
PLEXdb
NCBI GEO via
PLEXdb
NCBI GEO via
PLEXdb
(CV, coefficient of variation of the treatment means)
References:
Susceptibility of rice to the blast fungus, Magnaporthe grisea, Ribot C, Hirsch J,
Balzergue S, Tharreau D, Nottinghem JL, Lebrun MH, Morel JB.
J. Plant Physiol. 2008 Jan;165(1):114-24
2. The single functional blast resistance gene Pi54 activates a complex defence mechanism in
rice, Gupta SK, Rai AK, Kanwar SS, Chand D, Singh NK, Sharma TR.
J. Exp. Bot. 2012 Jan;63(2):757-72.
3. Genome-wide gene responses in a transgenic rice line carrying the maize resistance gene
Rxo1 to the rice bacterial streak pathogen, Xanthomonas oryzae pv. oryzicola, Zhou YL,
Xu MR, Zhao MF, Xie XW, Zhu LH, Fu BY, Li ZK. BMC Genomics 2010 Feb;11:78.
1.
5